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REJANE CORRÊA MARQUES

“AMAMENTAÇÃO EXCLUSIVA E ESTENDIDA COMO UM FATOR

DE PROTEÇÃO À EXPOSIÇÃO AO MERCÚRIO DERIVADO DE

VACINAS CONTENDO TIMEROSAL”

TESE SUBMETIDA À UNIVERSIDADE FEDERAL DO RIO

DE JANEIRO VISANDO A OBTENÇÃO DO GRAU DE

DOUTOR EM CIÊNCIAS

Universidade Federal do Rio de Janeiro

Centro de Ciências da Saúde

Instituto de Biofísica Carlos Chagas Filho

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Rejane Corrêa Marques

AMAMENTAÇÃO EXCLUSIVA E ESTENDIDA COMO UM FATOR DE PROTEÇÃO

À EXPOSIÇÃO AO MERCÚRIO DERIVADO DE VACINAS CONTENDO

TIMEROSAL

Tese apresentada ao Programa de Pós-

Graduação em Ciências Biológicas (Biofísica)

do Instituto de Biofísica Carlos Chagas Filho,

Universidade Federal do Rio de Janeiro, como

parte dos requisitos necessários à obtenção

do título de Doutor em Ciências.

Orientador: Prof. Dr. Olaf Malm

Co-orientador: Prof. Dr. José Garrofe Dórea

Rio de Janeiro

2007

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iii

FOLHA DE APROVAÇÃO

Rejane Corrêa Marques

AMAMENTAÇÃO EXCLUSIVA E ESTENDIDA COMO UM FATOR DE PROTEÇÃO

À EXPOSIÇÃO AO MERCÚRIO DERIVADO DE VACINAS CONTENDO

TIMEROSAL

Rio de Janeiro, 19 de dezembro de 2007.

________________________

Orientador: Prof. Dr. Olaf Malm

(IBCCF/UFRJ)

________________________

Co-orientador: Prof. Dr. José Garrofe Dórea

(DN/UnB)

________________________

Revisor: Prof. Dr. Jean Remy Davee Guimarães

(IBCCF/UFRJ)

________________________

Prof

. Dr

. Leny Alves Cavalcante

(IBCCF/UFRJ)

________________________

Prof. Dr. Wolfgang Christian Pfeiffer

(IBCCF/UFRJ)

________________________

Prof. Dr. João Paulo Machado Torres

(IBCCF/UFRJ)

DEDICATÓRIA

A Francisca (in memoriam), que me ensinou com

seu exemplo de perseverança e cuja compreensão

da vida e amor ao próximo transcende o limite das

palavras;

Ao meu companheiro, Jean, e meu filho, Franco

que me mostram todos os dias o quanto o amor

vale a pena.

AGRADECIMENTOS

Embora uma tese de doutorado seja uma obra coletiva, a responsabilidade de

escrevê-la e o estresse daí decorrentes são essencialmente individuais. Várias

pessoas contribuíram para que este trabalho. A todas elas minha incondicional

gratidão. Corro o risco de não fazer jus ao agradecimento. Será difícil demonstrar o

encanto das energias e impulsos trocados. Por tudo isso eu enfatizo, muito além do

protocolo, um significado: o da construção de uma verdadeira rede de afeto e

solidariedade. Devo contar que este não foi um percurso fácil. Por vezes pareceu

interminável, principalmente pelos percalços pessoais de toda ordem que me

atropelaram. Esses entraves, longe de ofuscarem a caminhada, acrescentaram-lhe

brilho. E, ao invés de me deterem, estimularam-me ainda mais. Nesses três anos

arrisquei-me em uma nova cidade, novo ambiente de trabalho, novos amigos. Dei

asas à imaginação, ao meu conhecimento, minha capacidade. Imagine o significado

disso para uma pessoa inquieta, indócil como eu. Gostei até de aprender que

existem pessoas que se identificam, ou não. Porém, o que eu mais gostei foi

aprender que o esforço vale à pena. Hoje eu sei. O mais importante é aprender a

aprender. Céus! Como existem coisas a serem aprendidas! Não sei se o que sinto

hoje é diferente de antes... Mas eu gostei muito.

Vamos aos agradecimentos...

Às mães e crianças participantes deste estudo, minha profunda gratidão.

À Universidade Federal de Rondônia (UNIR) e ao Instituto de Biofísica Carlos

Chagas Filho (IBCCF/UFRJ), pela oportunidade de crescimento intelectual.

Olaf Malm, pela orientação e oportunidade oferecida, pelo bom convívio nestes três

anos de trabalho. Com você tive a oportunidade de enriquecer meu conhecimento.

Obrigado pelas suas palavras sempre otimistas de certeza no nosso sucesso e,

sobretudo, pelo carinho e paciência.

José Garrofe Dórea, por sempre demonstrar que acredita no meu potencial, pelo

apoio dado nas discussões sobre os rumos deste trabalho, pelos ensinamentos

sobre arte e ciência, por todo o tempo dedicado a mim, por entender minhas dúvidas

e angústias; pelo esforço conjunto, e, principalmente, pela parceria, amizade,

carinho e orientação. Você é mais que um orientador. Muito obrigado.

Ao meu bom menino Franco, minha maior razão para todo esse esforço, meu

carinho, minha paixão e meu colo, que este seja motivo de orgulho e coragem na

sua transformação em um grande homem. Obrigado pela paciência com o meu

nervosismo, por compreender as minhas faltas, pelos inúmeros finais de semana

passados dentro de casa para me dar apoio, pela falta de assistência nas provas do

colégio e faculdade, pelos milhares de beijos que me deu e pelo amor que tem por mim.

Jean Remy, minha melhor companhia nas montanhas, florestas, rios e mar. Meu

porto seguro em todas as aventuras, até nas mais ousadas. Comungar com você na

crença de que a ciência e a ética devem caminhar juntas na construção de uma

sociedade mais íntegra, é uma dádiva. Obrigado por enfrentar ao meu lado todos os

obstáculos e pelo amor que nos une. Nenhuma palavra pode definir o sentido que

você tem na minha vida.

Minha família, meus queridos; por estar ao meu lado mesmo quando estou distante.

Pela rara paciência com a qual enfrentaram esta travessia, nem sempre tranqüila,

mas sempre cercada de apoio, compreensão e amor. Amo vocês.

Papai e mamãe (in memoriam), meus exemplos, luta, dignidade, amor, solidariedade

e respeito. Como vocês me fazem falta! O espírito de superação e sobrevivência dos

dois me acompanha.

Katiane Guedes Brandão, Rayson Corrêa Marques e Verusca Gomes dos Santos,

meus parceiros desde o início desse trabalho, quando ninguém, além de nós quatro,

acreditava nele. Por estarem sempre dispostos a me ajudar e pela maneira

carinhosa com que sempre o fizeram. Este trabalho não existiria sem vocês. Somos

uma grande equipe.

Aos meus amigos queridos, Eduardo, Roberto e Liliane, pela dedicação, pelo apoio

nos momentos mais difíceis e pela compreensão nas minhas ausências em

aniversários, jantares, cinemas e mesas de bar.

Wanderley Rodrigues Bastos pela amizade, parcerias, ensinamentos e ajuda no

trabalho; pela forma sua forma carinhosa de tratar todos a sua volta.

José Vicente Elias Bernardi, pela parceria, incentivo e ajuda (empolgada) com as

análises estatísticas que eu, invariavelmente, não entendia, mas você estava

sempre disposto a explicar.

Mauro César Geraldes, pelo carinho, acolhimento, proteção e compreensão nos

momentos cruciais desta jornada. Pelo modo alegre e irreverente com que leva a vida

e trata a todos a sua volta; pela amizade e parceria nos trabalhos, viagens e festas.

João Paulo Machado Torres e Márlon Freitas Fonseca, pela postura confiante e

receptiva com me receberam e pelos momentos de cooperação e amizade que

desfrutei. O doutorado teria sido mais difícil sem vocês.

Ao Departamento de Enfermagem (UNIR), pela liberação para realizar essa

maravilhosa jornada que não foi apenas de doutoramento, mas de crescimento

intelectual, pessoal, profissional e de formação de uma rede de parcerias e

verdadeiras amizades.

A UNESCO, Ministério da Saúde, CNPq e CAPES cujos apoios financeiros

possibilitaram a realização deste trabalho.

Aos Professores do IBCCF com quem tive o prazer de aprender, nas disciplinas,

seminários, reuniões e no dia-a-dia do doutorado.

Aos colegas do Laboratório de Radioisótopos, pelos excelentes momentos de

trocas: Renata, Dani Botaro e Kasper, Marianna, Larissa, Natasha, Cláudio, Márcio,

Rodrigo, Antonio, Sérgio, Laílson, Ronaldo, Bete, Ricardo, Madalena, Giselle...

SAUDADES!

vii

EPÍGRAFE

Aqui nessa casa ninguém quer a sua boa educação.

Nos dias que tem comida, comemos comida com a

mão.

E quando a polícia, a doença, a distância ou alguma

discussão nos separam de um irmão, sentimos que

nunca acaba de caber mais dor no coração.

Mas não choramos à toa.

Aqui nessa tribo ninguém quer a sua catequização,

falamos a sua língua, mas não entendemos o seu

sermão.

Nós rimos alto, bebemos e falamos palavrão, mas

não sorrimos à toa.

Aqui nesse barco ninguém quer a sua orientação.

Não temos perspectiva, mas o vento nos dá a direção.

A vida que vai à deriva é a nossa condução.

Mas não seguimos à toa.

Volte para o seu lar. Volte para lá!

ARNALDO ANTUNES

viii

RESUMO

MARQUES, Rejane Corrêa: Amamentação exclusiva e estendida como fator de

proteção à exposição ao mercúrio derivado de vacinas contendo timerosal. Rio

de Janeiro, 2007. Tese (Doutorado em Ciências Biológicas – Biofísica) – Instituto de

Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de

Janeiro, 2007.

Crianças são expostas ao mercúrio (Hg) proveniente das mães (através da

placenta e aleitamento materno), do ambiente (alimentos), e em muitas partes do

mundo, das vacinas contendo timerosal (VCT) recebidas durante a imunização. O

timerosal contém 49.6% etilmercúrio (EtHg) por peso. Devido às incertezas

associadas a um possível aumento de déficits neurodesenvolvimentais entre

crianças imunizadas, as vacinas conservadas com timerosal não são usadas desde

2004 nos Estados Unidos da América (EUA) e na União Européia (UE), mas são

amplamente produzidas e utilizadas em países em desenvolvimento, caso do Brasil.

Nós investigamos o impacto do timerosal no cabelo de crianças que receberam VCT

de acordo com o calendário de imunização preconizado pelo Ministério da Saúde do

Brasil. Usando o Hg total no cabelo como marcador de exposição pós-natal ao Hg

orgânico (Hg inorgânico e MeHg do leite materno; EtHg do timerosal) nós estudamos

sua associação com a escala de Gesell (QD) mensurada aos 6, 36 e 60 meses e a

influência da amamentação exclusiva no crescimento e desenvolvimento de crianças

de Porto Velho, Rondônia. As vacinas forneceram uma exposição EtHg de 12,5 μg

ao nascimento (mínima) a 37,5 μg aos seis meses (máxima), podendo perfazer um

total de 325 μg de Hg em cinco anos. Enquanto as mães apresentaram as

concentrações mais altas de Hg no parto, as crianças apresentaram valores mais

altos aos seis meses após completar o esquema de imunização com VCT para o

período. Posteriormente, a tendência descendente do Hg no cabelo demonstrado

pelas crianças coincidiu com o desmame e menos freqüentemente com o período de

vacinação. O tempo de amamentação e o Hg no cabelo foram, cada um,

significativamente correlacionados com QD, mas de maneiras opostas: o tempo de

amamentação foi positiva e significantemente associado com o QD aos cinco anos;

as concentrações de Hg no cabelo foram negativa e significativamente

correlacionadas com QD aos seis meses (r=-0.3329; p=0.0022) e cinco anos (r=-

0.8029; p=0.0106), mas não aos 36 meses (r=-0.1722; p=0.1218). O resultado dos

QD aos cinco anos dependeu do QD aos três anos, que por sua vez, foi influenciado

pelas variáveis do desenvolvimento e de exposição ao Hg. O QD aos seis meses foi

significantemente influenciado pela exposição pré-natal (Hg no cabelo materno e

infantil) e pós-natal aos seis meses. Nós acreditamos que o aleitamento materno é

uma importante estratégia para a proteção do SNC de crianças frente aos desafios

da exposição precoce ao Hg. O presente estudo foi financiado pelo Ministério da

Saúde do Brasil (MS) e Organização das Nações Unidas para Educação, Ciência e

Cultura (UNESCO), processo SC27824/2005/914-BRA2000-Decit-PRODOC.

Palavras-chave: Timerosal; etilmercúrio; vacinas; crianças; aleitamento materno;

neurodesenvolvimento; Escala de Gesell.

ABSTRACT

MARQUES, Rejane Corrêa: Amamentação exclusiva e estendida como fator de

proteção à exposição ao mercúrio derivado de vacinas contendo timerosal. Rio

de Janeiro, 2007. Tese (Doutorado em Ciências Biológicas – Biofísica) – Instituto de

Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de

Janeiro, 2007.

Children are exposed to Hg from mothers (via placenta and lactation),

environment (food), and in many parts of the world by thimerosal-containing vaccines

(TCV) during immunization. Thimerosal contains 49.6% Hg by weight. Because of

uncertainties associated with a possible rise in neurodevelopmental deficits among

vaccinated children, TCV have not been used since 2004 in the USA and the EU but are

widely produced and used in other developing countries. This longitudinal study (birth to

5 years) investigated the impact of thimerosal on the total Hg in hair of breast-fed infants

that received TCV according to the immunization schedule recommended by the

Ministry of Health of Brazil. Using total HHg as a marker of post-natal organic Hg

exposure (inorganic and methylmercury from breast milk, and ethylmercury from

thimerosal) we studied its association with Gesell schedules measured at 6m, 36m and

60m, and the influence of exclusive breastfeeding on growth of this sample of children

from Porto Velho, Western Amazon. TCV provided an ethylmercury (EtHg) exposure of

12.5 μgHg (minimum) up to 37.5 μgHg (maximum). The cumulative exposure by age 5

years could reach 325 μgHg. While mothers had the highest HHg concentrations at

childbirth, infants showed the highest HHg values at 6 months after the recommended

six shots of immunization with TCV; after that, the downward trend in HHg shown by

children coincided with both weaning and less frequent vaccination period (5 years).

Length of lactation and HHg were each significantly correlated with Gesell scores, but in

opposite ways: length of lactation was positive and significantly correlated with all Gesell

scores at 60m; HHg concentrations were negative and significantly correlated with

Gesell scores at 6m (r=- 0.3329; p=0.0022) and 60m (r=-0.8029; p=0.0106), but not at

36m (r=- 0.1722; p=0.1218). The DQ outcome at 60m depended on DQ at 36m that in

turn was influenced by infants developmental and Hg exposure variables. DQ at 6m was

significantly influenced by prenatal (maternal and infant HHg at birth) and post-natal Hg

exposure at 6 months. Until a more refined approach to recognize sensitive children to

TCV-EtHg exposure, the ND benefit of breastfeeding should suffice to recommend it in

situations of uncertainty. United Nations Educational, Scientific, and Cultural

Organization (UNESCO) and Ministry of Health of Brazil (MS) supported this work, grant

SC27824/2005/914BRA2000 Decit PRODOC.

Key words: Thimerosal, ethylmercury neurodevelopment, vaccines, Gesell scores,

breastfeeding.

LISTA DE ILUSTRAÇÕES

LISTA DE FIGURAS

FIGURA 1.ESTRUTURA QUÍMICA DO TIMEROSAL .................................................................................................. 20

FIGURA 2. ESTRUTURA QUÍMICA DO ETHG .......................................................................................................... 20

FIGURA 3.THE COW-POCK OR THE WONDERFUL EFFECTS OF THE NEW .......................................................... 25

FIGURA 4. CERTIFICADO DE VACINAÇÃO COMPULSÓRIA DE WALTER WILLIAN CHARLES VICENT. ..................... 26

FIGURA 5. CERTIFICADO DE VACINAÇÃO BEM-SUCEDIDA DE WALTER CHARLES WILLIAN VICENT. ................................................ 27

FIGURA 6. GRÁFICO DAS REIVINDICAÇÕES APRESENTADAS JUNTO AO PROGRAMA DE COMPENSAÇÕES AS

INJÚRIAS VACINAIS DOS ESTADOS UNIDOS (US VACCINE INJURY COMPENSATION PROGRAM), NO

PERÍODO DE 1989-2007. ............................................................................................................................ 30

LISTA DE QUADROS

QUADRO 1. DOSE DE MERCÚRIO EM VACINAS PARA USO INFANTIL .................................................................. 33

QUADRO 2. LIMITES DE EXPOSIÇÃO AO METILMERCÚRIO NA DIETA DE UM INDIVÍDUO ADULTO. ....................... 34

QUADRO 3. EXPOSIÇÃO AO MERCÚRIO DE VACINAS PEDIÁTRICAS CONSERVADAS COM TIMEROSAL. ................. 35

QUADRO 4. FONTES DE DADOS EPIDEMIOLÓGICOS PRIMÁRIOS INVESTIGANDO POSSÍVEIS ASSOCIAÇÕES

ENTRE VACINAS CONSERVADAS COM TIMEROSAL E APARECIMENTO DE SINTOMAS NEUROLÓGICOS. ....... 44

LISTA DE SIGLAS E ABREVIATURAS

AAP – AMERICAN ASSOCIATION OF PEDIATRICS

AIG – ADEQUADO PARA A IDADE GESTACIONAL

ANVISA – AGÊNCIA NACIONAL DE VIGILÂNCIA SANITÁRIA

ATSDR – AGENCY FOR TOXIC SUBSTANCES AND DISEASE REGISTRY

CDC – NATIONAL CENTER FOR DISEASE CONTROL AND HEALTH PROMOTION

CEP – COMITÊ DE ÉTICA EM PESQUISA COM SERES HUMANOS

DDA – DISTÚRBIO DO DÉFICIT DE ATENÇÃO

DHHS – DEPARTMENT OF HEALTH AND HUMAN SERVICES

dT – VACINA DUPLA ADULTO DIFTERIA-TÉTANO

DT – VACINA DUPLA INFANTIL DIFTERIA-TÉTANO

DTP – VACINA TRÍPLICE BACTERIANA (DIFTERIA, TÉTANO E COQUELUCHE)

DTP + HIB – VACINA QUÁDRUPLA BACTERIANA, ASSOCIANDO DTP À Hib

EDGA – ESCALA DESENVOLVIMENTAL DE GESELL E AMATRUDA

EtHg – ETILMERCÚRIO

EUA – ESTADOS UNIDOS DA AMERICA

FDA – FOOD AND DRUGS ADMINISTRATION

GIG – GRANDE PARA A IDADE GESTACIONAL

Hep B – VACINA CONTRA HEPATITE B

Hg – MERCÚRIO

Hib – Haemophilus Influenzae tipo b

IOM – INSTITUTE OF MEDICINE

ISRC – IMMUNIZATION SAFETY REVIEW COMMITTEE

LOAEL - NÍVEL MAIS BAIXO EM QUE EFEITO ADVERSO FOI OBSERVADO

MeHg – METILMERCÚRIO

SRC ou MMR – VACINA TRÍPLICE VIRAL (SARAMPO, RUBÉOLA E CAXUMBA)

MS – MINISTÉRIO DA SAÚDE

NCHS – NATIONAL CENTER FOR HEALTH STATISTICS

ND – NEURODESENVOLVIMENTAIS

OMS – ORGANIZAÇÃO MUNDIAL DA SAÚDE

OPAS – ORGANIZAÇÃO PAN-AMERICANA DE SAÚDE

xii

PC – PERÍMETRO CEFÁLICO

PIG – PEQUENO PARA A IDADE GESTACIONAL

PNI – PROGRAMA NACIONAL DE IMUNIZAÇÃO BRASILEIRO

PUFA – POLYUNSATURED FATTY ACIDS.

QD – QUOCIENTE DE DESENVOLVIMENTO

QI – QUOCIENTE DE INTELIGÊNCIA

RN – RECÉM-NASCIDO

SIDA – SÍNDROME DA IMUNO DEFICIÊNCIA HUMANA ADQUIRIDA

SNC – SISTEMA NERVOSO CENTRAL

TDAH – TRANSTORNO DE DÉFICIT DE ATENÇÃO E HIPERATIVIDADE

UE – UNIÃO EUROPÉIA/COMUNIDADE EUROPÉIA

UFRJ – UNIVERSIDADE FEDERAL DO RIO DE JANEIRO

UNESCO – ORGANIZAÇÃO DAS NAÇÕES UNIDAS PARA EDUCAÇÃO, CIÊNCIA

E CULTURA

UNIR – FUNDAÇÃO UNIVERSIDADE FEDERAL DE RONDÔNIA

VCT – VACINAS CONSERVADAS COM TIMEROSAL

VICP – VACCINE INJURY COMPENSATION PROGRAM

xiii

Sumário

DEDICATÓRIA .......................................................................................................................................... IV

AGRADECIMENTOS ................................................................................................................................. V

EPÍGRAFE ................................................................................................................................................ VII

RESUMO .................................................................................................................................................. VIII

ABSTRACT ................................................................................................................................................ IX

LISTA DE ILUSTRAÇÕES ......................................................................................................................... X

LISTA DE FIGURAS ....................................................................................................................................... X

LISTA DE QUADROS ..................................................................................................................................... X

LISTA DE SIGLAS E ABREVIATURAS .................................................................................................. XI

SUMÁRIO ................................................................................................................................................... XIII

1 INTRODUÇÃO ................................................................................................................................. 15

1.1 TIMEROSAL ..................................................................................................................................... 20

1.2 VACINAS .......................................................................................................................................... 22

1.2.1 Eventos Adversos e Vacinas Conservadas com Timerosal ................................................. 29

1.2.2 Quantificando o timerosal das vacinas pediátricas (Marques et al., 2007b) ........................ 32

1.3 LIMITES DE EXPOSIÇÃO AO MERCÚRIO: LIMITAÇÕES E CERTEZAS .................................................. 33

1.4 A PLAUSIBILIDADE BIOLÓGICA ........................................................................................................... 35

1.4.1 A farmacocinética e toxicologia do MeHg e do EtHg ............................................................ 38

1.4.2 Reações de hipersensibilidade após exposição a baixas doses de timerosal .................. 40

1.4.3 Evidências da relação dose-resposta na exposição a altas doses de EtHg ..................... 40

1.5 TIMEROSAL E DISTÚRBIOS DO DESENVOLVIMENTO NEUROLÓGICO ................................................. 41

1.5.1 Estudos Laboratoriais 42

1.5.2 Estudos Epidemiológicos 43

2 A HIPÓTESE .................................................................................................................................... 45

2.1 AS VACINAS SÃO SEGURAS? .............................................................................................................. 45

2.1.1 CONTROVÉRSIAS ATUAIS SOBRE A SEGURANÇA DAS VACINAS ......................................................... 49

2.2 DILEMA: COMUNGAR NO DOGMA OU SEGUIR O INSTINTO CIENTÍFICO? ............................................. 54

3 OBJETIVOS ..................................................................................................................................... 56

3.1 OBJETIVO GERAL ............................................................................................................................... 56

3.2 OBJETIVOS ESPECÍFICOS................................................................................................................... 56

4 MATERIAL E MÉTODOS ................................................................................................................ 57

4.1 CARACTERIZAÇÃO DA AMOSTRA ....................................................................................................... 57

4.2 A AVALIAÇÃO DO DESENVOLVIMENTO INFANTIL ................................................................................ 59

4.3 EXPOSIÇÃO ESTIMADA AO MERCÚRIO DAS VACINAS (TIMEROSAL INJETADO) E ALEITAMENTO

MATERNO .......................................................................................................................................................... 61

4.4 DETERMINAÇÃO DO NÍVEL DE EXPOSIÇÃO AO MERCÚRIO ................................................................. 61

4.5 ANÁLISE ESTATÍSTICA ........................................................................................................................ 62

4.6 CONSIDERAÇÕES ÉTICAS .................................................................................................................. 62

5 RESULTADOS ................................................................................................................................. 63

xiv

6 DISCUSSÃO .................................................................................................................................... 69

7 CONCLUSÕES ................................................................................................................................ 77

REFERÊNCIAS BIBLIOGRÁFICAS ..................................................................................................... 78

ANEXOS ...................................................................................................................................................... 90

ARTIGO 1. MATERNAL MERCURY EXPOSURE AND NEURO-MOTOR DEVELOPMENT IN BREASTFED INFANTS

FROM PORTO VELHO (AMAZON), BRAZIL......................................................................................................... 91

ARTIGO 2. HAIR MERCURY IN BREAST-FED INFANTS EXPOSED TO THIMEROSAL-PRESERVED VACCINES .. 101

ARTIGO 3. TIME OF PERINATAL IMMUNIZATION, THIMEROSAL EXPOSURE AND NEURODEVELOPMENT AT 6

MONTHS IN BREASTFED INFANTS .................................................................................................................... 107

ARTIGO 4. PRINCIPAL COMPONENT ANALYSIS AND DISCRIMINATION OF VARIABLES ASSOCIATED WITH PRE-

AND POST-NATAL EXPOSURE TO MERCURY ................................................................................................... 116

ARTIGO 5. CHANGES IN CHILDREN HAIR-HG CONCENTRATIONS DURING THE FIRST 5 YEARS: MATERNAL,

ENVIRONMENTAL AND IATROGENIC MODIFYING FACTORS.............................................................................. 125

ARTIGO 6. MATERNAL FISH CONSUMPTION IN THE NUTRITION TRANSITION OF THE AMAZON BASIN:

GROWTH OF EXCLUSIVELY BREASTFED INFANTS DURING THE FIRST 5 YEARS ............................................. 133

TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO .................................................................. 149

QUESTIONÁRIO MÃES E RECÉM-NASCIDOS ................................................................................................... 150

QUESTIONÁRIO PARA CRIANÇAS 6 MESES ................................................................................................... 152

TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO .................................................................. 154

QUESTIONÁRIO PARA CRIANÇAS DE 3 A 5 ANOS .......................................................................................... 155

ETAPAS DO DESENVOLVIMENTO INFANTIL SEGUNDO GESELL ...................................................................... 157

APÊNDICE .............................................................................................................................................. 158

CALENDÁRIO BÁSICO DE VACINAÇÃO DA CRIANÇA 2007 ........................................................................................... 158

1 INTRODUÇÃO

O mercúrio (Hg) é encontrado no ambiente em diversas formas químicas: Hg

elementar, inorgânico, e orgânico (etil-, metil-, alquil-, ou fenil-Hg). A química do Hg

modula sua toxicidade e metabolismo. Enquanto o Hg inorgânico atua principalmente

nos rins, o Hg metálico volátil e, sobretudo, o metilmercúrio (MeHg) afetam o sistema

nervoso central (SNC). As atuais questões sobre a toxicidade do Hg estão focadas

nos prováveis danos neurológicos em baixos níveis de exposição. Conquanto a

exposição a algum nível de Hg seja universal, a avaliação quantitativa mostrou que

os três maiores contribuidores da exposição ao Hg para a população geral são: dieta

(basicamente peixe), amálgama dental e alguns produtos farmacológicos (Clarkson

e Magos, 2006; Dórea, 2007; Marques et al., 2007a).

As formas químicas do Hg são cruciais no entendimento e na avaliação da

exposição infantil e do risco de toxicidade. A vulnerabilidade do cérebro humano em

desenvolvimento é a mais importante janela para os insultos dos compostos de Hg

(Castoldi, 2003; Dórea, 2007). A gênese e o desenvolvimento do SNC durante a

gravidez e lactação podem ser afetados por múltiplas causas, variando de nutrição

materna a exposição a substâncias tóxicas. Nesta janela de tempo específica, o Hg

pode afetar funções neurocomportamentais. A exposição moderada pode resultar

em sintomas de atraso (não observados ao nascimento) no andar e falar, além da

persistência de reflexos perinatais (Davidson et al., 1998; Grandjean et al., 1997;

WHO, 1976; 1990). Pondera-se que a exposição materna ao MeHg (do consumo de

peixes) pode afetar o neurodesenvolvimento, diminuir a inteligência e provocar

alteração do comportamento da prole (Clarkson, 1998; Grandjean et al., 2006). O

recém-nascido (RN) é particularmente vulnerável a estes efeitos porque seu SNC

ainda está se desenvolvendo.

Outra fonte de preocupação relacionada ao Hg é o seu uso, na forma de

timerosal, como agente anti-séptico de escolha para prevenir a contaminação

bacteriológica em vacinas, apresentando concentração de timerosal até 0,01%

(Amanna e Slifka, 2005). Como o etilmercúrio (EtHg) recebido na vacinação não é

parte da alimentação, ele pode atravessar a barreira e detoxificação do sistema

entero-hepático, não sendo fisiologicamente processado, podendo migrar para o

cérebro (Cory-Slechta, 2005; Dórea, 2007; Marques et al., 2007a). Devido a sua

lipossolubilidade, o EtHg pode se acumular em seres humanos e cruzar a barreira

hematoencefálica e placentária, apresentando uma meia-vida curta no sangue. Pode

depositar-se no SNC, onde é transformado posteriormente em Hg inorgânico

acumulando-se no cérebro animal e humano (Clarkson, 2002).

Em razão da limitada base de informação disponível, concluiu-se que os efeitos

nocivos do EtHg podem ser análogos àqueles do MeHg, outro organomercurial de

conhecida neurotoxicidade (Malm, 2001; Clarkson et al., 2007). É fato que o Hg é

tóxico para o cérebro humano em desenvolvimento, e, por isso, evitar a exposição

durante os períodos críticos do desenvolvimento do SNC tem sido alvo de estratégias

de saúde pública. Para proteger crianças dos efeitos nocivos do Hg há recomendações

para se evitar a ingestão de peixes durante a gravidez (Cohen et al., 2005, Dórea,

2007) e, apesar do uso de vacinas conservadas com timerosal (VCT) ter sido banido

nos Estados Unidos (EUA) e União Européia (UE), a Organização Mundial da Saúde

(OMS) consideram-nas seguras para uso pediátrico (CDC, 2001; WHO, 2000, 2007).

Sem uma sistemática síntese do conhecimento, as restrições para o consumo

de peixe se tornaram regra para prevenir os efeitos neurotóxicos do metilmercúrio

(MeHg) em recém-nascidos (Cohen et al., 2005; Dorea, 2007). Contudo, o consenso

precedeu as evidências no caso de exposição à EtHg de VCT, quando o Comitê de

Avaliação sobre Segurança da Imunização do Instituto de Medicina dos EUA

(Immunization Safety Review Committee – IOM/ISRC) concluiu que a hipótese de

que as VCT poderiam estar associadas com desordens neurodesenvolvimentais

(DN) era biologicamente plausível (US CDC, 1999; 2001). Apesar dos estudos

epidemiológicos não mostrarem associações entre VCT e autismo, há base para as

preocupações referentes a outras DN.

A capacidade de um recém-nascido de lidar com o Hg é modulada pelo seu

grau de imaturidade, ou seja, limitada pela produção da bile e função renal, vias

metabólicas subdesenvolvidas. Essas injúrias são acentuadas mais tarde por

diversos fatores associados com a exposição ao Hg: fonte (formas químicas de Hg

inorgânica e orgânica), rota (enteral/aleitamento materno versus parenteral/VCT) e

via (intrínseco na matriz protéica do leite humano vs. extrínseco na forma química

pura-EtHg) (Dorea, 2007).

RN e crianças (especialmente aquelas de países menos desenvolvidos)

representam a população de maior risco para DN (devido à imaturidade anatômica,

fisiológica e bioquímica) e aquelas mais expostas ao Hg – quando imunizadas com

VCT e após consumo de peixe materno (Marques et al., 2007b). Devido à grande

variabilidade na suscetibilidade infantil e circunstâncias modificáveis (desenvolvimento

anatômico e funcional), há um risco presumível de DN transitória sutil como seqüela

de VCT. Entretanto, como os efeitos sutis da exposição ao Hg sobre o SNC podem

se manifestar muito tempo depois da exposição e, porquanto este problema está

vinculado à ausência de marcadores apropriados (de exposição e efeito), há sérias

dificuldades em estabelecer causa e efeito (ou mesmo relações). Por isso, a conexão

entre DN e exposição precoce a esquemas de vacinação com VCT continua intricada

(Marques et al., 2007c; 2007d; 2007e). Portanto, isto é importante para distinguir

casos de baixo risco e alto risco (relacionados com a variação na imaturidade ou

suscetibilidade de crianças a termo).

A breve duração da amamentação é considerada um fator de risco para sinais

de distúrbio do déficit de atenção (DDA) e de hiperatividade. Presume-se que haja

um ganho de 3.2 pontos no quociente de inteligência – QI (após ajustamento para o

QI materno) entre seis meses e 15 anos (Anderson, et al., 1999; Dorea, 2007). O

risco irrefutável da dose de timerosal (0.01%) usado nas vacinas infantis é

mascarado pela insuficiência de conhecimento científico sobre a toxicocinética e

toxicodinâmica do EtHg em crianças com diferentes taxas de maturidade (Dorea,

2007, Marques et al., 2007e). Interações complexas são entraves para definir se a

segurança da dose de EtHg é superestimada devido a interação antagonista com o

aleitamento materno ou subestimada devido a alimentação alternada e outras co-

exposições em RN e crianças (Dórea, 2007; Marques et al., 2007d; 2007e).

Os estudos recentes associando exposição ao Hg e DN em crianças

dependeram das concentrações desse metal no cabelo. O cabelo é composto de

uma proteína complexa formada por aminoácidos que se ligam ao MeHg. Admite-se

que a ocorrência de DN é resultado do consumo de peixe contaminado com MeHg,

que rapidamente de liga ao cabelo (Cernichiari et al., 2007). Além disso, estudos

epidemiológicos direcionados para a exposição ao consumo de peixes têm usado

concentrações de Hg no cabelo como um indicador confiável.

Entretanto, durante os primeiros cinco anos de vida, crianças podem ser

expostas ao Hg nas mudanças das suas fontes de alimentação: do leite materno (ou

fórmulas) para dietas de desmame e, mais tarde, de outras fontes alimentares (que

podem incluir o peixe) quando passar a se alimentar com ―comida de adulto‖. Essas

crianças podem ser expostas, também, a outra forma de Hg: ao EtHg quando

imunizadas com vacinas conservadas com timerosal (Marques et al., 2007a, 2007e).

Crianças amazônicas são expostas a MeHg via transferência placentária e

leite materno (Barbosa et al., 1998; Barbosa e Dorea, 1998; Marques et al., 2007f).

Neste contexto, o aumento no risco de DN associado à MeHg naturalmente presente

nos peixes dos rios amazônicos foi fortemente sugerido (Grandjean et al., 1999). A

probabilidade do aumento de DN nestes estudos foi baseada nas concentrações de

Hg no cabelo (biomarcador de consumo de peixe). Nós levantamos a hipótese que,

pelo menos nos primeiros seis meses de vida, as concentrações de Hg no cabelo

podem ser influenciadas pelo pesado calendário vacinal (com vacinas conservadas

com timerosal) (Marques et al., 2007a). Além disso, durante os primeiros cinco anos,

a susceptibilidade a fatores modificadores permanece desconhecida, mas as

mudanças fisiológicas e alimentares (amamentação, desmame e dieta familiar

tradicional) provavelmente influenciam essas mudanças (Marques et al., 2007e).

As incertezas sobre a segurança das VCT e a controvérsia sobre a associação

entre o EtHg das vacinas e DN, incluindo autismo, são questões polêmicas que

envolvem tanto os cientistas quanto a sociedade (Bernard et al., 2001; Bigham e

Copes, 2005; Clements e Mcintyre; 2006; Coleman, 2003; COMED, 2007; Geier e

Geier, 2007a; Malm, 2001; Mutter et al., 2005; Marques et al., 2007b; 2007c; 2007d).

Por isso, vacinas VCT não são administradas em crianças norte-americanas desde

2002 e européias desde 2004. Contudo, por recomendação da OMS, o timerosal

ainda é amplamente utilizado como conservantes de vacinas produzidas para os

países em desenvolvimento, como o Brasil (Migowski, 2007; Marques et al., 2007b).

1.1 TIMEROSAL

O timerosal (ou tiomersal) é um composto organomercurial proveniente do sal

sódico do ácido o (etilmercuritio) benzóico que contém 49.6% etilmercúrio (EtHg) por

peso. É um pó cristalino de cor branca a amarelo-clara, solúvel em água e em

metanol, e insolúvel em éter e benzeno (Prado et al., 2004). É representado pela

fórmula estrutural C

HgNaO

S e sua estrutura química é a seguinte:

Figura 1.Estrutura Química do Timerosal

Também conhecido como: ―thiomersal‖ (Europa), ―thimerosol‖, ―thimerosal‖

(EUA), ―merthiolate‖, mertiolate, tiomersal, timerosol, etilmercúrico de sódio e

mertiolato de sódio, foi historicamente adicionado a vacinas como agente

antimicrobiano (0.005 a 0.01%). Transforma-se no organismo em EtHg (CH3-CH2-

Hg+) e tiosalicilato, sendo um composto químico altamente instável (Figura 2).

Figura 2. Estrutura química do EtHg

A fórmula patenteada pela Eli Lilly Company é utilizada como adjuvante

farmacotécnico com função de conservante em produtos farmacêuticos, sendo

amplamente empregada em vacinas e em líquidos protetores de lentes oftálmicas

(Geier et al., 2007; Prado et al., 2004; Migowski, 2007). Durante vários anos, este

fármaco foi usado como agente bacteriostático e fungistático tópico, sendo

apresentado na forma de tintura (solução hidroalcoólica a 0,1%), geralmente,

indicado para anti-sepsia de pequenas escoriações e ferimentos. A literatura é

significativa sobre as propriedades anti-sépticas do timerosal, comparando-o com os

outros compostos químicos empregados como anti-sépticos e concluindo que sua

ação é moderada (Litter, 1986; Prado et al.; 2004).

O timerosal, usado para uso tópico, chegou a ser um dos medicamentos de

maior venda livre, pelo fato de ser único no mercado e estar aliado a uma forte

propaganda que prometia muito mais eficácia do que realmente tinha (Ball et al.,

2001; Geier et al., 2007; Prado et al., 2004; Rodrigues e Saito; 1991; Valle et al.,

1991). A avaliação risco/benefício ficava evidente quando se comparava o

coeficiente fenólico do timerosal com os demais anti-sépticos. Litter (1986) cita que o

coeficiente fenólico da polivinilpirrolidonaiodo é 200, enquanto o timerosal apresenta

40. Prado et al. (2004) descreve que o fármaco tem pouca atividade contra bactérias

gram-negativas, sendo as Pseudomonas e Staphylococcus aureus resistentes aos

derivados de amônio quaternário.

No Brasil, em 8 de junho de 2001, o Diário Oficial da União publicou a

resolução nº 528, do Ministério da Saúde (de 17 de abril do mesmo ano), que

suspendeu a venda de produtos à base de timerosal (como o mertiolate e o

mercurocromo) em todo o país. A Agência Nacional de Vigilância Sanitária (ANVISA)

proibiu o uso desse composto alegando tratar-se de uma substância mercurial que,

segundo diretrizes internacionais, ofereceria risco de toxicidade aos usuários. Em

seu comunicado à imprensa, a ANVISA diz que a decisão foi tomada ―tendo em vista

a tendência mundial da diminuição da exposição de seres humanos a produtos à

base de derivados de mercúrio‖, e determina à imediata ―proibição da utilização de

derivados de mercúrio em medicamentos fabricados no Brasil, exceto vacinas‖. O

comunicado esclarece ainda que ―o tiomersal (derivado de mercúrio) não será usado

como remédio, mas apenas como conservante de vacinas, por recomendação da

Organização Mundial de Saúde‖. (grifo meu)

A retirada abrupta do timerosal por exigência da ANVISA veio trazer a público

uma série de questionamentos, entre eles, por qual razão este fármaco ficou tanto

tempo no mercado, se desde 30 de junho de 1980 já existia a Portaria n. 07/Dimed

(DO n. 125 de 7/7/1980), revogada pela Portaria 10/80, proibindo a fabricação de

medicamentos contendo substâncias compostas de Hg isoladas ou associadas e

dava prazo de 30 dias para as empresas modificarem a formulação sob pena de

revogação do respectivo registro. O fabricante do produto referência (Merthiolate), ao

tomar conhecimento da Resolução 528/2001 da ANVISA, imediatamente substituiu o

timerosal por um composto derivado de amônio quaternário, o cloreto de benzalcônio,

que segundo Prado et al. (2004) também apresenta fracas propriedades anti-sépticas.

1.2 VACINAS

Entre as intervenções de saúde pública que tiveram maior impacto sobre a

saúde das pessoas estão a água potável e as vacinas. Graças a pioneiros como

Edward Jenner e Louis Pasteur, as vacinas evitam doenças e morte de milhões de

pessoas no mundo inteiro, todos os anos. Porém, há ainda um longo caminho a

percorrer. Quando se discute a medicina moderna, poucos temas são tão polêmicos

quanto à vacinação. É uma história repleta de jogos políticos, grandes empresas, pais

desesperados, crianças doentes e morrendo, conflitos de interesse e a segurança

nacional – que envolve a controvérsia do aborto, uma vez que muitas vacinas são

criadas utilizando células de fetos abortados (Allen, 2007; Kennedy, 2005; Moreira,

2002, Temporão, 2003; Weisberg, 2007).

O assunto é polêmico.

A oposição às vacinas não é algo realmente "novo". E apesar de contestada

desde suas origens, a vacina acumula defensores entre a maioria dos profissionais

de saúde, cientistas e autoridades que atuam na esfera da saúde coletiva, bem

como uma quantidade significativa de ferrenhos adversários. Hoje, as vacinas e os

protocolos tradicionais de imunização estão sob fogo cruzado e as pessoas tem se

perguntado se os riscos são dignos dos benefícios que elas proporcionam. Contudo,

é inegável que as vacinas são a parte de benéfica de uma estratégia global de

saúde preventiva que reduziu grandemente as mortes por doenças infecciosas e

levou à erradicação da varíola, a eliminação da poliomielite na maioria dos países e

ao controle de muitas outras doenças, incluindo sarampo, rubéola, tétano e difteria

(Allen, 2007; Moulin, 2003; Ponte, 2003; Stewart e Devlin, 2006; Weisberg, 2007).

Então, não é surpreendente que haja um debate atual sobre a segurança e a

importância das vacinas, pois isso já acontecia antes mesmo da primeira vacina ser

introduzida. A vacina foi desenvolvida a partir de uma antiga técnica praticada na

Ásia – a ―variolização‖ – que consistia em infectar um paciente são com um fluido de

uma pústula de varíola humana. Isto geralmente reduzia a severidade da infecção,

pois causava uma manifestação moderada e controlada da varíola, tornando os

inoculados imunes à doença (Stewart e Devlin, 2006; Wilson e Marcuse, 2001).

Esta prática foi introduzida no Reino Unido, em 1721, por Lady Mary Wortley

Montagu, esposa de um diplomata britânico na Turquia. Lá, ela notou que algumas

senhoras não tinham cicatrizes da doença. Prontamente, ela inoculou seus dois

filhos e se tornou uma ativista da variolização (Stewart e Devlin, 2006; Weisberg,

2007; Wilson e Marcuse, 2001). A popularidade da variolização aumentou pouco a

pouco, apesar da forte oposição de muitos médicos que igualavam a técnica ao

assassinato de uma criança, ao expô-la a doença (Stewart e Devlin, 2006). Benjamin

Franklin inicialmente se opôs a variolização, mas mudou de idéia após seu filho de 4

anos, não vacinado, morrer em decorrência da varíola. Ele escreveu em sua

autobiografia que "lamentou amargamente" não ter vacinado seu filho (Stewart e

Devlin, 2006; Weisberg, 2007).

A vacinação foi, inicialmente, realizada em 1796 por Edward Jenner. Ele

observou que ordenhadores de vaca mostravam-se imune à varíola. Todas essas

pessoas tinham se contaminado com cowpox, uma doença do gado semelhante à

varíola, pela formação de pústulas, mas que não causava a morte dos animais. Após

uma série de experiências, constatou que estes indivíduos mantinham-se refratários

à varíola, mesmo quando inoculados com o vírus. Com o pus retirado de uma pústula

de uma ordenhadora que apresentava cowpox, ele inoculou o filho de seu jardineiro.

O garoto contraiu uma infecção benigna e, dias depois, estava recuperado. Meses

depois, expôs Phipps a um pus varioloso e o menino, felizmente, não desenvolveu a

doença (Jenner, 1798).

Era a descoberta da vacina.

Jenner publicou sua pesquisa em 1798, Variolae Vacciniae, mas persistia uma

grande oposição na classe médica e na população em geral (Stewart e Devlin, 2006;

Wilson e Marcuse, 2001). Grupos religiosos alertavam para o risco da degeneração

da raça humana pelo contágio com material bovino, apelidado de ―vacalização‖ ou

―minotaurização‖ (Fig. 3). Essa generalizada oposição a vacina obrigatória de Jenner

foi deflagrada por rumores e boatos que atribuíam as mais variadas complicações a

vacinação: de febres, amputação do braço e até mesmo a morte por ―envenenamento

do sangue meses mais tarde‖.

As semelhanças entre a oposição à vacina contra a varíola e as controvérsias

atuais em torno da vacinação são óbvias.

Figura 3.The Cow-Pock or The Wonderful Effects of the New

No mundo inteiro se criou leis para a vacinação obrigatória, como o ato do

parlamento inglês para promover a expansão e tornar compulsória a prática da

vacinação (Act to further extend and make compulsory the practice of vaccination),

de 1853. Este ato obrigava a toda criança ser vacinada dentro de três meses após o

nascimento. No registro de nascimento da criança, os pais eram notificados sobre a

obrigatoriedade da vacinação para se obter os registros de nascimento, casamento

e morte (Fig. 4). Se a vacinação fosse bem-sucedida, um certificado de vacinação

era entregue (Fig. 5).

De James Gillray (1757-1815), foi publicado na Inglaterra em 12 de junho de 1802 pela Anti-Vaccine Society. O

quadro mostra Edward Jenner entre pacientes no Small Pox and Inoculation Hospital, em St Pancras (Londres),

e sugere a transformação de indivíduos vacinados em vacas, após a vacinação.

Figura 4. Certificado de vacinação compulsória de Walter Willian Charles Vicent

Retirado de Stewart e Devlin, 2006.

Figura 5. Certificado de vacinação bem-sucedida de Walter Charles Willian Vicent.

Retirado de Stewart e Devlin, 2006.

O experimento clínico de Jenner de imunização com o vírus da varíola e a

publicação de sua pesquisa, deu origem a imunologia, mas nem um entendimento

das bases de sua eficácia, nem a aceitação universal da sua prática foi seguido. Ao

contrário, ceticismo e alarme público e científico é, sempre, a primeira reação (Allen,

2007; Bazin, 2001; Wilson e Marcuse, 2001).

A vacina jenneriana é um exemplo excepcional de empirismo científico, mas

também um símbolo sujeito a diversos significados e apropriações. A fluidez da

significação, traduzida na diferença entre discurso, regulamentação e prática, longe

dos rigores do laboratório bacteriológico que se consolidaram depois de 1880, faz da

vacinação um palco significativo de métodos e dinâmicas sociais inscritos nas

práticas da saúde (Porto e Ponte, 2003; Saavedra, 2004; Wilson e Marcuse, 2001).

Acusada de desviar a atenção e investimentos de problemas econômicos e

sociais básicos cujos impactos prevaleceriam sobre o quadro sanitário e qualidade

de vida da população, também é criticada por aqueles que defendem que o uso

continuado de uma substância biologicamente ativa é incerto e generalizante quando

se considera a multiplicidade e variabilidade do organismo humano (Ponte, 2003). O

debate envolve grupos religiosos, políticos e científicos, dos mais variados matizes

(Clements e Mcintyre, 2006; COMED, 2007; Geier e Geier, 2006a; Moulin, 2003;

Ponte; 2003; Temporão, 2003) e por vezes, ao longo da história, tem aproximado

oponentes de posições mais convergentes, quando grupos antivacinistas afirmam não

serem contra a vacinação e sim contra a vacinação sistemática, corroborando com

aqueles que, acreditando no uso das vacinas pelas autoridades de saúde pública

como algo imprescindível, admitem a flexibilização dos índices de cobertura vacinal,

reduzindo-os em alguns casos à vacinação de bloqueio (Moulin, 2003; Ponte, 2003).

As vacinas são, provavelmente, o principal instrumento de saúde pública já

posto à disposição dos tomadores de decisão, apesar da oposição que sofre desde

suas origens. O século passado foi ameaçado pelo fantasma das grandes epidemias

que atingiriam milhões de indivíduos devido à intensa movimentação das pessoas; à

devastação florestal, à eclosão de guerras e à possibilidade do uso de patógenos

em atentados terroristas e em conflitos entre nações (Ponte, 2003; Temporão, 2002;

Wilson e Marcuse, 2001).

Nos últimos anos houve um grande número de publicações sobre o

desenvolvimento e o uso de vacinas para evitar o surgimento epidemias cada vez

mais abrangentes (Clements e Mcintyre, 2006; COMED, 2007; Moulin, 2003, Ponte,

2003; Temporão, 2003; WHO, 2006c). Contribuem para esse processo previsões

bastante pessimistas de segmentos da epidemiologia que acreditam, por exemplo,

que o mundo será, em futuro próximo, assolado por uma pandemia de influenza que

ceifará milhares de vidas e arruinará a economia de muitos países. Verdadeiras ou

falsas tais previsões passaram a povoar o imaginário de setores da opinião pública,

fortalecendo a posição daqueles que defendem a ampliação do uso de vacinas.

Se Shakespeare tinha razão ao escrever que o mundo é um palco, então,

certamente há muitos atores neste palco. E eles estão freqüentemente interpretando

diferentes scripts. Na verdade, eles têm interesses muito diferentes.

1.2.1 Eventos Adversos e Vacinas Conservadas com Timerosal

A utilização de conservantes em vacinas foi instituída logo após a ocorrência

de acidentes trágicos de contaminação de frascos multidoses sem conservantes. Na

Austrália, em janeiro de 1928, durante a campanha de vacinação contra a difteria, de

21 crianças vacinadas por via subcutânea, 12 morreram nas 24-48 horas devido à

infecção por estafilococos (Zambrano, 2004). Em 1968 os EUA instituíram o uso de

conservantes (incluindo o timerosal) em frascos de vacinas multidoses, exceto as de

vírus vivos atenuado, como a SRC (tríplice viral, contra sarampo, rubéola e caxumba),

pólio oral e febre amarela (Bernier et al., 1981; Simon et al., 1993; Migowsk, 2007).

A vacina mais arrolada a eventos adversos pós-vacinais é o componente

pertussis da vacina tríplice bacteriana DTP ou quádrupla bacteriana que associa a

DTP à vacina Hib. O pertussis é responsável por eventos adversos, que, mesmo

raros, podem ser graves (Edlich et al., 2007; Fombonne, 2001; Geier e Geier, 2006a;

2007b; Maya e Luna, 2006). Evidências preliminares sugerem que o timerosal pode

estar associado a dificuldades na aquisição da linguagem, ao Transtorno de Déficit de

Atenção e Hiperatividade (TDAH) ou Distúrbio de Déficit de Atenção (DDA) (Blaxill,

2004; Doja e Roberts, 2006; Mutter et al., 2005). Milhares de famílias registraram

queixa junto ao Programa de Compensação as Injúrias Vacinais (VICP) do governo

norte-americano (figura 6) e, atualmente, todos os fabricantes e distribuidores de

VCT estão sendo processados nos EUA (Kennedy JR, 2005; Sugarman, 2007).

Figura 6. Gráfico das reivindicações apresentadas junto ao Programa de Compensações as Injúrias

Vacinais dos Estados Unidos (US Vaccine Injury Compensation Program), no período de 1989-2007

Retirado de Sugarman, 2007. Vacinações após 1988 (Post-1988) são aquelas que ocorreram em ou após 1 de

outubro de 1988; vacinações pré-1988 são aquelas que ocorreram antes de 1 de outubro de 1988 (pre-1988). Os

dados são da Administração dos Serviços e Recursos de Saúde dos EUA (US Health Resources and Services

Administration).

Desde o dia 11 de junho de 2007, em Washington, um Tribunal Federal de

Reclamações (Court of Federal Claims) está sendo confrontado com uma questão

controversa e altamente emocional: saber se vacinas podem ter causado autismo

em milhares de crianças norte-americanas. O processo envolve queixas de milhares

de famílias que atribuem às vacinas a responsabilidade pelo autismo diagnosticado

nos seus filhos. A maioria dessas famílias afirma que o timerosal das vacinas é o

responsável pelas disfunções na interação social e de comunicação de suas

crianças, fruto da alteração do espectro autista (COMED, 2007; Edlich, 2007; US

Vaccine Court, 2007).

Esse é um dos mais importantes processos judiciais na história médica norte-

americana. Ali, um júri especial composto de três juízes começou a ouvir as

evidências para apoiar – ou refutar – a hipótese de que o Hg em vacinas causou

autismo ou sintomas semelhantes ao autismo em crianças. É a primeira vez em que

a evidência sobre o dano autista das vacinas é analisada em uma corte legal.

Tecnicamente, este não é, de todo, um julgamento. É um "Autism Proceeding

Omnibus", sem culpados. Os juízes não são chamados de juízes, mas "Special

Masters"; as famílias demandantes e seus advogados são denominados de

―peticionários‖. E o réu, denominado "inquirido", não é uma droga potente, e sim, o

Departamento de Saúde e Serviços Humanos dos EUA (United States Department

of Health and Human Services – DHHS), representado pelos bem–pagos advogados

do Ministério de Justiça Norte-Americano.

Os três juízes do Tribunal de Vacinação (Vaccine Court) mergulharam nas

águas intensas e contraditórias da querela vacina-autismo, sabendo que devem

emergir do outro lado, cada um com sua própria decisão sobre o nexo causal. Por

fim, devem emitir juízo sobre algumas das 4900 queixas.

Em sua defesa o CDC apresentou um estudo onde foram avaliados quatro

anos da sua vasta base de dados sobre eventos adversos pós-vacinais e não se

encontrou qualquer ligação entre timerosal e autismo (US Vacinne Court, 2007). O

outro lado foi impedido de ver os dados brutos originais, de modo a reproduzir o que

os pesquisadores do CDC descobriram. A reprodução exata dos dados originais é

impossível porque, de algum modo, "desapareceram" e não estão mais disponíveis

para a re-análise, num provável crime de violação da lei federal norte-americana de

qualidade dos dados (COMED, 2007, Marques, 2008).

Conceitos importantes em vacinas (Bigham e Copes, 2005; Clements, 2004)

 ―Thimerosal-free‖ (livre de timerosal): Indica que a vacina não contém timerosal,

como conservante ou remanescente do processo de fabricação da mesma.

 ―Preservative-free” (livre de conservante): Indica que não se adicionou timerosal

como conservante na vacina, mas ele foi usado no processo de fabricação.

Podem estar presentes traços de Hg (< que 0,5 µg/0,5 mL) no produto final.

 Redução do timerosal: Indica que estão tentando diminuir expressivamente a

quantidade de timerosal. A vacina não pode ser considerada ―isenta de timerosal‖.

1.2.2 Quantificando o timerosal das vacinas pediátricas (Marques et al., 2007b)

O timerosal tem aproximadamente 50% de Hg por peso. Uma solução de

0,01% (1 parte por 10.000) de timerosal contém 50 μg de Hg por dose 1 mL ou 25

μg de Hg por dose de 0,5 mL (Quadro 1). Por exemplo, cada dose da vacina

combinada DTP+Hib disponível no calendário oficial de imunizações brasileiro

(Brasil, 2007) pode conter entre 25 a 50 µg de Hg/dose (0,5 mL), dependendo do

fabricante. A vacina pediátrica contra a hepatite B (Hep B) tem 12,5 a 25 µg de

Hg/0,5 mL, também a depender do laboratório fabricante.

QUADRO 1. Dose de mercúrio em vacinas para uso infantil

VACINA

Concentração de Timerosal

Hg μg/0.5 ml

DTaP

.01%

DTP

.01%

DTwP-Hib

.01%

Hib

.01%

Hep B

.005%

12.5

Influenza

.01%

Antimeningocócica

.01%

Antipneumocócica

.01%

1.3 Limites de Exposição ao Mercúrio: Limitações e Certezas

Atualmente não há limites de toxicidade estabelecidos para o EtHg. Os

estudos sobre toxicidade consideram que o MeHg e EtHg podem ser equivalentes

(Bigham e Copes, 2005; Clarkson, 2003, 2007; Magos, 1985; US ATSDR, 1999; US

EPA, 1997). Entretanto, os limites de toxicidade fixados para o MeHg se referem a

uma exposição crônica, por via alimentar e em população adulta, não para uma

administração subcutânea ou intramuscular em RN e crianças, população mais

sensível que a adulta para a exposição a metais pesados. Ou seja, estamos

utilizando limites de exposição crônica por via oral, provenientes de exposição

ambiental ao Hg, frente a exposições intermitentes por via subcutânea e

intramuscular de timerosal, que por sua vez, gera EtHg.

As exposições intermitentes e altas podem presumir um risco maior do que

baixas doses diárias. Por outro lado, se desconhece qual a quantidade de EtHg se

pode considerar segura em uma única dose (caso das VCT), ainda que Magos

(1985, 2001, 2003) tenha interpolado dados de relatos de caso publicados. Embora

Uma concentração de 1:10,000 é equivalente a concentração 0.01%. Uma concentração de 1:10,000 contém 25

microgramas de Hg por cada 0.5ml.

nenhum nível de ingestão diária tolerável tenha sido proposto para o EtHg, várias

agências publicaram limites máximos de exposição ao MeHg que fornecem uma

orientação para ―tomadores de decisão‖ no governo de populações cronicamente

expostas. No geral, esses limites são propostos com o objetivo de proteger o feto.

Os limites máximos de exposição são calculados das concentrações no cabelo

ou sangue que estão em um estado constante e são propostos para a aplicação em

longo prazo. Para fins de comparação, apresentamos no Quadro 2 os limites de

referência das seguintes Agências Internacionais de Saúde: OMS (WHO, 1996),

Environmental Protection Agency (US EPA, 1997), Food and Drugs Administration

(US FDA, 1982), Agency for Toxic Susbtances Disease Registry (US ATSDR, 1999).

QUADRO 2. Limites de exposição ao metilmercúrio na dieta de um indivíduo adulto.

Environmental Protection Agency (EPA)

0,1 µg/kg/dia

Agency for Toxic Susbtances Disease Registry (ATSDR)

0,3 µg/kg/dia

Food and Drug Administration (FDA)

0,4 µg/kg/dia

Organização Mundial da Saúde (OMS)

3,3µg/kg/semana=0,47µg/kg/dia

Os limites máximos de exposição não representam os níveis absolutos acima

do qual a toxicidade ocorre. Como estas recomendações são propostas para

exposições crônicas, de longo prazo, não para uma exposição máxima de um único

dia, deve-se tomar cuidado ao avaliar exposições calculadas sobre uma base

apropriada de tempo. Uma vez que as diretrizes para os limites de exposição são

derivadas de dados científicos similares, as diferenças entre agências refletem as

variadas suposições e fatores de incerteza que são aplicados na transformação dos

dados científicos em recomendações de políticas públicas. Após essas ressalvas,

atualmente a única possibilidade de estimar os limites de exposição ao EtHg das

VCT é utilizando os valores descritos para MeHg.

Não obstante diferirem em suas margens de segurança, os limites de

exposição ao MeHg reconhecidos pelas agências internacionais são todos da mesma

ordem de magnitude: microgramas/kilo/dia (µg/kg/dia). Para elucidar os cálculos de

exposição, adotamos como exemplo uma criança do sexo masculino, no percentil 50

de média de peso corporal, de acordo com a idade. Este cenário supõe que aos seis

meses a criança recebeu todas as vacinas do calendário preconizado pela OMS e

Ministério da Saúde do Brasil (MS), que corresponde a uma dose cumulativa de EtHg

de 187.5 µg

-1

. A exposição ao Hg do nascimento até os 18 meses pode chegar a

212,5 µg

-1

se todas as vacinas que contém timerosal forem administradas (Quadro 3).

QUADRO 3. Exposição ao mercúrio de vacinas pediátricas conservadas com timerosal.

Idade da criança/

Peso médio

Vacina

Timerosal/Hg

(μg/dose)

Exposição

ao Hg

OMS

ATSDR

EPA

FDA

0 meses/3,4 kg

Hep B

25/12,5

3,68

7,83

12, 27

36,80

9,20

1 mês/4, 5 kg

Hep B

25/12,5

2,78

5,91

9,27

27,80

6,95

2 meses/5,6 kg

DTP

50/25

4,46

9,49

14,87

44,60

11,15

2 meses/5,6 kg

Hib

50/25

4,46

9,49

14,87

44,60

11,15

Aos 2 meses a administração

da DTP e Hib é simultânea

37,5

6,70

14,26

22,33

67,00

16,75

4 meses/7 kg

DTP

50/25

3,57

7,60

11,90

35,70

8,93

4 meses 7 kg

Hib

50/25

3,57

7,60

11,90

35,70

8,93

Aos 4 meses a administração

da DTP e Hib é simultânea

37,5

5,36

11,40

17,87

53,60

13,40

6 meses/8,3 kg

Hep B

25/12,5

1,51

3,21

5,03

15,10

3,78

6 meses/8,3 kg

DTP

50/25

3,01

6,40

10,03

30,10

7,53

6 meses/8,3 kg

Hib

50/25

3,01

6,40

10,03

30,10

7,53

Aos 6 meses a administração da

Hep B, DTP, Hib é simultânea

62,5

7,49

15,94

24,97

74,90

18,73

18 meses/11,2 kg

DTP

50/25

2,23

4,74

7,43

22,30

5,58

Percentil 50;

μg de Hg/kg/dia;

Número de vezes que supera o limite de segurança estabelecido pelas agências em

μg/kg/dia;

no Brasil, desde 2002 foi implantada a vacina tetravalente DTP + Hib, com 50 μg de timerosal.

1.4 A Plausibilidade Biológica

Desde 1999, duas abordagens diferentes foram seguidas com relação às VCT

nos programas de imunização infantil. Os EUA, UE e outros países desenvolvidos

adotaram medidas para eliminar a exposição infantil ao Hg das vacinas. Desde

2004, nenhumas das vacinas monovalentes ou multivalentes de rotina

recomendadas e usadas para proteger crianças em idade pré-escolar nos EUA ou

na UE contêm timerosal. A eliminação do timerosal dos programas rotineiros de

imunização infantil nestas jurisdições foi alcançada, essencialmente, pelo uso

exclusivo da monodose (dose única), formato das vacinas sem conservantes (AAP,

1999; EMEA, 1999; 2000; IOM, 2004; US CDC, 1999; 2000).

Contudo, a maioria dos países ainda usa VCT em seus programas de

imunização infantil, uma vez que a OMS continua a defender o uso do timerosal nas

vacinas pediátricas, incluindo para a administração em crianças desnutridas,

prematuras ou com baixo peso. A base da posição da OMS é que os estudos

farmacocinéticos e epidemiológicos não apresentaram evidências convincentes da

toxicidade do EtHg pela exposição através das VCT. E argumenta que o uso destas

vacinas, particularmente nas regiões com elevadas cargas de doenças, provou-se

altamente eficaz na proteção de crianças (WHO, 2006a; 2007).

As duas diferentes abordagens quanto à questão do timerosal nas vacinas

continuam a gerar confusão entre os pais e trabalhadores de saúde que administram

vacinas. Mesmo nas jurisdições aonde timerosal foi eliminado das vacinas infantis

administradas rotineiramente, ainda há VCT que podem ser recomendadas para

algumas crianças de alto risco. Por exemplo, vacinas contra influenza, doença de

Lyme, doença pneumocócica invasiva ou contra a raiva humana (Fombonne et al.,

2006; Geier e Geier, 2007a; COMED, 2007).

Diversas publicações científicas relacionam o aumento no número de casos de

DN – tais como atraso na linguagem, alterações de conduta e síndrome autista – a

maior exposição que sofre a população infantil ao Hg orgânico das vacinas, devido a

um calendário de imunização cada vez mais repleto de VCT (Bernard, 2001; Blaxill,

2004; Blaxill et al., 2004; Doja e Roberts, 2006; Geier e Geier, 2006a; 2007b; Herman

et al., 2006; Maya e Luna, 2006; Mutter et al., 2005; Shevell e Fombonne, 2006).

O estudo realizado pelo IOM, publicado em 1 de outubro de 2001, concluiu

que a evidência científica era imprópria para aceitar ou descartar uma relação de

causa e efeito entre a exposição de crianças ao timerosal presente nas vacinas

infantis e transtornos do desenvolvimento neurológico como, síndrome autista,

transtornos de hiperatividade e atrasos da linguagem, pois seria necessário realizar

mais estudos para se estabelecer ou rechaçar uma relação causal (US CDC, 2001).

Contudo, concluíram que DN relacionadas à exposição ao Hg de VCT é uma hipótese

biologicamente plausível. Além disso, recomendou esforços para abolir o timerosal

das vacinas como medida preventiva de saúde pública para reduzir a exposição ao

Hg em RN e crianças. Dois anos depois um estudo epidemiológico mostrou um

incremento no risco relativo de sofrer de DN e enfermidades cardíacas com doses

maiores de Hg. Isto indicaria que quanto maior a dose de Hg maior a probabilidade

de sofrer danos durante o desenvolvimento neurológico (Geier e Geier, 2003a).

A plausibilidade da associação entre a exposição ao EtHg derivado das VCT

e efeitos para a saúde foi baseado no seguinte:

 Presumidas similaridades na farmacocinética e efeitos toxicológicos do EtHg e

do MeHg.

 Reações de hipersensibilidade após baixas doses de exposição a produtos

contendo timerosal.

 Aumentos mensuráveis de Hg no sangue de crianças após imunização com

VCT.

 Evidência de um efeito dose-resposta a altas doses de exposições ao EtHg tanto

alimentares quanto ocupacionais, agudas ou crônicas.

1.4.1 A farmacocinética e toxicologia do MeHg e do EtHg

Preocupações a respeito da segurança do timerosal (que contém EtHg) foram

baseadas em estudos que sugerem efeitos adversos em crianças com exposição in

utero ao MeHg em níveis antes considerados seguros (Grandjean, 1997; US CDC,

1999; 2000; WHO, 1991). A similaridade da farmacocinética e toxicológica foi

postulada em função das estruturas químicas e efeitos para a saúde similar em

doses elevadas. O metabolismo e mecanismos toxicológicos de ação do EtHg e

MeHg são complexos e diferenças significantes na farmacocinética entre esses dois

compostos estão sendo reconhecidas.

Duas diferenças importantes são a meia-vida significantemente mais curta do

EtHg no sangue e menor movimento de EtHg através da barreira hematoencefálica.

Magos (2003) estimou uma meia-vida corrigida alometricamente de 18 dias para o

Hg administrado como timerosal, que estava dentro de 10% dos níveis mensurados

de Hg no sangue relatados por Pichichero et al. (2002). Os dados apresentados por

Magos indicam que a acumulação transiente do Hg no sangue é resultado da

vacinação com VCT nos intervalos de dose de 4-6 semanas comumente indicados

para imunização infantil em países em desenvolvimento.

Os efeitos toxicológicos da exposição ao MeHg in utero são relevantes para o

EtHg?

Estudos de longitudinais são baseados na exposição ao MeHg. Esses estudos

acompanharam crianças cronicamente expostas no período pré-natal e pós-natal

(via alimentar) a baixas doses de Hg e foram realizados nas ilhas Seychelles

(Davidson et al., 1998; Myers, et al., 2003), nas ilhas Faroes (Grandjean et al., 1997,

1999, 2007), e na Nova Zelândia (Crump et al., 1998). Nas Seychelles, as exposições

crônicas in utero e em baixas doses resultaram de mães que tinham uma dieta

essencialmente baseada em peixes, enquanto o consumo de peixes das mães das

Ilhas Faroes mudava intermitentemente para o consumo de carne e gordura de

baleias piloto.

O estudo das Seychelles usou cabelo materno e da criança para avaliar a

exposição pré-natal e infantil ao Hg e escalas neurodesenvolvimentais para avaliar

os efeitos, não encontrando nenhum dano neurológico em crianças até os nove anos

de idade (Myers, et al., 2003). O estudo das Faroes usou sangue umbilical e cabelo

infantil para avaliar a exposição pré e pós-natal ao Hg, respectivamente, e testes

neurodesenvolvimentais de domínio específico para avaliar os efeitos, relatando

déficits neurológicos sutis em escores de memória, atenção e linguagem entre as

crianças testadas quando completaram sete anos de idade. Segundo os autores, a

exposição pós-natal ao Hg foi menos preditora desses efeitos do que a exposição

pré-natal. Os resultados dos testes neurocomportamentais não foram consistentes

para predizer disfunções tardias (Grandjean et al., 1997, 2007). O estudo da Nova

Zelândia correlacionou a exposição pré-natal ao MeHg – estimado de análises de

amostras maternas coletadas durante a gravidez – com resultados de testes

psicológicos e educacionais aplicados em crianças de seis e sete anos de idade. Um

possível efeito sutil do Hg foi detectado após a exclusão de um par mãe-filho “outlier”

das análises (Crump et al., 1998).

A exposição infantil ao Hg de VCT difere da exposição dos estudos das Ilhas

Faroes, Seychelles e Nova Zelândia em diversos aspectos-chaves. Primeiro, as

circunstâncias da exposição são diferentes da exposição pós-natal associada

apenas com a vacinação infantil. Em segundo lugar, a rota de exposição (parenteral

vs oral) é diferente, e terceiro, a exposição da vacinação é intermitente, enquanto

nesses três estudos é contínua.

1.4.2 Reações de hipersensibilidade após exposição a baixas doses de

timerosal

O timerosal é associado a reações alérgicas dérmicas de contato

(hipersensibilidade tardia). Entre 1% a 16% dos indivíduos testados exibiram alergia

dérmica. Hipersensibilidade imediata (anafilaxia) e desordens imunes

(glomerulonefrite) também foram relatadas em associação com exposição aos

produtos contendo timerosal (Bigham e Copes, 2005, Cox e Forsyth, 1988; Ellis,

1947; Forstrom et al., 1980; Geier et al., 2007).

1.4.3 Evidências da relação dose-resposta na exposição a altas doses de EtHg

Na China no início dos anos 1980, o consumo do arroz tratado com EtHg

causou uma multiplicidade de sintomas neurológicos incluindo fraqueza, vertigens,

torpor, parestesia e ataxia, identificados nas pessoas moderadamente afetadas em

níveis de exposição de 0.5

mg/kg de peso. Nascimento et al. (1990) não apenas

relatou uma morte em conseqüência da ingestão de timerosal, como também

advertiu sobre o onipresente perigo do envenenamento por timerosal. Magos (2001)

relatou que nenhum efeito adverso foi observado em níveis de Hg no sangue entre

140 e 650 ng/L em cinco adultos, avaliados 11-22 dias após exposição a doses

variadas de EtHg de alimentos contaminados, infusão ou aplicação tópica de

produtos farmacêuticos contendo EtHg. O LOAEL (níveis mais baixos em que efeitos

adversos foram observados) no sangue foi de 1000 ng/L. Embora as relações dose-

resposta tenham sido construídas de exposições pré-natal ao MeHg, nenhuma

relação dose-resposta foi estabelecida para exposições pós-natal a EtHg nas doses

utilizadas em vacinas.

1.5 Timerosal e Distúrbios do Desenvolvimento Neurológico

O Centro para Prevenção e Controle de Doenças dos EUA (CDC) entre o final

dos anos 1980 e durante os anos 1990 expandiu o número de doses de VCT a

serem administradas em crianças americanas. A vacinação de rotina foi

gradualmente ampliada de uma administração de cinco doses da vacina contra DTP

para, no final, incluir três doses da vacina hep B (a primeira dose administrada nas

primeiras 24h de vida), e de quatro doses da Hib. Adicionalmente, o CDC também

passou a indicar que três doses da vacina contra influenza fossem administradas a

populações infantis específicas (a primeira dose administrada aos seis meses de

idade). Logo, segundo o calendário de imunização americano, a exposição total ao

Hg das VCT poderia ter sido de 200 µg de Hg nos primeiros seis meses da vida

(Geier e Geier, 2003; Marques, 2008).

O IOM, por determinação do congresso americano, realizou no início dos anos

1990 um estudo que envolveu queixas contra a vacina contra coqueluche. O grupo

investigou dezoito tipos de efeitos adversos associados à vacina, entre os quais morte

súbita de bebês, agitação e espasmos, encefalite, meningite, autismo, anafilaxia e

diabetes. Após vinte meses avaliando estudos de casos, estatísticas epidemiológicas,

experiências com animais e estudos laboratoriais, a comissão descartou toda e

qualquer relação causal entre a vacina e autismo. Reconheceu evidências de que

ela pode provocar agitação, encefalite e choque anafilático e deixou sem resposta o

resto das perguntas alegando insuficiência de dados (COMED; Sugarman, 2007).

Vários autores afirmam que o papel do Hg nos danos ao sistema neurológico

e imunológico associados a atrasos de desenvolvimento já está comprovado (Doja e

Roberts, 2006; Holmes et al., 2003; Geier e Geier, 2006b; 2007a; 2007b; Mutter et

al., 2004, Walker et al., 2006). Mesmo assim, os laboratórios produtores continuam

enviando milhões de doses de VCT (sob os auspícios da OMS) para países em

desenvolvimento, onde a regulamentação e fiscalização de medicamentos não

proíbem o uso de timerosal como conservante (WHO, 2002; 2007).

1.5.1 Estudos Laboratoriais

Estudos em humanos: mudanças do Hg no sangue após o uso de VCT em crianças

Os estudos em Rochester (EUA) sugerem que a meia-vida do EtHg é de

apenas 7 dias – comparados aos 40-50 dias para o MeHg – e é excretado no

intestino, não se acumulando ativamente no corpo. Pichichero et al. (2002)

comparou amostras de fezes, sangue e urina de crianças entre 2 e 6 meses de

idade, expostas a VCT (DTP e Hep B, algumas receberam Hib) e controles que

receberam vacinas livres de timerosal. A administração de VCT não elevou os níveis

sanguíneos de Hg acima do limite considerado seguro. O EtHg parece ser eliminado

rapidamente do sangue através das fezes após injeção parenteral de VCT. Os

autores, porém, não conseguiram medir quanto do EtHg cruzou a barreira

hematoencefálica (impossibilidade virtual em humanos, sobretudo crianças) nem se

houve algum dano neurológico decorrente da imunização. Eles concluíram que o

timerosal nas doses usadas em vacinas oferece pouco risco para crianças a termo,

mas não deve ser administrado ao nascimento em crianças prematuras e de baixo

peso ao nascer (não incluídas no estudo).

Stajich et al. (2000) descreveram que a imunização com uma única dose da

vacina Hep B (12.5 µg/g

-1

de EtHg), resultaram em aumento no nível médio de Hg no

sangue de prematuros quando medido 2-3 dias após a vacinação. Ainda que os

prematuros tenham recebido doses mais altas (µg/kg) que as crianças a termo, as

concentrações sanguíneas de Hg pré-vacinação entre prematuros e a termo foram

mais altas que a razão de correspondência da concentração de Hg sanguíneo pós-

vacinação. Isto indicaria que os prematuros excretaram uma proporção maior da

dose de Hg/kg de peso do que crianças a termo. Para os autores, permanece incerto

se os níveis mais altos de Hg no sangue, subseqüentes a imunização com VCT,

detectados nas crianças prematuras ou de baixo-peso oferecem qualquer risco

toxicológico mensurável.

Pichichero e Stajich supõem que a menor massa corporal aumentaria em

proporção às concentrações de timerosal. Assim, é provável que todo o EtHg já

tenha sido excretado pelo corpo quando a dose vacinal seguinte for administrada; e

os níveis de pico são determinados pela quantidade de EtHg administrada. Logo, a

pergunta seguinte é qual a proporção (se houver) de Hg no sangue cruza a barreira

hematoencefálica, que níveis de EtHg no cérebro são alcançados, e se o nível é lesivo.

Estudos em modelos animais

Burbacher et al. (2005) mediram a distribuição sistêmica e no cérebro do Hg

total e inorgânico em macacos não expostos ao timerosal e compararam-nos com

macacos expostos ao EtHg. Os níveis de Hg no sangue em macacos evidenciaram

que a meia-vida do EtHg é muito mais curta quando comparado ao MeHg.

1.5.2 Estudos Epidemiológicos

A literatura anterior a 1999 consiste, principalmente, de estudos não

toxicológicos do MeHg e de síndromes clínicas associadas a aplicação tópica de

produtos farmacêuticos contendo timerosal. Desde 1999, houve um aumento

esperado nos estudos sobre avaliação de danos neurodesenvolmentais causados

pelo timerosal. Porém, apenas 12 publicações apresentaram dados novos

expressivos ou novas abordagens para dados existentes. Seis concluíram que não

há evidências de insulto neurológico ou psicológico relacionado ao Hg de VCT. Em

contraposição, Geier e Geier publicaram 6 artigos apontando que essa relação

existe. Como há poucos artigos originais e o debate científico em torno do assunto

se articula neles, cada um foi revisado e sumarizado no Quadro 4.

QUADRO 4. Fontes de dados epidemiológicos primários investigando possíveis associações

entre vacinas conservadas com timerosal e aparecimento de sintomas neurológicos.

Autor (s)

Tipo de Estudo

Ligação com alterações

neurodesenvolvimentais

Possíveis associações

Andrews

(2004)

Coorte

retrospectiva

Não confirmado

Convulsões, espasmos

Heron et al.

(2004)

Coorte

Prospectiva

Não confirmado

Nenhuma

Haviid et al.;

(2003)

Coorte

retrospectiva

Não confirmado

Nenhuma

Madsen

(2003)

Coorte

retrospectiva

Não confirmado

Nenhuma

Verstraeten et

al. (2003)

Coorte

retrospectiva

Inconclusivo: Não

confirmado

Desordens na linguagem,

na fala, déficit de atenção,

convulsões, espasmos.

Stehr- Green

(2003)

Ecológico

Não confirmado

Nenhuma

Geier (2003a;

2003b; 2004a;

2005; 2006a;

2006b)

Coorte ecológica

e retrospectiva

Ligação confirmada em

seis artigos

Diversas associações

confirmadas

2 A HIPÓTESE

2.1 As vacinas são seguras?

O método introduzido por Jenner, em 1796, veio a ser chamada de

―vacinação‖, de ―Vacca‖, a palavra latina para vacas e a substância usada para

vacinar foi chamada de ―vacina‖. Vacinas são injeções que contém uma parte ou o

todo de um patógeno que aumenta a resposta imune, mas não deve causar a doença.

São administradas para induzir o desenvolvimento da resposta imune e proteger o

indivíduo contra um patógeno ou toxina (Prescott, 1996); para trabalhar estimulando

o corpo a produzir anticorpos, proteínas que defendem o corpo de uma invasão de

genes perigosos. Agora, mais de 200 anos depois, avançamos de uma época em

que a vacinação era um evento raro, e as teorias de Jenner sobre a imunização não

eram amplamente aceitas, para os anos 2000, quando as vacinas se tornaram tão

banais que a maioria das crianças recebe várias delas antes de chegarem ao

primeiro aniversário. Seres humanos em todo o mundo, de todas as idades são

vacinados contra muitas doenças O resultado dessa vacinação em massa tem sido

uma acentuada diminuição das doenças que assolaram a população mundial.

Entretanto, nas últimas décadas um número significante pessoas tem se

preocupado com a segurança das vacinas. Este movimento vem da apreensão de

que algumas vacinas podem ter complicações, muitas vezes sérias, com seqüelas

permanentes. As cepas virais atenuadas usadas nas vacinas contra poliomielite,

sarampo, rubéola e varicela tem o potencial para transformar-se em cepas mais

virulentas e causar sérias enfermidades em crianças ou cuidadores susceptíveis.

Como qualquer vacina tem o potencial de aumentar uma resposta anafilática em

indivíduos alérgicos, essas preocupações têm levado muitos pais a recusarem

vacinar suas crianças. Somam-se a isso, as baixas taxas de vacinação em

populações que tem dificuldade de acesso aos serviços de saúde e uma fração

crescente de crianças que estão novamente suscetíveis a essas doenças.

A maioria das pessoas acredita que as vacinas são seguras e eficazes. Como

dito antes, elas certamente ajudaram a erradicar ou reduzir a ocorrência de doenças

letais, como a varíola, pólio e coqueluche. Contudo, devem-se creditar às vacinas o

declínio dessas doenças ou elas são ineficazes e a ausência ou diminuição destas

foi devido à melhoria nas condições de vida?

Esta é a uma das controvérsias que o mundo enfrenta hoje.

Médicos, microbiologistas e outros profissionais da saúde irão atribuir o

declínio nos casos relatados de doenças ao aumento da vacinação em massa. Para

eles, é muito melhor vacinar e correr o risco de complicações de uma vacina que não

vacinar. Os ativistas contra a imunização dizem que isso não vale à pena, pois os

índices de mortalidade já tinham começado a declinar significativamente antes das

vacinas se tornarem disponíveis. E defendem que injetar um patógeno letalmente

conhecido em um hospedeiro saudável é como colocar um revólver e puxar o gatilho

esperando que ele não dispare.

O que nos é divulgado é que o controle das epidemias nos dias atuais é

resultado da vacinação em massa. Em 1967, a OMS recebeu registro de 131.000,00

casos de varíola, em 42 países. Em maio de 1980 a varíola foi oficialmente

considerada erradicada no mundo como resultado das campanhas de imunização

em massa (Scheibner, 1993). Com esta descoberta nós fomos aconselhados a

encerrar os programas de vacinação contra a varíola. A utilidade das vacinas foi

comprovada e o sucesso alcançado.

Mas foi isso mesmo?

As estatísticas de muitos países indicam que a varíola já estava declinando

antes dos programas de vacinação tornarem-se obrigatórios (Miller, 1993). Isto pode

ser atribuído à melhoria na educação, renda, condições sanitárias e em outras

reformas governamentais que levaram a melhoria na qualidade de vida das pessoas.

Não obstante, uma vez que os programas de imunização se tornaram compulsórios,

mortes por doenças evitáveis por vacina diminuíram.

Todavia, vejamos o exemplo da varíola: a probabilidade de a varíola ser fatal

é cinco vezes maior tanto em indivíduos vacinados quanto em não vacinados

(Scheibner, 1993). A vacina não deveria supostamente proteger contra a doença?

Similarmente à varíola e a poliomielite, a ocorrência de coqueluche também

teve uma diminuição nos casos e mortes associadas à doença (Moulin, 2003). A

doença é causada por uma bactéria que afeta o sistema respiratório, acarretando

severos ataques de tosse, febre e circulação inadequada de oxigênio (Miller, 1993).

Muitos acreditam que a incidência da coqueluche diminuiu devido à melhoria nas

condições de vida, mais que ao uso de uma vacina eficaz. A vacina contra a

coqueluche, chamada DTP, protege também contra a difteria e o tétano. Diversos

estudos afirmam que a DTP pode causar danos cerebrais. Entretanto, O CDC e a

associação médica norte-americana continuam afirmando que não há evidências de

que a DTP produza danos cerebrais (Freed et al., 1996, US CDC, 1999; 2000; 2001).

Se vacinas são eficazes por que as estatísticas mostram um aumento nas

taxas de ocorrência de muitas doenças após o início da vacinação? E se as vacinas

são ineficazes, por que a varíola foi totalmente erradicada e a incidência de outras

doenças foi grandemente reduzida desde o início do desenvolvimento das vacinas?

Quem está correto?

A controvérsia sobre a vacinação tem origem em um conflito conceitual na

área da saúde que marcou o século XIX e remonta aos famosos debates entre dois

notáveis cientistas: Louis Pasteur e Claude Bernard. Bernard formulou a teoria

segundo a qual a causa de uma doença estava em elementos ambientais, externos

e internos, não passando de uma perda de equilíbrio do corpo gerada por diversos

fatores. Para ele, o corpo é um "terreno" onde os microorganismos podem ou não

atuar de forma lesiva, dependendo das condições que encontram. A doença seria

um sinal do esforço do organismo para reequilibrar-se. Pasteur é autor da teoria

segundo a qual cada doença possui uma causa única, um vírus ou bactéria que

invade o organismo e ali produz um tipo especifico de devastação (Allen, 2007;

Bordenave, 2003; Capra, 2001; Moulin, 2003).

Pasteur venceu a disputa. Ele foi um debatedor talentoso e soube tirar proveito

do surgimento das epidemias daquele tempo para comprovar a coerência do seu

conceito de causação específica. Desde então, todo um modelo centrado na

microbiologia e, recentemente, na biologia molecular, fundamentou os procedimentos

de saúde modernos, incluindo às vacinações em massa. Capra (2001) afirma que,

mais tarde, Pasteur reconheceu a importância do "terreno" para as enfermidades,

tendo ressaltado a influência dos fatores ambientais e dos estados mentais na

resistência às infecções.

Pasteur ou Bernard? No mundo atual, ao que tudo indica, ficou ainda mais

difícil resolver velhas incertezas. Os muitos interesses que envolvem o assunto

gerarão ainda mais questionamentos e críticas mordazes antes que se chegue a

algum acordo. A resposta aos nossos questionamentos não chegará até que haja

pesquisas suficientes e imparciais em relação aos efeitos reais das vacinas.

2.1.1 Controvérsias atuais sobre a segurança das vacinas

A comunidade científica tem sido confrontada com as acusações de encobrir

provas de que o Hg em vacinas infantis causa autismo (Allen, 2007; Bernard et al.,

2001; Blaxill et al., 2003; Colgrove e Bayer, 2003; Kennedy Jr, 2005; Nature

Neuroscience, 2001). O timerosal, um conservante de vacinas à base de Hg, está

sob vigilância e investigação pública e profissional – e seu uso questionado – devido

à crescente conscientização da sua presença em vacinas infantis (Dórea, 2007;

Fombrone, 2006; Geier e Geier, 2007a; Marques et al., 2007b). A principal alegação

é que o composto exporia crianças aos prováveis efeitos neurológicos atribuídos ao

Hg. O uso do mercúrio – um conhecido neurotóxico – em vacinas não é novo. Ele é

usado para evitar contaminação por fungos e bactérias desde os anos 30, reduzindo

o risco de contaminação na abertura e manipulação dos frascos multidoses (Edlich

et al., 2007; Geier e Geier, 2006; US CDC, 1999; 2000; WHO, 2003).

Nos últimos tempos parece ter aumentado – ou pelo menos se tornado mais

visível – a ocorrência de eventos adversos de determinadas vacinas, como DPT e

Hep B (Zetterstro, 2004; Geier, 2004; Geier e Geier, 2007, Maya e Luna, 2006). Os

registros de eventos adversos variam da simples irritabilidade ao desenvolvimento

da doença que se pretendia evitar. Há registro de casos extremos em que a

vacinação resultou em morte (Brasil, 2007; Edilich et al., 2007; Geier e Geier, 2007b,

Sanford e Kimmel, 2002).

O sítio do governo americano para relatos de eventos adversos pós-vacinais

recebeu 108.000 queixas entre janeiro e outubro de 2000, encaminhadas para

averiguação técnica. A maioria dos relatos dizia respeito a desconfortos leves, como

febres e indisposição passageiras, que os cientistas costumam desconsiderar.

Mesmo assim, as referências a complicações colaterais graves, inclusive mortes, em

14% das denúncias levou o Serviço de Saúde dos EUA a redobrar a vigilância sobre

os fabricantes de vacinas e a interferir nas normas de produção (US CDC, 1999; US

DHHS, 2007). Outros países também fecharam o cerco às vacinas nos últimos anos,

baixando medidas preventivas (EMEA, 1999; 2000).

Um número crescente de estudos independentes associa a exposição ao Hg a

problemas neurológicos em milhões de pessoas. De reduções imperceptíveis do QI

a DN mais debilitantes (Geier e Geier, 2007b; Maya e Luna, 2006; Mutter et al., 2004;

Trasande et al., 2005). Por outro lado, uma série de outros estudos ligados ao

establishment da saúde garante que a apreensão é infundada (Clements e Mcintyre,

2006; Fombonne et al., 2006; Migowsk, 2007; Pichichero et al., 2002; Shevell e

Fombonne, 2006; Thompson et al., 2007; Trollfors et al., 2006), já que a pequena

quantidade contida nas vacinas seria facilmente eliminada pelo organismo.

É difícil acreditar que níveis de mercúrio 60 a 250 vezes mais elevados do

que os níveis de resíduos perigosos (ATSDR, 2001; EPA, 1997; FDA, 1982; WHO,

2006) sejam chamados de "minúsculos", como querem os defensores do timerosal.

É notório: todas as diretrizes publicadas para exposição ao Hg foram extrapoladas.

Os EUA e países europeus já proibiram o uso do timerosal em vacinas. E

apesar da OMS declarar que não há nível seguro para Hg, continua a recomendar sua

utilização nas fórmulas vacinais. Agora, se ―quantidade‖ é a palavra-chave, convém

lembrar que a quantidade de VCT foi triplicada no começo dos anos 1990, por

recomendação da própria OMS (WHO, 2006; 2007). Em 1999, quando a Associação

Americana de Pediatria (AAP) e o Departamento de Saúde dos EUA (DHHS)

reuniram-se para revisar e avaliar os riscos do timerosal em vacinas infantis a

hipótese inicial era: se o EtHg e MeHg são quimicamente similares, por conseguinte

devem ter os mesmos efeitos toxicológicos (Magos, et al., 1985; Clarkson, 2007).

Assim, a pergunta que precisava ser respondida era: ―O EtHg comporta-se da

mesma maneira que o Hg elementar e o MeHg, se acumula e causa os mesmos

efeitos ou efeitos similares?‖ A pergunta é crucial – pois, se a resposta for ―sim‖, então

não há argumentos: vacinas não devem conter Hg. Pode-se contra-argumentar que a

pergunta não poderia ser respondida por que dados sobre a toxicidade da exposição

humana a baixas doses de EtHg não estavam disponíveis em 1999. Contudo, foram

presumidos como similares àqueles causados por outros compostos organomercuriais

(Ball et al., 2001; Bigham e Copes, 2005; Blaxill et al., 2004; Halsey, 1999; Halsey e

Goldman, 2003).

A controvérsia do timerosal começou de maneira inesperada com uma

―epifania

‖ que os limites de exposição para MeHg foram excedidos nas vacinas

infantis. Após a apreensão inicial dos prováveis riscos para a saúde das crianças

relacionados ao Hg nas vacinas, veio a ―constatação‖ de que o que se sabia sobre a

toxicologia do EtHg era insuficiente, a despeito do considerável conhecimento

disponível sobre o MeHg. A literatura sobre o assunto estava limitada a casos de

timerosal aplicado topicamente em doses que excediam, em muito, quaisquer vacinas

(Cox e Forsyth, 1988; Fagan, 1997). Os epidemiologistas e ―cientistas de bancada‖ logo

começaram a publicar seus artigos trazendo a luz diferenças entre o perfil toxicológico

do MeHg e do EtHg, e a comprovação da segurança do conservante nas vacinas.

Ainda assim, a opinião pública permaneceu incrédula. Um conjunto de circunstâncias

– desde 1999 – conspirou para criar um ambiente de descrença na população.

O ―mainstream‖ científico – sobre imunização e Hg – foi exaustivamente citado

pelos grupos de pressão que eram contrários a remoção do produto das vacinas,

sempre assegurando que não existia comprovação científica da relação entre DN e

Epifania: manifestação ou percepção da natureza ou do significado essencial de uma coisa (dicionário Houaiss).

o Hg das vacinas. E embora os críticos do timerosal tenham proposto que alguns

dos sintomas do autismo e outras DN fossem análogos aos sintomas associados a

exposições ao Hg, peritos como Nelson e Bauman (2003) discordaram, indicando

que as diferenças clínicas e neuropatológicas entre o autismo e a intoxicação por Hg

extrapolam as similaridades e são limitadas a sintomas não específicos, tais como

ansiedade, depressão e medos irracionais. As conclusões dos ―experts‖ ofereciam

fortes evidências contra a plausibilidade biológica e epidemiológica da causação.

A reação da opinião pública foi contrária a da comunidade científica.

Mas, se timerosal não tem relação causal com o aumento dos casos de

autismo (Coury e Nash, 2003; Clements e Mcintyre, 2006; Doja e Roberts, 2006;

Thompson et al., 2007), ele causa o que? Coury e Nash (2003) afirmam que a

epidemiologia das desordens do espectro autista mudou. Um aumento na

prevalência foi notado durante as duas décadas passadas. O que não está claro é a

causa deste aumento. Múltiplos fatores parecem ser responsáveis. Entre eles às

mudanças nos critérios diagnósticos e maior consciência por parte dos cuidadores e

pais. Portanto, mais pesquisas são necessárias para ajudar a esclarecer a incidência

e prevalência das DN e sua etiologia. Neste ínterim, epidemiologistas e

toxicologistas precisam centrar atenção em assegurar que todas as evidências

possíveis sejam examinadas e que possam acrescentar novas descobertas sobre a

segurança ou não do timerosal em vacinas.

Halsey declarou em 1999 que muitos dos argumentos contra as vacinas estão

fundamentados em hipóteses não comprovadas ou em elos causais com provas

insuficientes, mas que gradualmente está se dando conta de que existe um risco

real para as crianças; e Clarkson, no mesmo documento, admite que talvez tenha

que rever os resultados de seus estudos sobre Hg em crianças de Seychelles,

levando em consideração as VCT administradas, a época, nas crianças avaliadas

por seu grupo (COMED, 2007).

Geier e Geier (2006a; 2006b; 2006c) afirmam que há uma relação direta entre

o Hg nas vacinas das crianças e autismo, contradizendo afirmações do governo norte-

americano de que não há relação comprovada entre os dois. Os dados mostram que

desde que o Hg foi removido das vacinas infantis, o aumento nas taxas registradas

de autismo e outras DN nas crianças não só parou como caiu drasticamente: até 35%

nos EUA. Utilizando o próprio banco de dados do governo, os cientistas analisaram

casos registrados de DN em crianças, inclusive autismo, antes e depois da remoção

timerosal. Esses resultados contradizem diretamente as recomendações de 2004 do

IOM, que reavaliou as informações acerca da segurança das vacinas do programa

de vacinação norte-americano. Ainda que sem disposição de excluir ou corroborar

uma relação causal entre Hg e autismo, o comitê interdisciplinar do IOM concluiu que

não há necessidade de mais estudos (IOM, 2004).

Então lembremos. Se já é consenso científico (corroborado pela OMS) que:

a) o mercúrio é tóxico;

b) nos primeiros meses de vida crianças são mais suscetíveis a interferências

no desenvolvimento neurológico causado pela exposição ao mercúrio; e

c) prevenir exposição ao mercúrio durante os períodos críticos do

desenvolvimento do SNC deve ser objeto de estratégias de saúde pública;

Causa estranheza a posição da OMS, que avaliza as recomendações para

restrição ao consumo de peixes durante a gravidez, com o intento de proteger

crianças dos efeitos danosos do mercúrio, mas ―permite‖ injetar a substância em

crianças. Ora, se parece razoável minimizar a exposição de fetos e crianças a fontes

de exposição ao mercúrio, outras duas questões permanecem sem resposta: por

que continuar usando vacinas com mercúrio quando existem alternativas? E a

vacina hepatite B, administrada nas primeiras horas de vida, não representa um

risco ainda maior para recém-nascidos?

Atualmente a maioria das vacinas Hep B disponíveis para países em

desenvolvimento – inclusive em preparações monodoses – contém timerosal

(Clements, 2004; Geier e Geier, 2007; Marques et al.; 2007b). O mesmo se dá em

relação às vacinas DTP e Hib, administradas em três doses a partir do segundo mês

de vida. Não obstante a OMS garanta a segurança do uso desse composto à base

de Hg em vacinas pediátricas e para grávidas, em janeiro de 2005, começou a

promover estudos em modelos animais para avaliar este aspecto (Clements e

Mcintyre, 2006; WHO, 2005).

A luz dos fatos há uma quantidade substancial de crianças que recebem doses

de mercúrio das vacinas infantis conservadas com timerosal por uma quantidade de

anos, já que o composto ainda é adicionado rotineiramente às vacinas administradas

a crianças no mundo inteiro. Nos EUA e UE, por exemplo, a vacina influenza, bem

como as dT e DT (difteria-tétano), meningite, e tétano monovalente continuam

usando o conservante, a despeito das medidas de eliminação do uso do composto

em vacinas (Geier e Geier, 2007c).

2.2 Dilema: comungar no dogma ou seguir o instinto científico?

Críticas as vacinas devem ser feitas com cuidado e responsabilidade, a fim de

evitar a um problema maior de saúde pública: a queda nos índices de vacinação.

Usar os efeitos nocivos do timerosal para clamar contra as campanhas de vacinação

fere o bom senso, pois colocaria em risco um programa de saúde bem-sucedido,

trazendo muitos prejuízos à população, como o retorno das epidemias do passado.

É fato. Por isso, será necessário mais tempo até que todas as dúvidas sejam

esclarecidas e as opiniões hoje antagônicas afluam para um novo entendimento. Não

há resposta fácil. Mas a questão é: há dúvidas e desconfiança onde antes parecia só

haver certezas e tranqüilidade.

O que deve se deixar claro é que nem todos os que fazem restrições às

vacinas querem abolir o seu uso. O ponto consensual é o de que está na hora dos

centros tradicionais de pesquisas se disporem a investigar os problemas relacionados

às vacinas e reavaliar as práticas atuais nessa área. E é essa uma das maiores

complicações.

Sabemos que as vacinas têm benefícios globais. Quem nos diz o que seríamos

hoje se não fosse à descoberta das vacinas? Do que reconheço, talvez eu não

pudesse estar hoje, aqui, escrevendo sobre esta questão. Mas, isto não compensa o

fato de que muitas crianças são mortas ou lesadas após receber vacinas. Há muitas

vacinas seguras disponíveis para uso atualmente, porém, elas não são utilizadas. E

por quê? Porque a comunidade médica continua a defender as vacinas antigas

porque elas são menos dispendiosas e as empresas farmacêuticas – produtoras das

vacinas – vão perder dinheiro, visto que as vacinas mais seguras são mais caras e

levam mais tempo para serem produzidas.

Para manter a confiança do público nas vacinas, devemos garantir que a sua

segurança é levada a sério e que, quando indicado, ações são tomadas no seu

devido tempo para reduzir o potencial de risco.

E o debate está apenas começando.

3 OBJETIVOS

3.1 Objetivo Geral

Avaliar as implicações da exposição ao mercúrio derivado de vacinas

conservadas com timerosal (VCT) e da dieta (incluindo aleitamento materno) nos

atrasos desenvolvimentais de crianças.

3.2 Objetivos Específicos

 Avaliar a exposição pré e pós-natal ao mercúrio sobre o desenvolvimento

neuropsicomotor de crianças.

 Avaliar qual o papel da dieta no processo de acumulação do mercúrio no

cabelo de crianças.

 Verificar se durante o calendário de vacinação recomendado no Brasil pode-

se superar os limites de exposição diária ao mercúrio estabelecido pela

Organização Mundial de Saúde e outras agências internacionais.

 Analisar os níveis de exposição ao mercúrio no cabelo de crianças.

 Verificar se a exposição ao Hg das vacinas conservadas com timerosal afetou

o crescimento e desenvolvimento de crianças.

4 MATERIAL E MÉTODOS

4.1 Caracterização da Amostra

Os dados iniciais deste estudo não foram coletados com o objetivo de avaliar

o impacto da imunização nas DN. O protocolo de pesquisa inicial foi concebido para

avaliar o crescimento e desenvolvimento das crianças expostas ao Hg no período

pré-natal e até o sexto mês de vida através da alimentação materna (pré-natal) e

aleitamento (MARQUES, 2002). Quando o trabalho de investigação foi iniciado não

estávamos cônscios da questão das VCT (anexo 1 e 2). A amostra inicial constituiu-

se de cem crianças nascidas nas maternidades do Hospital de Base Ary Pinheiro,

Hospital Pan-Americano e Regina Pacis, e suas respectivas mães. Os referidos

estabelecimentos de saúde estão localizados na cidade de Porto Velho, Rondônia.

Foram selecionadas todas as grávidas que concordaram em participar do

estudo. As crianças deveriam alimentar-se exclusivamente com leite materno até os

seis meses de idade. Os dados da ficha dos pacientes foram obtidos com a mãe ou

responsável legal após prévia autorização por escrito (anexo 7) e nos registros

hospitalares. Na mãe, avaliamos hábitos alimentares, antecedentes pessoais (pré-

natal, parto, abortos, natimortos, recém-nascidos malformados, doenças) e familiares

(passado em garimpo, ingestão de alimentos potencialmente contaminados, e outros),

na busca de fatores de risco para exposição ao Hg ou que poderiam afetar o

desenvolvimento de seus filhos (anexos 8 e 9).

Os recém-nascidos (RN) foram submetidos a exame de rotina ao nascer,

realizados pelo pediatra e/ou enfermeira presentes no momento do parto para

verificação: 1) do índice de Apgar

no 1º e 5º minutos (avaliação da vitalidade); 2) da

presença de reflexos, 3) da maturidade (prematuro, a termo ou pós-maduro); 4) do

peso, estatura, perímetro torácico e cefálico; 5) classificação quanto ao peso: pequeno

para a idade gestacional (PIG), adequado para a idade gestacional (AIG) e grande

para a idade gestacional (GIG); 6) ausculta do tórax e coração e verificação de

anormalidades grosseiras e malformações congênitas.

Os dados antropométricos, peso, estatura e perímetro cefálico (PC), foram

comparados aos dados tabulados pela OMS, enquadrados nos z-escores e percentis

apropriados (WHO, 2006a), utilizando-se o programa ANTRHO 2005. Os gráficos de

crescimento têm sido usados para traçar o crescimento físico de crianças desde 1977

e passaram por duas amplas revisões (WHO, 2000; 2006b). Consistem de uma série

de curvas, ilustrando a distribuição de crianças de acordo com as medidas do corpo

selecionadas. São considerados os mais apropriados para aplicação clínica,

representando um instrumento fundamental na avaliação do crescimento de crianças

(Kuczmarski et al., 1998; WHO, 2006a).

Aos seis meses de idade, 86 crianças compareceram ao exame clínico e

neuropsicomotor programado, quando amostras de cabelo foram novamente

coletadas. Para fins de avaliação usamos apenas 82 pares de mãe-filho. Maiores

detalhes aparecem nos anexos 1 e 2. A avaliação foi realizada nos meses de maio e

junho de 2001, aos sábados, no horário das 8 às 12 horas, e das 14 às 18 horas. As

mães foram convidadas a levar suas crianças ao prédio central da Fundação

Universidade Federal de Rondônia (UNIR) para a realização do exame físico e do

A maioria dos hospitais utiliza um sistema de índices (classificação) elaborado pela Dra. Virgínia Apgar em 1952,

para fazer uma avaliação clínica das condições físicas do recém-nascido com 1 minuto após o nascimento e

novamente com 5 minutos. Essa avaliação inclui freqüência cardíaca, esforço respiratório, tônus muscular,

irritabilidade reflexa e cor. Cada sinal pontua de 0 a 2 pontos. A contagem de 7 a 10 indica RN vigoroso, de 4 a 6

RN moderadamente deprimido, de 0 a 3 gravemente deprimido (Ziegel e Cranley, 1986).

desenvolvimento neuropsicomotor. Ao chegar ao local, mãe e filho (a) eram levados

a uma sala preparada com balança, mesas e cadeiras, mesa de exame, fitas métricas,

tesouras inox, brinquedos, material para acondicionamento do cabelo coletado, fichas

dos gráficos de crescimento e desenvolvimento e questionários de cada criança. Nos

meses de novembro e dezembro de 2003 e 2005, as crianças submeteram-se à nova

avaliação física para verificação dos dados antropométricos e do desenvolvimento

neuropsicomotor. Novos questionários, readequados para a idade, foram aplicados.

Os dados contidos nos questionários incluem dados sobre hábitos alimentares,

situação vacinal, antecedentes pessoais e familiares, e foram obtidos após nova

autorização (Anexos 10 e 11).

Nessa fase, a coleta foi realizada de modo a atender a disponibilidade de

cada mãe. Inicialmente, as avaliações foram realizadas na Policlínica Hamilton

Raulino Gondim, bairro Tancredo Neves, visando atender as mães residentes em

bairros próximos. A seguir, as moradoras dos bairros centrais foram convidadas a

levar suas crianças ao prédio central da UNIR. Para as mães que faltaram à

avaliação foi realizado visita domiciliar. Em cada avaliação, novas amostras de

cabelo eram coletadas para estimativa da exposição ao mercúrio.

4.2 A Avaliação do Desenvolvimento Infantil

Na avaliação do desenvolvimento neuropsicomotor, consideramos como

padrão de normalidade a Escala Desenvolvimental de Gesell e Amatruda (EDGA)

(Gesell, 2003; Gessel e Amatruda, 2002). No estudo da conduta motora avaliamos

as reações posturais, prensa palmo-plantar, locomoção e coordenação dos

movimentos. Na conduta adaptativa testamos à capacidade construtiva, que é

influenciada pelo desenvolvimento motor. Na conduta linguagem, avaliamos todas

as formas de visíveis e audíveis de comunicação. Na conduta pessoal-social

avaliamos as reações individuais da criança frente às pessoas e estímulos,

dependendo do temperamento da criança e condições ambientais. A EDGA faz uso

de tabelas que permitem obter um ―quociente de desenvolvimento‖ (QD) em cada

um dos quatro campos avaliados e um geral. O QD é a relação entre a idade

maturacional (derivada do desempenho comportamental da criança nas provas) e a

idade real (cronológica), expressa em proporção:

QD = IDADE MATURACIONAL X 100

IDADE CRONOLÓGICA

As tabelas são usadas para checar a presença ou a ausência de

comportamentos significativos, representados por sinais de positivo (+) e negativo (–),

respectivamente. Os dados são comparados à escala elaborada a partir de condutas

padrão apresentadas por crianças em determinadas faixas etárias. Deste modo, ao

realizarmos comparações quantitativas e qualitativas, comparamos o estado de

desenvolvimento com o comportamento adequado para a idade e determinamos se

e em que grau ele se desvia. O QD de uma criança perfeitamente média é 100.

Valores entre 85 e 68 são considerados limítrofes e tem implicações em termos de

acompanhamento. QD abaixo de 68 indica atraso significativo em uma ou mais áreas

do desenvolvimento.

Os materiais necessários foram confeccionados ou comprados, considerando

as especificações descritas na escala. O tempo necessário para a aplicação das

provas foi 20 a 30 minutos, aproximadamente, dependendo da colaboração da

criança. As idades – chaves com os comportamentos esperados para os quatros

setores do teste de Gesell são apresentadas no anexo 12.

4.3 Exposição estimada ao mercúrio das vacinas (timerosal injetado) e

aleitamento materno

A exposição ao Hg derivado de vacinas baseou-se no Calendário de

Imunização do PNI. Até os seis meses todos os lactentes receberam o esquema de

imunização completo. Após esse período as mães foram orientadas sobre a

importância da vacinação e a seguir as recomendações dos serviços de saúde. Entre

as vacinas tomadas pelas crianças durante os 5 anos do estudo algumas foram

conservadas com 0,01% timerosal. A concentração de Hg nas doses recebidas

através de vacinas foi de 12,5 (hep B) ou 25 µg Hg/0.5 mL (outras vacinas).

O esquema de vacinação com VCT e respectivas dosagens de Hg, como

indicado pelo fabricante, é apresentado nos anexos 2 a 4. Nós usamos os dados de

concentrações de Hg no leite (adaptado de Dórea, 2004), para estimar a exposição ao

Hg durante o período em que as crianças estavam sob amamentação exclusiva –

média do peso infantil × média da ingestão diária de leite materno (140 mL/kg) ×

número de dias × média da concentração de Hg no leite materno (1,9 µg/L) – e

discutimos os resultados nos anexos 2, 4, 5 e 6.

4.4 Determinação do nível de exposição ao mercúrio

Os níveis de Hg total foram verificados em amostras de cabelo (5 a 10 g) da

mãe e filho (nascimento, seis meses, três e cinco anos), sangue materno (4,5 mL),

placenta e cordão umbilical (5 g/peso úmido), coletadas na época do parto. As

amostras foram analisadas pelo método de espectrofotometria de absorção atômica

pela técnica de vapor frio. As técnicas e metodologias analíticas aplicadas no estudo

estavam de acordo com as do Laboratório de Biogeoquímica Ambiental da UNIR

(Bastos et al., 1998). Mais detalhes aparecem nos anexos 1 a 6.

4.5 Análise Estatística

A análise e interpretação dos dados foram feitas através de estatística

multivariada (regressão logística, análise dos componentes principais, por

correspondência e prospecção de dados) e os testes de significância baseados em

permutação. Para testar as diferenças entre grupos aplicamos métodos apropriados

para cada caso. Os dados serão apresentados na forma de tabelas, ilustrações e

gráficos, e comparados à bibliografia existente sobre o assunto. Detalhamento deste

tópico é apresentado nos anexos 1 a 6.

4.6 Considerações Éticas

Para a primeira fase do estudo, o protocolo deste projeto foi aprovado pelo

Comitê de Ética em Pesquisa com Seres Humanos do Núcleo de Medicina Tropical

da Universidade Federal do Pará (CEP/NMT/UFPA) para o período de 2000 a 2001.

Para as fases seguintes, a pesquisadora submeteu novo protocolo de pesquisa ao

Comitê de Ética em Pesquisa com Seres Humanos do Núcleo de Saúde da

Fundação Universidade Federal de Rondônia – CEP/NUSAU/UNIR, tendo sido

aprovado para o período de 2003 a 2007. Não houve danos à dimensão física,

psíquicas, morais, intelectuais, sociais, culturais ou espirituais dos participantes, em

qualquer fase da pesquisa e dela decorrente, ou mesmo dano associado ou

decorrente deste estudo científico. O estudo cumpriu todas as exigências da

resolução CNS 196/96.

5 RESULTADOS

Os dados descritivos das mães e RN são apresentados no anexo 1. Os dados

demográficos mostram uma ampla variação do status socioeconômico. A maioria

das mulheres foi recrutada de um hospital público (n: 61; Hospital de Base) enquanto

39% vieram de dois hospitais privados (n:13, Hospital Panamericano; n: 26, Hospital

Regina Pacis). Uma proporção substancial (39%) dessas mães eram primíparas.

Para verificar alterações do desenvolvimento intra-uterino avaliamos as características

físicas, habilidade funcional e sinais neuromusculares dos RN.

Na avaliação estatística dos parâmetros antropométricos pelo teste Anova,

verificou-se a homogeneidade dos resultados entre os dois sexos. Os dados

antropométricos de mães e RN são apresentados nos anexos 1, 2, 3 e 6. Nove RN

estavam abaixo das curvas de referência do NCHS para o peso. Após seis meses de

aleitamento materno exclusivo, quatro crianças apresentaram sobrepeso e apenas

três meninos apresentaram estatura pequena para idade.

Os dados de exposição ao Hg são apresentados nos anexos 1 a 6. O

consumo de peixe materno (Hg no cabelo) não dependeu da renda mensal ou

educação (anos). Os níveis de Hg no cabelo materno variaram amplamente (0.2

µg.g

-1

to 62.4 µg.g

-1

), refletindo extremos de consumo de peixe. Mulheres que

relataram baixo consumo de peixe (<2 refeições/semana) apresentaram

concentrações medianas de Hg no cabelo mais baixas (3.5 µg.g

-1

) que o outro grupo

(5.7 µg.g

-1

). A maioria das amostras de cabelo (57%) apresentou níveis de Hg

abaixo de 6 µg.g

-1

. A concentração mediana de Hg no cordão umbilical (7.5 ng/g)

estava próxima à mediana de Hg na placenta. A análise de Hg no cabelo do RN

mostrou que 92% apresentavam concentrações <6 µg.g

-1

. Os coeficientes de

correlação entre variáveis maternas e dos RN estão sumarizados no anexo 1.

As avaliações do desenvolvimento neuropsicomotor das crianças aos seis

meses são apresentadas nos anexo 1, 2, 4 e 5. A maioria das crianças (74%)

apresentou desenvolvimento normal e 26% atraso desenvolvimental em ou mais

setores da EDGA: 1% apresentou atraso motor, 9% déficit na linguagem, e 16%

tiveram atraso em dois ou mais setores (7% motor, linguagem, adaptativo, e 9%

motor, linguagem). Após a identificação (e remoção) de variáveis redundantes, foi

aplicada uma Análise de Correspondência para identificar grupos de variáveis

associadas. A associação (co-ocorrência) é caracterizada pela posição similar nos

dois planos dimensionais apresentados na Fig. 1, anexo 1 (Marques et al., 2007a).

A exposição pós-natal ao Hg derivada da amamentação e das VCT é ilustrada

na tabela 1, anexo 5. Houve uma grande evolução nos perfis de desenvolvimento

quando as crianças estavam mais velhas. Comparando com as avaliações dos seis

meses, o mesmo número de crianças permaneceu com QD abaixo de 75% em três

anos, mas os centis elevaram-se (anexo 5). A relação entre PC e QD não foi

significantemente correlacionada (Tabela 3, anexo 4).

Apresentamos os QD (aos 6 meses) relacionando-os com a dose de timerosal

ao nascimento nos anexos 2, 3, 4, e 5; não houve correlação significativa. Porém, os

QD foram significativamente correlacionados com duração do aleitamento materno

(anexo 5). Os resultados das análises de regressão múltipla são apresentados nas

Tabelas 3 a 5, anexo 4. Os modelos demonstraram que os atrasos desenvolvimentais

foram transitórios.

Este estudo extraiu informações da assimetria associada com mudanças nas

concentrações de Hg no cabelo materno e infantil em amostragens específicas:

nascimento (exposição fetal), seis meses de aleitamento materno exclusivo, 36

meses (desmame) e 60 meses (pré-escola). A distribuição do Hg no cabelo em

lactentes seguiu um padrão diferente de suas mães (Fig. 1 a 4, anexo 5).

Mudanças metabólicas pós-parto, desenvolvimento infantil e dietas de transição

e, provavelmente, Hg das VCT contribuem para a assimetria das mudanças do Hg

no cabelo entre mães e crianças. É importante notar que a exposição à VCT a partir

da primeira vacina tomada ao nascimento (0 dia) é a maior e a mais desafiadora das

doses. Neste momento a pequena massa corporal dos neonatos tem um impacto

equivalente ao dobro das doses (da DTP, hep B) aos seis e doze meses de idade. A

estimativa da exposição ao Hg do leite materno só foi possível até o sexto mês,

quando as mães foram cuidadosamente monitoradas e psicologicamente apoiadas a

manter o aleitamento materno exclusivo. Contudo, não houve associação significante

entre o Hg do cabelo e a duração do aleitamento, tanto para as mães quanto para as

crianças. A maior média de Hg no cabelo aos seis meses coincidiu tanto com a

exposição ao Hg, ao leite materno, quanto com o pesado cronograma vacinal. Além

disso, a distribuição de Hg em lactentes seguiu um padrão diferente de suas mães

(figura 2, anexo 5).

Após seis meses, crianças em aleitamento exclusivo receberam uma carga de

imunização (cinco tipos de vacinas; até 150 µg de Hg) que mostrou um incremento

das concentrações de Hg superiores ao nascimento, aos três e cinco anos de idade.

As concentrações de Hg em suas respectivas mães foram mais altas no parto. A

freqüência de consumo de peixes é apresentada e discutida nos anexo 1, 4 e 5.

Usando o Hg total no cabelo como marcador de exposição pós-natal ao Hg

orgânico (Hg inorgânico e MeHg do leite materno; EtHg do timerosal) nós estudamos

sua associação com a escala de Gesell mensurada aos seis meses, três e cinco

anos de idade. O Hg no cabelo aos seis meses respondeu aos eventos relacionados

à exposição ao Hg e amamentação.

A maioria dos atrasos desenvolvimentais observados aos seis meses foram

superados com o crescimento; aos cinco anos 87% das crianças apresentavam QD

adequados (>85). O tempo de amamentação e o Hg no cabelo foram, cada um,

significantemente correlacionados com QD, mas de maneiras opostas: o tempo de

amamentação foi positiva e significantemente associado com o QD aos cinco anos; as

concentrações de Hg no cabelo foram negativa e significativamente correlacionadas

com QD aos 6 meses (r=-0.3329; p=0.0022) e cinco anos (r=-0.8029; p=0.0106), mas

não aos 36 meses (r=-0.1722; p=0.1218). O resultado dos QD aos 5 anos dependeu

do QD aos 3 anos, que por sua vez, foi influenciado pelas variáveis do desenvolvimento

e de exposição ao Hg. O QD aos 6 meses foi significantemente influenciado pela

exposição pré-natal (Hg no cabelo materno e infantil) e pós-natal aos seis meses.

Enquanto as mães apresentaram as mais altas concentrações de Hg no cabelo

à época do parto, as crianças apresentaram os mais altos valores aos seis meses,

coincidindo com período em que o calendário vacinal com VCT é mais intenso. Depois

disso, a tendência de diminuição no cabelo da criança coincidiu tanto com o período

do desmame quanto com o período de vacinação menos freqüente (cinco anos). A

amamentação estendida (até 36 meses) não foi significantemente associada com o

Hg no cabelo materno ou infantil. Porém, foi observada associação significante

(Spearman’s r) entre as concentrações de Hg no cabelo da mãe e da criança ao

nascimento (r = 0.3534; P = 0.001), seis meses (r = 0.4793; P < 0.0001), três anos (r

= 0.0122; P = 0.012) e cinco anos (r = 0.0357; P = 0.005). Mudanças metabólicas

maternas no pós-parto, desenvolvimento infantil, dietas de transição e provável Hg

de VCT contribuem para a assimetria das mudanças entre mães e crianças.

É importante notar que a exposição ao Hg a partir da primeira VCT recebida ao

nascimento (0 dia) é a maior e a mais desafiadora das doses. Neste momento a

pequena massa corporal dos neonatos recebe um impacto equivalente ao dobro das

doses (DTP e hep B) aos seis e 12 meses. A estimativa da exposição ao Hg do leite

materno só foi possível até 6 meses, quando mães foram cuidadosamente

monitoradas e psicologicamente apoiadas para manter-se em aleitamento materno

exclusivo. Na verdade, a maioria das mães (66%) amamentou por 12 meses e

algumas relataram amamentar até 60 meses (figura 1, anexo 5).

Não encontramos associação estatisticamente significativa entre o Hg no

cabelo (mãe e filho) e a duração do aleitamento. A média de Hg no cabelo aos seis

meses coincidiu tanto com a exposição ao Hg do leite materno quanto com o pesado

cronograma de vacinação. A distribuição do Hg no cabelo de lactentes seguiu um

padrão diferente de suas mães (figura 2, anexo 5). Houve uma grande melhoria nos

QD quando as crianças estavam mais velhas. Comparando com as medidas dos seis

meses, o mesmo número de crianças permaneceu abaixo de 75% aos três anos, mas

os centis subiram. Nenhuma correlação significativa foi observada entre Hg no cabelo

da mãe (exposição pré-natal) e QD aos seis meses. Entretanto, a exposição pós-natal

(Hg no cabelo da criança) foi significantemente correlacionada com os QD aos seis

meses (r=-0.3329; P=0.0022) e 60 meses (r=-0.8029; P=0.0106), mas não aos 36

meses (r=-0.1722; P=0.1218).

Entre os seis e 36 meses, não houve somente mudanças nas práticas de

alimentação (desmame) associadas às mudanças na exposição ao Hg, mas também

nas doses e intervalos de vacinação.

Nós investigamos o crescimento do PC e sua associação com os QD. O

percentual de aumento do PC e QD não foram significantemente correlacionados

(Tabela 1 e 3, anexo 4). O mecanismo de crescimento somático que rege o PC foi

independente do neurodesenvolvimento funcional. O crescimento cerebral durante

diferentes períodos de alimentação (aleitamento materno exclusivo e desmame) e a

intensidade da exposição ao EtHg não foram significativamente correlacionadas.

Atrasos no neurodesenvolvimento ocorreram independentemente da exposição pré -

natal ao Hg ou do percentual de aumento pós – natal do PC, mas foram

influenciados pela amamentação e concentrações Hg no cabelo.

No anexo 3 e 6 apresentamos a escala de Gesell (aos seis meses) em função

da dose de timerosal ao nascimento e não houve correlação significativa. No entanto,

os QD foram significativamente correlacionados com a duração da amamentação

(figura 5, anexo 6). Correlações positivas e significantes foram observadas para o QD

motor (r= 0.3118; P=0.0044), linguagem (r= 0.3093; P=0.0047), QD adaptativo (r=

0.3246; P=0.0029), QD pessoal social (r= 0.3272; P= 0.0027) e para o QD total

(r=0.3479; P=0.0014).

Aos seis meses o QD foi significantemente influenciado pelo pré-natal (Hg no

cabelo materno e do RN) e exposição pós-natal aos seis meses. Neste modelo as

interações de variáveis relacionadas às concentrações de Hg ao nascimento (pré-

natal) e aos cinco anos, bem como a duração da amamentação, idade gestacional e

peso ao nascer, não foram afetados significativamente pelo QD total. Além disso,

também é possível inferir que os níveis de Hg no cabelo aos seis meses podem

responder a eventos relacionados à exposição ao Hg, ou seja, aleitamento materno

e VCT (anexos 4 e 5).

6 DISCUSSÃO

Assumindo que o cabelo é o melhor integrador da exposição passada ao

MeHg, um dos achados deste estudo é que o marcador de consumo de peixe (Hg no

cabelo materno) foi significantemente correlacionado com Hg no cabelo da criança

coletado no nascimento. Essa significante correlação permaneceu até os seis meses.

Contudo, o atraso motor observado em crianças com altos níveis de Hg no cabelo

não indicaram uma relação dose-resposta: os mais altos valores foram encontrados

em crianças do grupo considerado normal. Na figura 1, anexo 1, os mais altos níveis

de renda e educação estão posicionados no grupo #2. Esses achados sugerem a

ocorrência de algumas condições protetoras que não estão presentes em famílias

desprivilegiadas. A situação de pobreza e baixo nível educacional da família

provavelmente requerem suporte para o desenvolvimento ideal da mãe e filho. A

média de Hg de 7.4 µg/g

-1

no cabelo dessas mulheres é mais baixa que os valores

relatados para mulheres ribeirinhas (8.3–9.4 µg/g

-1

) de outros estudos apresentados

e discutidos no anexo 1.

A amostra de mulheres acompanhadas neste estudo, não é homogênea, nem

cultural nem socioeconomicamente. Entretanto, como não houve insultos pré-natais

severos que pudessem distorcer os padrões iniciais do neurodesenvolvimento, os

atrasos observados aos seis meses estavam dentro do esperado para a população

brasileira (Paine e Pasquali, 1983). Mesmo em populações homogêneas há variações

nos resultados neurocomportamentais. Dodge et al. (1975) discutiram as limitações

de confiabilidade de uma única função como avaliadora de um insulto sobre o

desenvolvimento normal. Quando se levam em consideração as avaliações dos três

e cinco anos, observam-se atrasos no desenvolvimento não correlacionados com

parâmetros socioeconômicos ou culturais. Estudos associando Hg e resultados

neurodesenvolvimentais de crianças algumas vezes não fazem referência ao status

do aleitamento materno. Por causa do papel fundamental da amamentação no

desenvolvimento neuropsicomotor é importante ressaltar esses estudos.

Componentes específicos como os ácidos graxos poliinsaturados da série

Omega 3 (PUFA), presentes no leite materno, mas não em fórmulas, que são

essenciais para o desenvolvimento e organização neuronal tem explicado escores

cognitivos e desenvolvimentais favoráveis ao aleitamento materno (Agostoni et al.,

2001). Por isso, quando a amamentação é considerada em estudos de exposição ao

Hg, há inequívocos benefícios para o desenvolvimento das crianças amamentadas

(Grandjean et al., 1994; Jensen et al., 2005; Marques et al.; 2007a).

Em estudos neurodesenvolvimentais com crianças durante o período de

amamentação, freqüentemente não levam em conta a exposição iatrogênica das

vacinas. Redwood et al. (2001) revisou exposição ao Hg devido ao timerosal (EtHg).

O timerosal contribui com até 25 µg/g

-1

por dose; 12.5-40% dependendo da vacina.

Durante o esquema de imunização, crianças podem receber vacinas ao nascimento

(12.5 µg/g

-1

), um (12,5 µg/g

-1

), dois (62.5 µg/g

-1

), quatro (50 µg/g

-1

) e seis (62.5 µg/g

-1

)

meses que podem expô-la a um total de 207.5 µg/g

-1

de EtHg durante o primeiro

semestre de vida (Marques et al., 2007b; 2007c).

O impacto causado pelo EtHg injetado num organismo imaturo é um ponto

importante a se considerar. Especula-se que a remoção do timerosal das vacinas não

produziria mais que 50% de redução na exposição de Hg infância (Bigham e Copes,

2005). Esta redução só poderia ocorrer no leite humano com concentrações de Hg

superiores ao valor médio (figura 1, anexo 2) e mais freqüentemente com fórmulas,

que geralmente contêm concentrações de Hg mais elevadas (Dórea e Donangelo,

2006). Além do mais, este cenário assume uma discutível equivalência de um bolus

(Hg injetado) versus a integração (de seis meses) do Hg no leite ingerido e não

contempla os princípios da neurotoxicidade do Hg: a forma química, a dose, a via de

administração, os fatores atenuantes de neurotoxicidade do aleitamento e os fatores

neurotóxicos do período perinatal (peso ao nascer/prematuridade).

As esperadas implicações metabólicas dessas rotas de exposição e formas

químicas de Hg extremamente diferentes (EtHg injetado vs Hg ingerido no leite), bem

como as doses de Hg são difíceis de decifrar. Temos a alteração nas interações

fisiológicas de uma dose diária de Hg intrínseco auto – ajustada (inorgânico e MeHg)

na amamentação contra o impacto total de uma dose extrínseca de EtHg. No que se

refere à amamentação, o Hg do leite ocorre num contexto de benefícios comprovados

em uma ampla gama de circunstâncias atenuantes da neurotoxicidade. Neste estudo,

nós observamos um aumento relativo do Hg no cabelo. Este achado reforça uma

ligação entre o EtHg do timerosal e aumento de Hg no cabelo, sustentando à

utilização do Hg no cabelo de crianças vacinadas como um fator de confundimento

da exposição ao Hg de fontes maternas.

A possível associação entre VCT e autismo está ganhando atenção científica.

Mutter et al. (2004, 2005) discutiram a epidemiologia e o mecanismo das DN

associadas ao Hg e sugerem que efeitos do MeHg encontrado em peixes são menos

tóxicos quando comparados a fontes iatrogênicas de Hg. É necessário, novamente,

advertir que estudos epidemiológicos relacionando autismo e VCT não têm levado em

consideração o aleitamento materno. Aliás, há um estudo sugerindo que o início do

desmame precoce pode contribuir para a etiologia do autismo (Tanoue e Oda, 1989).

Os perfis de distribuição das concentrações de Hg no cabelo materno e infantil

durante os primeiros cinco anos revelaram diferenças no tipo de exposição. A

cronologia da amostragem de Hg no cabelo da criança correspondeu aos eventos

associados à dieta (que incluiu amamentação) e calendário de imunização. Nós

encontramos uma clara tendência do Hg no cabelo da criança coincidindo com o

calendário vacinal, mas não podemos provar uma relação causa-efeito. A ocorrência

do EtHg nas VCT e de dietas de transição do desmame podem ter contribuído para

o padrão de mudanças do Hg no cabelo infantil. A média nos níveis de Hg no cabelo

infantil aos 5 anos é equivalente à materna, refletindo provavelmente exposição às

mesmas fontes dietéticas de Hg. Uma vez que os lactentes foram expostos a doses

mais altas de EtHg durante os primeiros seis meses, o aumento nos níveis de Hg no

cabelo da criança pode ser resultado das taxas mais elevadas de transferência do

EtHg no cabelo, em comparação com períodos subseqüentes da amostragem.

A flutuação do Hg no cabelo materno é uma questão importante, com

implicações para políticas de saúde pública. Além disso, nós identificamos a entrada

de outras fontes orgânicas de Hg. Contudo, nossa limitada capacidade analítica para

determinar o EtHg no cabelo derivado das VCT é uma importante limitação na

interpretação desses aspectos dos resultados.

Uma variedade de fatores intervenientes associados à imaturidade infantil e ao

tempo de exposição complica a avaliação dos efeitos da exposição precoce ao Hg

sobre neurodesenvolvimento. Os efeitos no SNC respondem a fatores relacionados

ao hospedeiro (susceptibilidade genética, estado nutricional e condição do SNC), a

exposição (dose, freqüência e duração) e a formas químicas específicas de Hg

(inorgânicos e orgânicos). Essas interações governam a reatividade química, a

biotransformação e, eventualmente, a toxicocinética e toxicodinâmica do EtHg em

lactentes. Ainda que não possamos ter certeza se o Hg no cabelo das crianças veio

das vacinas ou de fontes maternas, estudos em animais já mostraram que o EtHg foi

incorporado ao crescimento do cabelo de forma semelhante ao MeHg (Fang e Fallin,

1973; 1976; Zareba, 2007).

Um problema comum frente aos efeitos tóxicos do Hg ou à susceptibilidade em

níveis subclínicos é a dificuldade de reconstruir com precisão respostas às fontes após

a exposição. Isto é especialmente o caso com lactentes e acompanham mudanças

nas dietas integradas com fontes de Hg (que podem incluir EtHg extrínseco), como no

presente estudo. Além disso, existem as considerações éticas relacionadas com a

amamentação e vacinação que limitam comparações de grupo (sem tais

características), tornando estudos de exposição particularmente difíceis de realizar.

A associação transitória entre dosagem de EtHg das VCT e DN observados

baseia-se no pressuposto que: a) a amamentação estava igualmente presente

durante um período mínimo de seis meses em todas as crianças e exerceu um efeito

protetor do SNC; b) durante o aleitamento materno exclusivo todas as vacinas foram

igualmente administradas a todas as crianças; c) a exposição a todas as formas de

Hg podem ter como resultado que só a formas orgânicas podem ser encontradas no

cabelo (e incluir o EtHg). Nós usamos Hg total no cabelo como um substituto para as

três possíveis formas de exposição ao Hg: do leite (Hg inorgânico e MeHg) e

imunização (EtHg das VCT). Nossos modelos de regressão foram bem sucedidos

em diferenciar os efeitos negativos da exposição ao Hg dos resultados do

neurodesenvolvimento (talvez relacionados com a amamentação).

Usando a análise dos componentes principais (PCA) foi possível identificar

hierarquias e posições de variáveis inter-relacionadas que identificaram riscos

variáveis de exposição associados com escalas de DN aos seis meses. Deste modo,

nossos resultados podem explicar o aparente conflito relatado por Thompson et al.

(2007): seu modelo estatístico foi insuficiente para discriminar resultados

contrastantes de danos e benefícios no mesmo sistema neurológico, levando à

associações inconsistentes entre a exposição ao timerosal das vacinas e os testes

neuropsicológicos.

Embora não haja evidência de associação entre autismo e VCT, permanecem

as questões relativas a outras DN. Andrews et al. (2004) estudaram crianças inglesas

retrospectivamente no que se refere à DTP/DT recebida em três e quatro meses: foram

observados resultados significativos para o aumento dos riscos de espasmos com o

aumento das doses de VCT. Também foram observadas associações negativas

significantes para atrasos desenvolvimentais inespecíficos, atraso geral e déficit de

atenção em relação à exposição VCT.

Os estudos retrospectivos de crianças norte-americanas mostraram resultados

diferentes (Geier e Geier, 2003; 2006; Verstraeten et al.; 2003). Estes estudos foram

baseados em dados projetados para eventos pós-vacinais e coletados no sistema de

notificação de efeitos adversos do governo norte-americano (Vaccine Adverse Effects

Reporting System-VAERS) e no Sistema de Ligação dos Dados Vacinais (Vaccine

Data Link System-VDS) e utilizaram estratégias diferentes de investigação.

Quando os efeitos protetores do aleitamento materno estão ausentes,

insuficientes ou diminuídos no período do desenvolvimento, os riscos de atrasos ou

DN são aumentados. Portanto, é surpreendente que estudos sobre VCT associando

efeitos neurodesenvolvimentais e práticas de aleitamento materno não tenham sido

realizados. Na verdade, apenas Heron e Golding (2004) ajustaram diversas variáveis,

incluindo amamentação em seu estudo epidemiológico. Após este ajustamento, eles

interpretaram os resultados como ausência de associação entre VCT e DN. Até agora,

estudos epidemiológicos retrospectivos (de países que suprimiram a utilização de

VCT em crianças) têm fornecido pistas de possíveis relações entre VCT e DN. Estas

pistas devem ser seguidas com estudos que sejam direcionados para fatores de

modificação relacionados a práticas de alimentação materna (exposição pré-natal e

dieta) e infantil, especialmente nos países que continuam a utilizar timerosal.

A imunização conferida pelas VCT está no centro do controle e prevenção

das doenças infecciosas (salvando vidas e evitando sofrimento desnecessário). Esta

proteção imprescindível é reforçada quando a criança é amamentada. O aleitamento

materno tem demonstrado um efeito imuno-modulador (Pabst et al., 1997) que confere

significativamente níveis mais altos de anticorpos da DTP ao lactente do que para

aquelas crianças que são alimentadas com fórmula (Hahn-Zoric et al., 1990; Silfverdal

et al., 2007). Os estímulos imunológicos moderados das vacinas podem causar febre

e respostas anoréticas, assim, o aleitamento materno protege contra ingestão de

energia diminuída, essencial para o desenvolvimento infantil (Lopez-Alarcon et al.,

2007). A amamentação parece aliviar o trauma da dor causada pelas inoculações

(Efe e Ozer, 2007).

Incertezas sempre devem beneficiar as crianças. Países ricos (geralmente com

baixas taxas de amamentação), que podem pagar vacinas livres de timerosal optaram

pela retirada VCT. Nos países que utilizam VCT, a abordagem do ―benefício da

dúvida‖ deve incluir práticas protetoras do SNC contra DN transitórias associadas com

exposição pós-natal ao Hg. Neste contexto a estratégia de apoio ao aleitamento

materno exclusivo deveria acontecer junto com a vacinação infantil.

Ademais, nossos resultados mostram que atrasos no desenvolvimento

neuropsicomotor podem estar associados às iniqüidades em saúde e situação

socioeconômica desprivilegiada. Cory-Slechta (2005) discutiu o baixo status

socioeconômico como um fator de risco para resultados adversos à saúde e

disfunções comportamentais tanto em adultos quanto em crianças. Baixo status

socioeconômico está associado com altos níveis de retardo mental, desordens do

aprendizado e déficits de atenção e linguagem em crianças. Rice (2005) argumentou

que a mais alta incidência de doença e disfunções que acompanham baixo status

socioeconômico é devido ao alto estresse ambiental vivenciado por tais populações:

uma injúria crônica associada a dificuldades econômicas resultando em prolongada

elevação dos níveis de cortisol.

7 CONCLUSÕES

1. A dose e via parenteral da exposição ao etilmercúrio derivado das vacinas

modulam o aumento relativo do mercúrio no cabelo de crianças amamentadas

aos seis meses de idade.

2. Mudanças nos metabolismo materno pós-parto, desenvolvimento infantil e

dietas de transição, e o mercúrio das vacinas conservadas com timerosal

contribuem para a assimetria das mudanças nas concentrações de mercúrio

entre mães e crianças.

3. Em crianças amamentadas, diferenças na exposição inicial ao etilmercúrio

das vacinas conservadas com timerosal, não podem predizer atrasos clínicos

do neurodesenvolvimento aos seis meses. Nós acreditamos que o aleitamento

materno foi importante para a proteção do SNC das crianças frente aos

desafios da exposição precoce ao mercúrio.

4. As desordens neurodesenvolvimentais em crianças imunizadas com vacinas

conservadas com timerosal são sensíveis à duração da amamentação e

exposição ao mercúrio (que inclui fontes maternas). Portanto, controlar a

amamentação é um aspecto importante nos estudos epidemiológicos sobre

vacinas conservadas com timerosal e desordens neurodesenvolvimentais.

5. Por fim, sugerimos que o aleitamento materno exclusivo deve ser parte

complementar de uma estratégia preventiva contra possíveis atrasos

transitórios do neurodesenvolvimento associados com exposição pós-natal ao

mercúrio nas quais as vacinas conservadas com timerosal representam uma

carga adicional.

REFERÊNCIAS BIBLIOGRÁFICAS

AGOSTONI, C., MARANGONI, F., GIOVANNINI, M., GALLI, C., RIVA, E. Prolonged

breast-feeding (six months or more) and milk fat content at six months are associated

with higher developmental scores at one year of age within a breast-fed population.

Adv. Exp. Med. Biol. 501, 137-141. 2001.

AMANNA I, SLIFKA MK. Public fear of vaccination: separating fact from fiction. Viral

Immunol 18:307–315. 2005.

ALLEN, A. Vaccine: The Controversial Story of Medicine’s Greatest Lifesaver.

Norton: 2007. 512 pp.

AMERICAN ACADEMY OF PEDIATRICS (AAP). Thimerosal in vaccines—an

interim report to clinicians. Disponível em: http://www.aap.org/new/thimpublic.htm.

acessado em 18 de outubro de 2005.

AMERICAN PSYCHIATRIC ASSOCIATION (APA). Diagnostic and statistical manual

of mental disorders. 4

ed. Washington D.C: American Psychiatric Association; 1994.

ANDERSON JW, JOHNSTONE BM, REMLEY DT. Breast-feeding and cognitive

development: a meta-analysis. Am J Clin Nutr 70:525-535. 1999.

ANDREWS N, MILLER E, GRANT A, STOWE J, OSBORNE V, TAYLOR B:

Thimerosal exposure in infants and developmental disorders: a retrospective cohort

study in the United Kingdom does not support a causal association. Pediatrics.

114(3):584-591. 2004

BALL, L.K Ball R, Pratt RD. An assessment of thimerosal use in childhood vaccines.

Pediatrics, 107:1147–54. 2001.

BASTOS, W. R., MALM, O.; PFEIFFER, W. C., CLEARY, D., Establishment and

analytical quality control of laboratories for Hg determination in biological and

geological samples in the Amazon, Brasil Ciência Cultura: 50, 255-260. 1998.

BARBOSA,A C; DÓREA,JG. Indices of mercury contamination during breast feeding in

Amazon Basin. Environmental Toxicology and Pharmacology, v.6, p.71-79, 1998.

BARBOSA, AC.; SILVA, SRL; DÓREA, JG. Concentration of mercury in hair of

Indigenous mothers and infants from the Amazon Basin. I. Arch. Environmental

Contam. Toxicology, 34:100-105, 1998.

BAZIN, H. The ethics of vaccine usage in society: lessons from the past. Curr. Opin.

Immunol. 13, 505–510. 2001.

BERNARD, S., ENAYATI, A., REDWOOD, L., ROGER, H., BINSTOCK, T. Autism: A

novel form of mercury poisoning. Med. Hypoth. 56:462–471. 2001

BERNIER RH, FRANK JA, NOLAN TF JR. Abscesses complicating DPT vaccination.

Am J Dis Child. 135(9): 826-8. 1981.

BIGHAM, M; COPES, R. Thiomersal in Vaccines Balancing the Risk of Adverse

Effects with the Risk of Vaccine-Preventable Disease. Drug Safety, 28 (2). 2005.

BLAXILL, M. F. What’s going on? The question of time trends in autism. Public

Health Rep. 119:536–551. 2004

BLAXILL, M. F., REDWOOD, L., BERNARD, S. Thimerosal and autism? A plausible

hypothesis that should not be dismissed. Med. Hypoth. 62:788–794. 2004.

BORDENAVE, G. Review: on the shoulders of giants. Louis Pasteur (1822–1895).

Microbes and Infection, 5:553–560. 2003

BRASIL. MINISTÉRIO DA SAÚDE. Secretária de Políticas de Saúde. Saúde da

Criança. Acompanhamento do Crescimento e Desenvolvimento Infantil. Série

cadernos de atenção básica n.11. Série A Normas e Manuais Técnicos n. 173.

Brasília 2002 100p. Acesso em: 11 de abril, 2007. Disponível on-line:

http://dtr2001.saude.gov.br/bvs/publicacoes/crescimento_desenvolvimento.pdf.

BRASIL. MINISTÉRIO DA SAÚDE. Secretaria de Vigilância em Saúde. Programa

Nacional de Imunizações: 30 anos. Editora MS. Brasília: Ministério da Saúde, 2003.

BRASIL. MINISTÉRIO DA SAÚDE. Secretária de Políticas de Saúde. Manual de

Vigilância Epidemiológica de Eventos Adversos Pós-Vacinação. 2007. Brasília,

DF 160p. (no prelo). Acessado em junho de 2007. Disponível em:

http://portal.saude.gov.br/portal/arquivos/pdf/manual_pni_consulta.pdf.

BURBACHER; T.M., SHEN, D.D.; LIBERATO, N.; GRANT, K.S.; CERNICHIARI E,

CLARKSON; T: Comparison of Blood and Brain Mercury Levels in Infant Monkeys

Exposed to Methyl mercury or Vaccines Containing Thimerosal. Environmental

Health Perspectives. National Institutes of Environmental Health Sciences; 2005.

CAPRA, F. O Ponto de Mutação Editora. Cultrix. Ed. 22, 447p. 2001

CASTOLDI, A.F., COCCINI, T., MANZO, L. Neurotoxic and molecular effects of

methylmercury in humans. Rev. Environ. Health. 18, 19-31. 2003.

CERNICHIARI, E, MYERS, GJ, BALLATORI, N., ZAREBA, G, VYAS, J, CLARKSON,

T, 2007. The biological monitoring of prenatal exposure to methylmercury.

Neurotoxicology. doi:10.1016/j.neuro.2007.02.009, in press.

CLARKSON, T.W. Methylmercury and fish consumption: weighing the risks. CMAJ,

158: 1465-6, 1998.

CLARKSON TW. The three modern faces of mercury. Environ Health Perspect.

110 (suppl 1):11-23. 2002

CLARKSON TW, MAGOS L, MYERS GJ. The toxicology of mercury: current

exposures and clinical manifestations. N Engl J Med; 349 (18): 1731-7. 2003

CLARKSON TW; MAGOS L. The Toxicology of Mercury and Its Chemical

Compounds. Critical Reviews in Toxicology, 36:609–662, 2006.

CLARKSON, TW; VYAS, JB; BALLATORI, N. Mechanisms of Mercury Disposition n

the Body. Am. J. Ind. Med. Oct;50(10):757-64.2007.

CLEMENTS CJ. The evidence for the safety of thiomersal in newborn and infant

vaccines. Vaccine. 22(15-16):1854-1861. 2004

CLEMENTS CJ, BALL LK, BALL R, PRATT RD: Thiomersal in vaccines: is removal

warranted? Drug Saf. 24(8):567-574. 2001

CLEMENTS, CJ; MCINTYRE, PB. When science is not enough – a risk/benefit profile

of thiomersal-containing vaccines. Expert Opin. Drug Saf. 5(1):17-29. 2006.

COALITION FOR MERCURY-FREE DRUGS (COMED). Citizen petition requesting

certain actions with respect to vaccines and other drug products, containing

added mercury, in order to reduce the health risk to susceptible fetuses, newborns,

children, adolescents and adults. Washington, US. Docket N

- 2007p. 2007.

COLEMAN, E. Ethylmercury in Vaccines. Pediatrics;111;922-923. 2003.

COLGROVE,J; BAYER, R. ―Could It Happen Here? Vaccine Risk Controversies and

the Specter of Derailment,‖ Health Affairs 24, no. 3 (2005): 729–739. 2005.

CORY-SLECHTA, D.A. Studying Toxicants as Single Chemicals: Does this Strategy

Adequately Identify Neurotoxic Risk? Neurotoxicology. 26, 491–510. 2005.

COURY DL, NASH PL: Epidemiology and etiology of autistic spectrum disorders

difficult to determine. Pediatr. Ann. 32(10):696-700. 2003.

COX NH, FORSYTH A. Thimerosal allergy and vaccination reactions. Contact

Dermatitis. 18(4):229-233. 1988.

CRUMP KS, KJELLSTROM T, SHIPP AM, et al. Influence of prenatal mercury

exposure upon scholastic and psychological test performance: benchmark analysis

of a New Zealand cohort. Risk Anal 1998; 18 (6): 701-13.1998.

DAVIDSON PW, MYERS GJ, COX C et al.: Effects of prenatal and postnatal

methylmercury exposure from fish consumption on neurodevelopment. Outcomes at

66 months of age in the Seychelles Child Development Study. JAMA 280(8):701-

707. 1998

DEER, B. Revealed: MMR research scandal. The Sunday Times (London). 2004

DODGE, P.R., PRENSKY, A.L., FEIGIN, R.D. Nutrition and the Developing Nervous

System. The C.W. Mosby Company, Saint Louis, pp. 1–538. 1975.

DOJA A, ROBERTS W. Immunizations and autism: a review of the literature. Can J

Neurol Sci. 2006 Nov; 33(4):341-6. 2006.

DOREA, J.G. Fish are central in the diet of Amazonian riparians: should we worry

about their mercury concentrations? Environ. Res. 92, 232-244. 2003.

DOREA, J.G. Cassava cyanogens and fish mercury are high but safely consumed in

the diet of native Amazonians. Ecotoxicol. Environ. Saf. 57, 248–256. 2004.

DOREA, J.G. Mercury and lead during breast-feeding. Br J Nutr 92:21–40. 2004

DOREA, J.G., MOREIRA, M.B., EAST, G., BARBOSA, A.C. Selenium and mercury

concentrations in some fish species of the Madeira River, Amazon Basin, Brazil. Biol.

Trace Elem. Res. 65, 211-220. 1998.

DOREA, J.G., BARBOSA, A.C., FERRARI, I., SOUZA, J.R. Mercury in hair and in

fish consumed by riparian women of the Rio Negro, Amazon, Brazil. Int. J. Environ.

Health Res. 13, 239–248. 2003.

DOREA, J.G., SOUZA, J.R., RODRIGUES, P., FERRARI, I., BARBOSA, A.C. Hair

mercury (signature of fish consumption) and cardiovascular risk in Munduruku and

Kayabi Indians of Amazonia. Environ. Res. 97, 208–218. 2005a.

DOREA, J.G., BARBOSA, A.C., FERRARI, I., SOUZA, J.R. Fish consumption (Hair

Mercury) and nutritional status of Amazonian Amer-Indian Children. American

Journal of Human Biology. 17, 507-514. 2005b

DOREA, J.G. Exposure to Mercury during the First Six Months via Human Milk and

Vaccines: Modifying Risk Factors. American Journal of Perinatology, v. 24, p.387-

400, 2007.

DURSTON S, HULSHOFF POL HE, CASEY BJ, GIEDD JN, BUITELAAR JK, VAN

ENGELAND H. Anatomical MRI of the developing human brain: what have we

learned? J Am Acad Child Adolesc Psychiatry; 40:1012–1020. 2001.

EDLICH, RF; OLSON,DM; OLSON,BM; GREENE,JA; GUBLER,KD; WINTERS,KL;

KELLEY,AR; BRITT, LD; LONG, WB. Update on the national vaccine injury

compensation program. The Journal of Emergency Medicine, Vol. 33, No. 2, pp.

199–211, 2007

EFE E, OZER ZC. The use of breast-feeding for pain relief during neonatal

immunization injections. Appl Nurs Res 20:10-16. 2007

ELLIS, F. A. The sensitizing factor in Merthiolate. J. Allergy, 18:212–213. 1947.

FAGAN DG, PRITCHARD JS, CLARKSON TW, GREENWOOD MR: Organ mercury

levels in infants with omphaloceles treated with organic mercurial antiseptic. Arch.

Dis. Child. 52:962-964. 1977.

FANG SC, FALLIN E. Uptake, distribution, and metabolism of inhaled ethylmercuric

chloride in the rat. Arch Environ Contam Toxicol;1:347–361. 1973

FREED, GARY L., MD, MPH; KATZ, SAMUEL L., MD; CLARK, SARAH J., MPH.

Safety of Vaccinations. Journal of the American Medical Association.

276(23):1869-1872. 1996

FOMBONNE E. Is there an epidemic of autism? Pediatrics. 107:411-2. 2001.

FOMBONNE E, ZAKARIAN R, BENNETT A, MENG L, MCLEAN-HEYWOOD D.

Pervasive developmental disorders in Montreal, Quebec, Canada: prevalence and

links with immunizations. Pediatrics. Jul;118(1):e139-50. 2006.

FORSTROM, L., HANNUKSELA, M., KOUSA, M., LEHYMUSKALLIO, E. Merthiolate

hypersensitivity and vaccination. Contact Dermatitis 6:241–245. 1980.

GEIER DA, GEIER MR. Thiomersal in Childhood Vaccines, Neurodevelopment

Disorders, and Heart Disease in the United States. Journal of American Physicians

and Surgeons Vol 8 Number 1 Spring. 2003a

GEIER DA, GEIER MR. An assessment of the impact of thimerosal on childhood

neurodevelopmental disorders. Pediatr. Rehabil. 6(2):97-102. 2003b

GEIER DA, GEIER MR: Neurodevelopmental disorders after thimerosal containing

vaccines: a brief communication. Exp Biolo Med. 228(6):660-4. 2003c

GEIER DA, GEIER MR. Neurodevelopmental disorders following thiomersal-

containing childhood immunizations: a follow-up analysis. Int. J. Toxicol. 23(6):369-

376. 2004a.

GEIER DA, GEIER MR: A comparative evaluation of the effects of MMR

immunization and mercury doses from thimerosal-containing childhood vaccines on

the population prevalence of autism. Med. Sci. Monit. 10(3):PI33-PI39. 2004b.

GEIER DA, GEIER MR. A two-phaseepidemiological study of the safety of

thimerosalcontaining vaccines: a follow-up analysis. Med. Sci. Monit. 11(4):CR160-

CR1170. 2005.

GEIER DA, GEIER MR. An assessment of downward trends in neurodevelopmental

disorders in the United States following removal of thimerosal from childhood

vaccines. Med Sci Monit. 12(6): CR231-239. 2006a.

GEIER, D. A., AND GEIER, M. R. A clinical trial of combined anti-androgen and anti-

heavy metal therapy in autistic disorders. Neuro. Endocrinol. Lett. 27:833–838. 2006b.

GEIER, D. A., AND GEIER, M. R. A meta-analysis epidemiological assessment of

neurodevelopmental disorders following vaccines administered from 1994 through

2000 in the United States. Neuro. Endocrinol. Lett. 27:401–413. 2006c

GEIER, D. A., GEIER, M. R. A prospective assessment of porphyrins in autistic

disorders: a potential marker for heavy metal exposure. Neurotoxicol. Res. 10:57–

64. 2006d

GEIER, D. A., GEIER, M. R. A case-series of children with apparent mercury toxic

encephalopathies manifesting with clinical symptoms of regressive autistic disorders.

J. Toxicol. Environ. Health A. 70:837–851. 2007a.

GEIER, D. A., GEIER, M. R. A prospective study of Thimerosal containing Rho(D)-

immune globulin administration as a risk factor for autistic disorders. J. Matern. Fetal

Neonatal Med. 20:385–390. 2007b.

GEIER, D. A., SYKES, L. K; GEIER, M. R. A review of thimerosal (merthiolate) and

its ethylmercury breakdown product: specific historical considerations regarding

safety and effectiveness. Journal of Toxicology and Environmental Health, Part

B, 10:575–596, 2007.

GESELL A. A criança de 0 a 5 anos. 6rd ed., Martins Fontes, São Paulo, pp. 20-

300. 2003

GESELL, A., AMATRUDA, C.S. Psicologia do Desenvolvimento – do Lactente à

criança pequena, bases neuropsicológicas e comportamentais. 1rd ed.,

Atheneu, São Paulo, pp. 30-106. 2000.

GRANDJEAN, P., JORGENSEN, P.J., WEIHE, P. Human milk as a source of

methylmercury exposure in infants. Environ. Health Perspect. 102, 74–77. 1994.

GRANDJEAN, P; WHEIHE, P; WHITE, R. F; DEBES, F; ARAKI, S;YOKOYAMA, K;

MURATA, K; SORENSEN, N; DAHL, R.; JORGENSEN, P.J. Cognitive deficit in 7-

yearsold children with prenatal exposure to methylmercury. Neurotoxicol. Teratol.

n.19, p.417-428, 1997.

GRANDJEAN, P.; BUDTZ – JORGENSEN, E.; WHITE, R. F. et al. Methylmercury

exposure biomarkers as indicators of neurotoxicity in children aged 7 year. American

Journal of Epidemiology, v.150, n.3, USA, p.301-304, 1999.

GRANDJEAN,P; BELLINGER,D; BERGMAN, A; CORDIER, S; DAVEY-SMITH, G;

ESKENAZI, B; GEE, G; GRAY,K; HANSON, M; HAZEL, P; HEINDEL, JJ; HEINZOW,

B; HERTZ-PICCIOTTO, I; HU, H, HUANG, T T-K; JENSEN, K; LANDRIGAN, PJ;

MCMILLEN, C; MURATA, K; RITZ, B; SCHOETERS, G; SKAKKEBÆK, NE;

SKERFVING, S; WEIHE P. The Faroes Statement: Human Health Effects of

Developmental Exposure to Chemicals in Our Environment. Basic & Clinical

Pharmacology & Toxicology,102, 73–75. 2007.

HALSEY, NA. Limiting Infant Expousure to Thiomersal in Vaccines and other sources

of Mercury. JAMA, November 10, Vol 282, N

18. 1999.

HALSEY NA, GOLDMAN LR. Mercury in infants given vaccines containing

thiomersal. Lancet. 361(9358):698-699. 2003.

HAHN-ZORIC M, FULCONIS F, MINOLI I, MORO G, CARLSSON B, BOTTIGER M,

RAIHA N, HANSON LA. Antibody responses to parenteral and oral vaccines are

impaired by conventional and low protein formulas as compared to breast-feeding.

Acta Paediatr Scand. 79:1137–1142. 1990

HVIID A, STELLFELD M, WOHLFAHRT J, MELBYE M. Association between

thimerosal-containing vaccine and autism. JAMA 2003; 290 : 1763-6.

HERMAN, LM; GERBERT, DA; LARSON, LW; LÉGER, MM; MCNELLIS, R;

O'DONOGHUE, DL; ULSHAFER, C; VAN DYKE, EM. Vaccines, thimerosal, and

neurodevelopmental outcomes. Journal of American Academy of Physician

Assistants. vol.19, n. 1. 2006.

HERON J, GOLDING J: ALSPAC STUDY TEAM. Thimerosal exposure in infants and

developmental disorders: a prospective cohort study in the United Kingdom does not

support a causal association. Pediatrics 114(3):577-583. 2004.

HOLMES, AS; BLAXILL, MF; HALEY, BE. Reduced Levels of Mercury in First Baby

Haircuts of Autistic Children. International Journal of Toxicology, 22:277–285, 2003.

HORNIG M, CHIAN D, LIPKIN WI. Neurotoxic effects of postnatal thimerosal are

mouse strain dependent. Mol Psychiatry 9:833–845. 2004

HVIID A, STELLFELD M, WOHLFAHRT J, MELBYE M: Association between

thimerosalcontaining vaccine and autism. JAMA. 290(13):1763-1766. 2003.

INHAMUNS, A.J., FRANCO, M.R.B. Composition of total, neutral, and phospholipids

in Mapara (Hypophthalmus sp.) from the Brazilian Amazonian area. J. Agric. Food

Chem. 49, 4859-4863. 2001

INSTITUTE OF MEDICINE (IOM). Immunization safety review: vaccines and

autism. Washington (DC): National Academies Press, 2004.

JENNER, E. An enquiry into the causes and effects of the varriolae vaccinae; a

disease discovered in some of the western counties of England, particularly

Gloucestershire, and known by the name of the cowpox; 1798. The Jenner

Museum. Disponível: www.jennermuseum.com; acessado: agosto de 2007.

JENSEN, T.K., GRANDJEAN, P., JORGENSEN, E.B., WHITE, R.F., DEBES, F.,

WEIHE, P. Effects of breast feeding on neuropsychological development in a

community with methylmercury exposure from seafood. J. Expo. Anal. Environ.

Epidemiol. 15, 423–430. 2005.

JOINT STATEMENT OF THE AMERICAN ACADEMY OF PEDIATRICS (AAP) AND

THE UNITED STATES PUBLIC HEALTH SERVICE (PHS). Timerosal in Vacinnes.

Pediatrics. 104:568-569. 1999.

KENNEDY JR, RF. Deadly Immunity. Rolling Stone, 2005. Disponível em:

http://rollingstone.com/politics/story . Acessado em 20 de junho de 2006.

KUCZMARSKI, R.J., OGDEN C.L., GRUMMER-STRAWN, L.M., FLEGAL, K. M.,

GUO, S. S., WEI, R., MEI, Z., CURTIN, L.R., ROCHE, A.F., JOHNSON, C. L. CDC

growth charts: United States. Advance data from vital and health statistics.

Hyattsville, Maryland: National Center for Health Statistics. 314, 1-28. 2000.

LITTER, M. Antiinfecciosos locales. Anti-sépticosfungicidas e parasiticidas

externos. In: Farmacologiaexperimental y clinica. 7. ed. Buenos Aires: El Ateneo,

1986. p. 1397-1443

LOPEZ-ALARCON M, GARZA C, DEL PRADO M, GARCIA-ZUNIGA PA, BARBOSA

L. Breastfeeding's protection against illness-induced anorexia is mediated partially by

docosahexaenoic acid. Eur J Clin Nutr. 2007.

LOWE, I., SOUTHERN, J. 1994. The antimicrobial activity of phenoxyethanol in

vaccines. Lett. Appl. Microbiol. 18:115–116.

LUMAN, ET; FIORE, AE; STRINE, TW; BARKER, LE. Impact of Thimerosal-Related

Changes in Hepatitis B Vaccine Birth-Dose Recommendations on Childhood

Vaccination Coverage Journal of American Physicians and Surgeons v 11, n1. 2004

MADSEN KM, LAURITSEN MB, PEDERSEN CB et al.: Thimerosal and the

occurrence of autism: negative ecological evidence from Danish population-based

data. Pediatrics. 112(3 Pt 1):604-606. 2003

MAGOS L. Review on the toxicity of ethylmercury, including its presence as a

preservative in biological and pharmaceutical products. J Appl Toxicol; 21: 1-5. 2001.

MAGOS L. Neurotoxic character of thimerosal and the allometric extrapolation of

adult clearance half-time to infants. J Appl Toxicol; 23: 263-9. 2003.

MAGOS L, CLARKSON TW. Overview of the clinical toxicity of mercury. Ann Clin

Biochem; 43: 257–68. 2006.

MAGOS L, BROWN AW, SPAROW S, et al. The comparative toxicology of ethyl- and

methylmercury. Arch Toxicol 1985; 57 (4): 1985

MALM, O. Autismo é associado a uso excessivo de vacinas. Ciência Hoje. vol. 30 nº

175. 2001.

MARQUES RC Uma casa dividida: preocupações públicas e a segurança das

vacinas. Ciência Hoje. In press. 2008.

MARQUES RC, DOREA JG, BASTOS WR, REBELO MF, FONSECA MF, MALM O.

Maternal mercury exposure and neuro-motor development in breastfed infants from

Porto Velho (Amazon), Brazil. Int J Hyg Env Health. 210:51-60. 2007a.

MARQUES, R.C., DOREA, J.G., BASTOS, W.R., FONSECA, M.F., MALM, O. Hair

mercury in breast-fed infants exposed to thimerosal preserved vaccines. Eur. J.

Pediatr., 166:935-941. 2007b

MARQUES RC, DOREA JG, MANZATTO AG, BASTOS WR, BERNARDI JVB, MALM

O. Time of perinatal immunization, thimerosal exposure and neurodevelopment at six

months in breastfed infants. Acta Paediatr. Acta Paediatr 96:864-868. 2007c.

MARQUES RC, BERNARDI JV, DOREA JG, BASTOS WR, MALM O. Principal

component analysis and discrimination of variables associated with pre- and post-

natal exposure to mercury. Int J Hyg Env Health (in press). 2007d

MARQUES, R.C., DOREA, J.G., BASTOS, W.R., MALM, O. Changes in children hair-

Hg concentrations during the first 5 years: Maternal, environmental and iatrogenic

modifying factors. Regulatory Toxicology and Pharmacology. 49. 17–24. 2007e

MARQUES RC, DOREA JG, BERNARDI JVB BASTOS WR; MALM O. Maternal fish

consumption in the nutrition transition of the Amazon Basin: Growth of exclusively

breastfed infants during the first 5 years. Annals of Human Biology. In press. 2007f

MARQUES, R. C. Avaliação da exposição ao mercúrio e seus compostos sobre

o desenvolvimento neuropsicomotor em crianças de Porto Velho-RO. Belém,

2002, 102p. Dissertação (Mestrado em Doenças Tropicais), Núcleo de Medicina

Tropical, Universidade Federal do Pará, Brasil.

MAYA,L; LUNA; F. El timerosal y las enfermedades del neurodesarrollo infantil. An

Fac Med., Lima; 67(3). 2006.

MIGOWSKI, E. Vacinas: riscos e benefícios. Um guia prático e rápido. São

Paulo. Editora BSB, 2007. 260p

MILLER, NEIL Z. Vaccines: Are they really safe and effective? New Atlantean

Press: Santa Fe, NM. 32, 45. p 17-20, 1993.

MILLER, NEIL Z. Immunization: Theory vs. Reality. New Atlantean Press, Santa Fe,

NM, p 131.1996.

MOREIRA, MS. Política de Imunização no Brasil: processo de introdução de

novas vacinas. Dissertação de Mestrado. Rio de Janeiro. Escola Nacional de

Saúde Pública. Fundação Oswaldo Cruz. Ministério da Saúde, 2002.

MOULIN, A. M. A hipótese vacinal: por uma abordagem crítica e antropológica de

um fenômeno histórico. História, Ciências, Saúde. Manguinhos, vol. 10 (supl. 2):

499-517, 2003.

MURCH SH, ANTHONY A, CASSON DH, MALIK M, BERELOWITZ M, DHILLON

AP, et al. Retraction of an interpretation. Lancet. 2004; 363:750.

MUTTER, J, NAUMANN, J, SADAGHIANI, C, SCHNEIDER, R, WALACH, H.

Alzheimer Disease: Mercury as pathogenetic factor and apolipoprotein E as a

moderator. Neuroendocrin. Lett. 25, 275-283. 2004

MUTTER, J., NAUMANN, J., SADAGHIANI, C., WALACH, H., DRASCH, G. Mercury

and autism: Response to the letter of K. E. v. Muhlendahl [Int. J. Hyg. Environ. Health

208 (2005) 435]. Int. J. Hyg. Environ. Health. 208, 437–438. 2005

MYERS GJ, DAVIDSON PW, COX C, SHAMLAYE CF, PALUMBO D, CERNICHIARI

E, SLOANE-REEVES J, WILDING GE, KOST J, HUANG LS, CLARKSON TW:

Prenatal methylmercury exposure from ocean fish consumption in the Seychelles

child development study. Lancet, 361:1686-1692. 2003.

NASCIMENTO, LO; LORENZI FILHO, G; ROCHA-ADOS, S. Lethal mercury poisoning

due to ingestion of Merthiolate. Rev. Hosp. Clin. Fac. Med. Sao Paulo 45: 216–218.

1990.

NATURE NEUROSCIENCE. Editorial: A mercurial debate over autism. v.8, n.9,

september, 2005.

NELSON KB, BAUMAN ML. Thiomersal and autism? Pediatrics. 111:674-679. 2003.

PABST HF, SPADY DW, PILARSKI LM, CARSON MM, BEELER JA, KREZOLEK

MP. Differential modulation of the immune response by breast- or formula-feeding of

infants. Acta Paediatr 86:1291-1297. 1997.

PATRICK, L. Mercury toxicity and antioxidants: Part 1: role of glutathione and alpha-

lipoic acid in the treatment of mercury toxicity. Altern. Med. Rev. 7, 456-471, 2002.

PICHICHERO, ME; CERNICHIARI E; LOPREIATO J; TREANOR J. Mercury

concentrations and metabolism in infants receiving vaccines containing thiomersal: a

descriptive study. Lancet 360:1737-1741. 2002

PONTE, C. F. Vacinação, controle de qualidade e produção de vacinas no Brasil a

partir de 1960. História, Ciências, Saúde. Manguinhos, vol. 10 (suplemento 2): 619-

53, 2003.

PORTO, A; PONTE, CF. Vacinas e campanhas: as imagens de uma história a ser

contada. História, Ciências, Saúde. Manguinhos, vol.10 suppl.2, p.725-742. 2003.

PRADO, MJVP; OLIVEIRA, RC; FERREIRA, SRF; VIEIRA; RCPA; PINHO; JJRG. A

retirada do timerosal do mercado farmacêutico e sua substituição pelos derivados de

amônio quaternário. Avaliação do risco/benefício. Lecta, v. 22, n. 1/2, p. 37-47,

jan./dez. 2004.

PRESCOTT, HARLEY, AND KLEIN. Microbiology (Third Edition), Wm. C. Brown

Publishers, Chicago. 1996. p 588.

REDWOOD, L., BERNARD, S., BROWN D. Predicted Mercury Concentrations in Hair

From Infant Immunizations: Cause for Concern. Neurotoxicology. 22, 691-697, 2001.

RICE, D.C. Assessing the effects of environmental toxicant exposure in developmental

epidemiological studies: issues for risk assessment. Neurotoxicology. 26, 483-489.

2005.

RODRIGUES, M.; SAITO, T. Avaliação da eficácia antimicrobiana do timerosal e

fenol em alguns produtos imunobiológicos. Rev. Farm. Bioq. Univ. São Paulo, v.

27, n. 2, p. 127-136, 1991.

SAAVEDRA, M. Percursos da vacina na Índia portuguesa — séculos XIX e XX. Hist.

cienc. saude-Manguinhos, vol.11 suppl.1 Rio de Janeiro 2004

SANFORD R. KIMMEL, M.D., Vaccine Adverse Events: Separating Myth from

Reality. American Family Physician. 1, v. 66, n. 11. 2002.

SCHEIBNER, VIERA. Vaccination. New Atlantean Press, Santa Fe, NM. p 205-210.

SHEVELL M, FOMBONNE E. Autism and MMR vaccination or thimerosal exposure:

an urban legend? Can J Neurol Sci. Nov;33(4):339-40. 2006

SIMON PA, CHEN RT, ELLIOT JA, SCHWARTZ B. Outbreak of pyogenic abscesses

after diphteria and tetanus toxoids and pertussis vaccinnation. Pediatr Infect Dis J;

12(5): 368-71. 1993.

SILFVERDAL SA, EKHOLM L, BODIN L. Breastfeeding enhances the antibody

response to Hib and Pneumococcal serotype 6B and 14 after vaccination with

conjugate vaccines. Vaccine. 25:1497-1502. 2007

STAJICH GV, LOPEZ GP, HARRY SW, et al. Iatrogenic exposure to mercury after

hepatitis B vaccination in preterm infants. J Pediatr. 136: 679-681. 2000.

STEHR-GREEN P, TULL P, STELLFELD M, MORTENSON PB, SIMPSON D:

Autism and thimerosal containing vaccines: lack of consistent evidence for an

association. Am. J. Prev. Med. 25(2):101-106. 2003

STEUERWALD, U; WEIHE, P; JORGENSEN, PJ; BJERVE, K; BROCK, J;

HEINZOW, B; BUDTZ-JORGENSEN, E; GRANDJEAN, P. Maternal seafood diet,

methylmercury exposure, and neonatal neurologic function. The Journal of

Pediatrics, v.136 (5) p.599-605, 2000.

STEWART, AJ; DEVLIN, PM. The history of the smallpox vaccine. Journal of

Infection. 52, 329–334. 2006.

SUGARMAN, S.D. Cases in Vaccine Court — Legal Battles over Vaccines and

Autism. n engl j med 357;13. september 27, 1275-7p. 2007

TANOUE, Y., ODA, S. Weaning time of children with infantile autism. J. Autism. Dev.

Disord. 19, 425-434. 1989.

TAYLOR B, MILLER E, FARRINGTOM CP, PETROPOULOS MC, FAVOT-MAYAUD

I, LI J. et al. Autism and measles, mumps and rubella vaccine: no epidemiological

evidence for a causal association. Lancet. 353:2026-29; 1999.

TEMPORÃO, JG. O Complexo Industrial da Saúde: Público e Privado na

Produção e Consumo de Vacinas no Brasil. Tese de Doutorado, Rio de Janeiro:

Instituto de Medicina Social, Universidade do Estado do Rio de Janeiro. 2002.

TEMPORÃO, JG. O mercado privado de vacinas no Brasil: a mercantilização no espaço

da prevenção. Cad. Saúde Pública, Rio de Janeiro, 19(5):1323-1339, set-out, 2003.

THE EUROPEAN AGENCY FOR THE EVALUATION OF MEDICINAL PRODUCTS

(EMEA). Human Medicines Evaluation Unit. EMEA public statement on thiomersal

containing medicinal products: 8 July 1999; EMEA/20962/99. Disponível em:

http://www.eudra.org/emea.html. Acessado em: dezembro de 2006.

THE EUROPEAN AGENCY FOR THE EVALUATION OF MEDICINAL PRODUCTS

(EMEA). Human Medicines Evaluation Unit. EMEA Position Statement – Recent

developments concerning thiomersal in vaccines: 29 June 2000; EMEA/CPMP1578/

00. Disponível em: http://www.eudra.org/emea.html. Acessado em: dezembro de 2006.

THE NATIONAL ADVISORY COMMITTEE ON IMMUNIZATION (NACI). Thimerosal:

updated statement .Canada Communicable Disease Report;33(ACS-6):1-13. 2007

THOMPSON WW, PRICE C, GOODSON B, SHAY DK, BENSON P, HINRICHSEN

VL, LEWIS E, ERIKSEN E, RAY P, MARCY SM, DUNN J, JACKSON LA, LIEU TA,

BLACK S, STEWART G, WEINTRAUB ES, DAVIS RL, DESTEFANO F. Early

thimerosal exposure and neuropsychological outcomes at 7 to 10 years. N Engl J

Med. Sep 27;357(13):1281-92. 2007

TRASANDE L, LANDRIGAN PJ, SCHECHTERS C. Public health and economic

consequences of methylmercury toxicity to the developing brain. Env Health Persp;

113:590-6. 2005.

TROLLFORS, B; KNUTSSON, N; TARANGER, J.; MARK, A; BERGFORS, A; SUNDH,

V; LAGERGA,T. Diphtheria, tetanus and pertussis antibodies in 10-year-old children

before and after a booster dose of three toxoids: implications for the timing of a booster

dose. Eur J Pediatr; 165: 14–18. 2006.

US CENTERS FOR DISEASE CONTROL AND PREVENTION (US CDC).

Thimerosal in vaccines: a joint statement of the American Academy of Pediatrics and

Public Health Service. MMWR Morb Mortal Wkly Rep; 48 (26): 563-5. 1999.

US CENTERS FOR DISEASE CONTROL AND PREVENTION. (US CDC). Notice to

readers: summary of the joint statement on thimerosal in vaccines. MMWR Morb

Mortal Wkly Rep; 49 (27): 622, 631. 2000.

US CENTERS FOR DISEASE CONTROL AND PREVENTION (US CDC). Joint

statement: AAP and PHS. Concerning removal of thimerosal from vaccines. 2001.

Disponível em: www.cdc.gov/nip/vacsafe/concerns/thimerosal/joint_statement_00.htm.

Acessado em: 21 de setembro de 2007]

US DEPARTMENT OF HEALTH AND HUMAN SERVICES (US DHHS). NATIONAL

VACCINE INJURY COMPENSATION PROGRAM (VICP). Rockville, M. D: Health

Resources and Services Administration. Acessado em setembro de 2007. Disponível

em: http://www.hrsa.gov/vaccinecompensation/.

US FOOD AND DRUG ADMINISTRATION (FDA). Center for Biologics Evaluation

and Research. Thimerosal in vaccines. Acessado em abril de 2006. Disponível em:

http://www.fda.gov/cber/vaccine/thimerosal.html.

US FOOD AND DRUGS ADMINISTRATION (US FDA). Mercury Containing Drug

Products for Topical Antimicrobial Over-the-Counter Human Use; Establishment

of a Monograph. Federal Register. 47(2): 436-442 (January 5 1982). 1982.

US COURT OF FEDERAL CLAIMS. VACINNE COURT. OFFICE OF SPECIAL

MASTERS. Omnibus autism proceeding. Claims for vaccine injuries resulting in

autism spectrum disorder or a similar neurodevelopmental disorder. Various

petitioners. Washington, D.C. United States. 2007. Acessado no período de maio a

outubro de 2007. Disponível em: ftp://autism.uscfc.uscourts.gov/autism/index.html

VALLE, LBS; OLIVEIRA-FILHO, RM; DELUCIA,R; OGA, S. Farmacologia integrada.

In: DEMUSI, M. Antissépticos, desinfetantes, esterilizantes. São Paulo: Atheneu. v.

2, p. 575-606. 1991.

VERSTRAETEN T, DAVIS RL, DESTEFANO F, LIEU TA, RHODES PH, BLACK SB,

SHINEFIELD H, CHEN RT; VACCINE SAFETY DATALINK TEAM. Safety of thimerosal-

containing vaccines: a two-phased study of computerized health maintenance

organization databases. Pediatrics 112:1039-1048. 2003.

WALKER, SJ; SEGAL, J; ASCHNER, M. Cultured lymphocytes from autistic children

and non-autistic siblings up-regulate heat shock protein RNA in response to

thimerosal challenge. NeuroToxicology 27 685–692. 2006.

WEISBERG, SS. Vaccine preventable diseases: current perspectives in historical

context, Part I. Dis Mon; 53(9): 422-66. 2007.

WILSON, CB; MARCUSE, EK. Vaccine safety–vaccine benefits: science and the

public’s perception. Nature Reviews. Immunology. v. 1, p.160-4. 2001.

WORLD HEALTH ORGANIZATION (WHO). Environmental Health Criteria 1:

Mercury. Geneva, Switzerland, WHO, 1976.

WORLD HEALTH ORGANIZATION (WHO). Environmental Health Criteria 101 –

Methylmercury. Geneva. WHO, 99p. 1990.

WORLD HEALTH ORGANIZATION (WHO). Group on the Growth Reference

Protocol, 1998. A growth curve for the 21st century: the WHO multicentre

growth reference study. WHO, Geneva. 1998.

WORLD HEALTH ORGANIZATION (WHO). Global Advisory Committee on Vaccine

safety, 20-21 June 2002. Wkly Epidemiol Rec; 77(47): 389-90. 2002.

WORLD HEALTH ORGANIZATION (WHO). Global Advisory Committee on Vaccine

Safety: statement on thiomersal, 2003. Acessado em fevereiro de 2006. Disponível

em: http://www.who.int/vaccine_safety/topics/thiomersal/statement200308/en/index.html.

WORLD HEALTH ORGANIZATION (WHO). Thiomersal: neuro-behavioural studies

in animal models. Wkly. Epidem. Rec.80: 3-7. 2005

WORLD HEALTH ORGANIZATION (WHO). WHO Multicentre Growth Reference

Study Group. WHO Child Growth Standards: length/height-for-age, weight-for-

age, weight-for-length, weight-for-height and body mass index-for-age: methods

and development. Geneva: World Health Organization, 2006a.

WORLD HEALTH ORGANIZATION (WHO). WHO Multicentre Growth Reference

Study Group. WHO Child Growth Standards based on length/height, weight and age.

Acta Paediatr Suppl. 450:76–85. 2006b

WORLD HEALTH ORGANIZATION (WHO). WHO vaccine-preventable diseases

monitoring system, 2006 global summary. Expanded Programme on

Immunization of the Department of Immunization, Vaccines and Biologicals. WHO

Document Production Services, Geneva, Switzerland. 2006c.

WORLD HEALTH ORGANIZATION (WHO). Statement on Thiomersal. WHO Global

Advisory Committee on Vaccine Safety. Geneva, World Health Organization. 2006d.

Disponível em: http://www.who.int/vaccine_safety/topics/thiomersal. Acessado em:

15 de fevereiro de 2007.

WORLD HEALTH ORGANIZATION (WHO). Preventing disease through healthy

environments. Exposure to mercury: a major public health concern. WHO

Document Production Services, Geneva, Switzerland, 2007.

YOSHIDA, H. Problems with eradicating polio – World Health Organization's Global

Polio Eradication Initiative – Brief Article. Science News. Nov 25, 2000.

ZAMBRANO, B. Consideraciones generales sobre el mercurio, el timerosal, y su uso

en vacunas pediátricas. Rev Med Uruguay. 20: 4-11. 2004.

ZAREBA G, CERNICHIARI E, HOJO R, NITT SM, WEISS B, MUMTAZ MM, JONES

DE, CLARKSON TW. Thimerosal distribution and metabolism in neonatal mice:

comparison with methyl mercury. J Appl Toxicol. 27:511-518. 2007.

ZETTERSTRO, R. Flawed reports of immunization complications: consequences for

child health. Acta Pædiatr. 93. 2004.

ZIEGEL, E.E.; CRANLEY, M. S. Enfermagem obstétrica. 8ª ed., Guanabara Koogan,

Rio de Janeiro, 1986. 696p

ANEXOS

Int. J. Hyg. Environ.-Health 210 (2007) 51–60

Maternal mercury exposure and neuro-motor development in breastfed

infants from Porto Velho (Amazon), Brazil

Rejane Correˆ a Marques

a,b,Ã

, Jose

Garrofe Do

rea

, Wanderley Rodrigues Bastos

Mauro de Freitas Rebelo

,Ma

rlon de Freitas Fonseca

, Olaf Malm

Fundac¸a˜o Universidade Federal de Rondo

nia, Porto Velho, RO, Brazil

Laborato

rio de Radioisotopos Eduardo Penna Franca, Instituto de Biofı

sica Carlos Chagas Filho, Universidade Federal do Rio de

Janeiro, CCS, Bl, G, IBCCF, Cidade Universitaria, Ilha do Funda˜o, 21941-900 Rio de Janeiro, RJ, Brazil

Universidade Federal de Brası

lia, Brası

lia, DF, Brazil

Received 15 March 2006; received in revised form 14 August 2006; accepted 14 August 2006

Abstract

Fish is an important item in the diet of Amazonians, and per se is their best single source of essential nutrients.

Rapid urbanization and migration are bringing changes in dietary habits of Amazonians. Exposure to ﬁs h-Hg during

pregnancy and lactation were studied in 100 women and newborns from Porto Velho. Tissue-Hg concentrations and

neurodevelopment (Gesell Developmental Schedules) were asses sed at birth and at 6 months in exclusively breastfed

infants. Maternal mean frequency of ﬁsh consumption was low (o2 meals/week; range 0–47 meals/week) compared

to Amazonian standards. Women consuming o2 ﬁsh meals/week showed less median hair-Hg (3.5 mgg

À1

) than

women that consumed X2 ﬁsh meals/week (5.7 mg g). Median total Hg in maternal hair (5.4 mgg

À1

) was higher than in

newborns (1.6 mgg

À1

). Signiﬁcant correlation was observed between maternal hair-Hg and infant hair-Hg at birth

(r ¼ 0:353; po0:01) and at six months (r ¼ 0:510; po0:01). Placenta-Hg was also signiﬁcantly correlated to mate rnal

hair-Hg (r ¼ 0:321; po0:01), newborn hair-Hg (r ¼ 0:219; po0:05), mate rnal blood-Hg (r ¼ 0:250; po0:01) and to

umbilical cord-Hg (r ¼ 0:857; po0:01). Most infants (74%) had normal Gesell Schedules but among the 26% showing

neuro-motor development delays only six (7%) had multiple (motor, language, and adap tative) delays. The infants

with multiple delays were born from mothers with range of hair-Hg comparable to mothers of normally developed

infants. Coincidentally, mothers of infants with multiple delays also showed the lowest range of income and level of

education. Fish consumption, income, and level of education varied greatly among these breastfeeding urban mothers.

It seems that development delays of exclusively breastfed infants are a component of the health inequalities that

accompanies socioeconomic disadvantages.

Keywords: Amazon; Fish consumption; Neuro-motor development; Breastfeeding; Hair-Hg

Introduction

Mercury is listed among the most toxic substances in

industrialized countries. One-third of emissions are

estimated to originate from natural sources and the

ARTICLE IN PRESS

www.elsevier.de/ijheh

doi:10.1016/j.ijheh.2006.08.001

Corresponding author. Laborato

rio de Radioisotopos Eduardo

Penna Franca, Instituto de Biofı

sica Carlos Chagas Filho, Universi-

dade Federal do Rio de Janeiro, CCS, Bl. G. IBCCF, Cidade

Universita

ria, Ilha do Funda

o, 21941-900 Rio de Janeiro, RJ, Brazil.

Tel./fax: +55 21 2561 5339.

E-mail address: [email protected] (R.C. Marques).

other two-thirds from anthropogenic sources (Patrick,

2002). Environmental Hg is found in various chemical

forms: elemental Hg, inorganic Hg, and organic Hg

(ethyl-, methyl-, alkyl-, or phenyl-Hg). The chemistry of

Hg modulates its toxicity and metabolism. While

inorganic Hg acts mainly on the kidneys, volatile

metallic Hg and especially methylmercury (MeHg)

primarily affect the central nervous system (CNS). The

vulnerability of the developing human brain is the most

important window for harmful Hg compounds (Castoldi

et al., 2003). Dietary intake of ﬁsh and seafood products

is the main source of MeHg exposure. Fish MeHg is

easily absorbed by gastrointestinal tract and rapidly

enters the blood stream. It is distribut ed throughout the

body within 3–4 days. It is estimated that 5% of dietary

MeHg is found in the blood and 10% in the brain,

with a half-life ranging between 45 and 70 days

(Castoldi et al., 2003).

Fish is an important item in the diet of Amazonians,

and per se is their best source of essential nutrients. Fish

is a fundamental complement for native Amazonians on

protein-poor starchy food diets (70–80% of dietary

energy from cassava); its protein content is well digested

and has a high biological value (Dorea, 2004). Further-

more, besides its nutrient content, ﬁsh ﬂesh can enhance

absorption of Zn and Fe (Dorea, 2004). According to

Inhamuns and Franco (2001), Amazonian ﬁsh contain

omega-3 polyunsaturated fatty acids (PUFA; decosa-

hexanoic [22:6] acid and eicosapentaenoic [20:5] acid)

essential for infant neurodevelopment. Decosahexanoic

acid is an essential component of nervous system cell

membranes that is delivered to the fetus and post-natally

into milk. However, ﬁsh is also a bioconcentrator of

natural MeHg. Fortunately, Amazonian ﬁsh are, on the

other hand, a good source of selenium (Dorea et al.,

1998), known to counteract the toxic effects of Hg;

signiﬁcant correlations between Hg and Se in hair were

reported in Amazonians (Vasconcellos et al., 2000;

Campos et al., 2 002).

A legitimate concern arising from the large amounts

of metallic Hg (used to extract gold) discarded in the

Amazonian environment prompted ﬁsh-H g studies that

were summarized by Dorea (2003). Amazon ﬁsh

bioconcentrate MeHg originated from naturally occur-

ring Hg in the rainforest (Barbosa et al., 2003). It is now

known that Amazon soils are rich in Hg and those

constitute a natural source of Hg for methylation

(Fadini and Jardim, 2001; Bastos et al., 2006). However,

while deforestation (along with agricultural projects)

and alluvial-gold extraction have brought about vast

changes in the environment of West-Amazonia, urban

development has brought changes in the traditional

lifestyles of human populations.

Fish consumption is part of the cultural adaptation of

indigenous ‘‘ribeirinhos ’’ (riverine populations). Amazo-

nian women (especially Rio Negro ribeirinhos ) depend-

ing heavily on ﬁsh consumption have showed hair-Hg

that is amon g the highest of the world (Barbosa et al.,

1998). Despite this no neurological problems involving

ﬁsh consumption or other neurotoxic substance in foods

like cassava products have been described (Dorea,

2004). Neuro-behavioral tests suggested that riverine

children of East Amazonia presented alterations asso-

ciated with high hair-Hg concentrations (Grandjean

et al., 1999), but a recent study reported no signiﬁcant

difference (Tavares et al., 2005). In the French-

Guyanese Amazon, Cordier et al. (2002) rep orted no

major neurologic signs in Amerindian children of three

different levels of ﬁsh-Hg exposure (regions with high,

intermediate and low ﬁsh consumption). Nevertheless,

but they observed that after adjusting for some potential

confounders, there were dose-dependent (regions) ef-

fects, which were increased deep tendon reﬂexes, poor

coordination of the legs, and decreased performance in

the Stanford-Binet Copying score.

Tissue accumul ation and toxicity are not equivalent in

the case of Hg; MeHg accumulation is greater in the

kidney than in the brain, but the brain appears to be the

key target (Cory-Slechta, 2005). It is a point of fact that

the CNS formation and development during pregnancy

and lactation can be affected by multiple causes, ranging

from maternal nutrition to neurotoxic-substance expo-

sure. In this speciﬁc time window, Hg exposure can

affect neurobehavioral functions. Mild exposure may

result in delayed symptoms (not observed at birth), such

as difﬁculty in walking and talking, and persistence of

abnormal perinatal reﬂexes (World Health Organization

(WHO), 1990; Myers and Davidson, 1998).

Our objectives were: (a) to study ﬁsh-Hg exposure of

urban Amazonian mothers; (b) to associate maternal

exposure (hair-Hg) with tissue-Hg and factors relevant

to neuro-motor development of breastfed infants; and

dimensions of infant neurodevelopment and maternal

socioeconomic and Hg exposure features.

Materials and methods

Porto Velho is the capital of the state of Rondonia

(West Amazonia). Until the 1960s it was a traditional

Amazonian city but after agricultural projects in the

southwest region of the state followed the opening of

roads, there was also a great inﬂux of prospectors for

exploring alluvial gold along the banks of the Rio

Madeira basin. Since then, Porto Velho has experienced

signiﬁcant demographic changes with people coming

from many other Brazilian regions.

The research protocol was approved by the Ethics

Committee of Studies for Humans of the Universidade

Federal de Rondonia. Pregnant mothers were intro-

duced to the study and invited to participate by a nurse

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–6052

during their routine visits to the Pre-natal Clinics of

three hospitals of Porto Velho: Hospital de Base,

Hospital Panamericano and Hospital Regina Pacis.

Potential participants received plain-language informa-

tion about the study and a written consent form was

presented and signed by the volunteering mother; the

written consent stated that participation was voluntary,

their conﬁdentiality was assured and that they could

withdraw from the study at any time. Mothers were

selected among those in good health, reporting no illness

or complaints at the time of the study and who were

willing to breast feed. Excluding factors were occupa-

tional exposure to toxic chemicals and hereditary

neurological illnesses. One hundred mother s between

the ages of 15 and 45 years were recruited among those

that manifested the intention of exclusively breastfeed-

ing up to 6 months of age.

For each mother a complete clinical evaluation was

obtained from medical records and at the time of the

ﬁrst interview a questionnaire was applied to assess

socioeconomic and educational status. The question-

naire also evaluated food habits, frequency of ﬁsh

consumption, and intention of breastfeeding. While at

the maternity wards, we co llected samples of cord blood,

placenta and hair from mothers and respective infants

(fetal hair). Maternal blood (4.5 mL), placenta (three

different aliquots) and umbilical cord sample (5–10 g)

were stored in nitric-acid clean vessels, refrigerated and

taken to the laboratory and frozen at À20 1C until

analysis. Hair strands were cut from the occipital region

and placed in plast ic bags, with the root end stapled on a

paper sheet.

The newborns were clinicall y examined with special

attention to vitality, perinatal reﬂexes, maturity, and

congenital malformations; weight, length, head circum-

ference, and Apgar scores were recorded. Anthropo-

metric data at birth (weight, length and head

circumference) were compared with data tabulated by

US National Center for Health Statistics—(NCHS)

after its adaptation by the WHO for world-wide use

(World Health Organization (WHO) Group on the

Growth Referen ce Protocol, 1998; Kuczmarski et al.,

2000). The software for calculating pediatric anthro-

pometry (ANTHRO 1.02, 1999) from the Centers for

Diseases Control and Prevention was used.

At 6 months of age, only 86 of the 100 original

mother–infant pairs reported for the programmed

clinical and neurobehavioral examination when hair

samples were collected again. Five mothers had moved

out of the state, ﬁve did not report and could not be

found, and four bab ies had died within the ﬁrst month.

Because two mothers developed gestational diabetes and

two others developed pre-eclamptic toxem ia, we used

only data from 82 mother–infant pairs. Children

received the full immunization scheme in accordance

with Brazilian vaccination program.

The infants’ development assessment was conducted

at the age of 6 months by trained professionals using the

Gesell Developmental Schedules (Gesell, 20 03; Gesell

and Amatruda, 2000). The Gesell Schedules included all

reactions (voluntary, spontaneous or learned) and

reﬂexes. We also evaluated postural reactions, hand

pressure, locomotion and coordination, constructive

ability (which is inﬂuenced by motor development),

visible and audible communication, individual reactions

regarding people and stimulations (depending mainly on

the temperament of the child and the surroundings). The

results were expressed as developmental scores for the

Motor Skills, Language Development, Adaptive Beha-

vior, and Personal Social behaviors.

Hg determination

Sample preparation and Hg determination were done

according to routine procedures previously established

at the Universidade Federal do Rio de Janeiro (Bastos et

al., 1998). We followed routine procedures of the

laboratory after adaptation of analytical protocol used

for Hg determ ination in previous studies analyzing

blood, hair and ﬁsh-ﬂesh matrices (Bastos et al., 1998;

Malm et al., 1989, 1998). Brieﬂy, the hair samples were

comminuted with stainless steel scissors, weighed, and

digested before analysis. Blood samples were digested

with concentrated HNO

(3 mL) and KMnO

(5%;

6 mL) using a microwave oven system for 35 min (CEM-

Coorporation, MDS 2000, Matthews, NC, USA).

Placenta and umbilical cord samples were weighed and

digested with HNO

(1:1; 5mL) and KMnO

(5%; 4 mL) using a digestion block at 80 1C for 1 h

(Tecnal Ltd., Piracicaba, Sa

o Paulo, Brazil). Human

hair samples were washed with EDTA 0.01%, dried in

an oven at 50 1C, weighed and digested with 5 mL of

HNO

(1:1) and 4 mL of 5% KMnO

using a

digestion block at 80 1C for 40 min. The determination

of total Hg in the digested samples was done by cold

vapor atomic absorption spectrometry with a ﬂow

injection system-FIMS (CV-AAS, Perkin-Elmer—FIMS

400, Ueberlingen, Germany). All glasswa re used in the

analytical protocol was washed clean, rinsed with 5%

EDTA and double distilled, and left to rest in 5% HNO

overnight. Then it was rinsed again in double-distilled

water, and dried at 100 1C for 12 h. Precision an d

accuracy of Hg determinations were assured by the use

of internal standards, use of triplicate analyses of

samples and certiﬁed reference materials (IAEA-085

and 086, Vienna, Austria) with recoveries of 92%.

Statistical analysis

A multivariate correspondence model was used to

analyze maternal factors (tissue Hg, socio-economic

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–60 53

status) that might affect the infant’s social and adaptive

abilities, gross motor ability or the ﬁne motor ability;

this step was done with statistical software (Statsoft,

2002, Tulsa, AZ, USA). Variables used for the analysis

are listed in Table 1. Afterwards, correlation analysis

was used to compare the tissue-Hg concentrations

between mothers and respective neonates (SPSS for

Windows 14.0, 2005, Chicago, USA).

Results

Descriptive data of mothers and infants are presented

in Tables 1–5. The demographics summarized in Table 1

show a wide range of socioeconomic status (SES). Most

of the 100 mothers were enrolled from a public hospital

(n ¼ 61, Hospital de Base) while 39% were from

corporate middle-class hospitals (n ¼ 13, Hospital Pa-

namericano; n ¼ 26, Hospital Regina Pacis); a substan-

tial proportion (39%) of these mothers were primiparas.

The median income was US$125, and 64% did not have

indoor plumbing, thus indicating that the majority of

mothers were socioeconomically underprivileged. Irre-

spective of income and education, 57% reported that

they consumed ﬁsh up to once a week, 4% reported

more than 7 ﬁsh servings a week and only 5% reported

not consuming ﬁsh (Table 1). Anthropometric data of

mothers and infants are shown in Table 2. Nine

newborns were below and two were above NCHS

reference cu rves for weight. After 6 months of exclusive

breastfeeding four children were overweight and only

three boys presented short length.

Mercury exposure data are presented in Tables 3–5.

Tissue Hg concen trations as a function of reported

ARTICLE IN PRESS

Table 1. Socioeconomic characteristics of the 100 mothers enrolled in the study

Characteristics Minimum Median Maximum Percentage

Mother education (years) 0 8 18

Income (US$)/m 16.67 125.00 1250.00

Type of home

Owned 57

Rental 14

Living with relatives 29

Persons/household 2 5 14

No electricity 20

Water supply

Running water 36

Well 61

Local rivers 3

Fish-eating habits

Fish meals (week) 0 1 14

0–1/week 57

2–7/week 43

Fish-eating sources

Market ﬁsh 53

Local rivers 42

No ﬁsh 5

Table 2. Biodata of the 82 mother–infant pairs

Minimum Median Maximum

Maternal data

Pre-natal

Age (years) 15 22 40

Weight(kg) 43 55 68.5

Height (m) 1.51 1.61 1.7

BMI (kg m

À2

) 17.40 21.19 26.56

Gestational age (w) 36 39.5 43

Pregnancy, n

128

At the end of pregnancy

Weight(kg) 53 69 85

BMI (kg m

À2

) 22.35 26.53 32.39

Infant data

Birth

Weight (g) 2200 3200 4370

Length(cm) 46 50 55

Head circumference(cm) 30 34 39

Six months

Weight (g) 6110 7000 8500

Length(cm) 61 68 73

Head circumference (cm) 40 43 45

Primiparas ¼ 39.

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–6054

frequency of ﬁsh consumption are shown in Table 3.In

this study, maternal ﬁsh consumption (hair-Hg) did not

depend on monthly income or years of education; mean

income and education were very close between the

groups. The maternal hair-Hg concentrations varied

widely (0.2–62.4 mgg

À1

) reﬂecting extremes of reported

ﬁsh consumption. Indeed mothers that reported low

ﬁsh-consumption ( o2 servings a week) showed a lower

median hair-Hg concentrations (3.5 mgg

À1

) than the

other group (5.7 mgg

À1

). Most of the maternal hair-Hg

concentrations (57%) were below 6 mg g; 34% of hair

samples showed Hg concentrations between 6 and

15 mgg

À1

and 9% showed hair-Hg greater than

15 mgg

À1

. The statistics of Hg concentrations in mothers

and infants are shown in Table 4. The median umbilical

cord-Hg concentration (7.5 ng g

À1

) was close to the

median placenta-Hg concentration. Fetal hair-Hg of the

82 newborns showed that 92% were o6 mg g; 7% were

between 6 and 15 mgg

À1

and only 1% was 415 mgg

À1

Correlation coefﬁcients among maternal and infant Hg-

variables are summarized as follows: signiﬁcant non-

parametric correl ations (Spermans) were observed

between maternal hair-Hg and infant hair-Hg at birth

(r ¼ 0:353; po0:01) and at 6 months (r ¼ 0:510;

po0:01). Placenta-Hg was also signiﬁcantly correlated

with maternal hair-Hg (r ¼ 0:321; po0:01), newborn

hair-Hg (r ¼ 0:219; po0 :05), maternal blood-Hg

(r ¼ 0:250; po0:05), and with umbilical cord-Hg

(r ¼ 0:857; po0:01).

The assessment of neuro-motor development of

breastfed 6-month-old infants is summarized in Table 5.

Most infants (74%) showed normal schedules and 21

children (26%) exhibited developmental delay in one or

more features of the Gesell Schedules: 1% showed

motor impairment, 9% had language deﬁcits, and 16%

had multiple impairments (7% motor, language, adap-

tative, and 9% motor, language). There were no

children who presented developmental delay in the

personal social behavior. There were no children who

presented developmental delay in the social ability. The

26% of infants showing neuromotor delays showed

higher median hair-Hg values; these infants were born

from mothers that had median hair-Hg concentrations

also higher than mothers of normally developed infants.

However, infants with multiple delays were born from

mothers that also showed the lowest median income.

The infants with higher median fetal hair-Hg showed

even higher median hair-Hg at 6 months (Table 5).

Multivariate analysis was performed to iden tify

groups of related variables. After identiﬁcation (and

removal) of redundant variables by Cluster Analysis,

Correspondence Analysis was applied to identify groups

of associated variables. Association (co-occurrence) is

characterized by similar position in the two-dimensional

(2D) plane depicted in Fig. 1. The ﬁrst dimension is

characterized by the infant groups which showed the

most delayed development (adaptive, motor and lan-

guage) and the greatest hair-Hg concentrations (group

#1 and group #3). In contrast with a group of normal

children from high income families and more educated

mothers (group #2). The second dimension is mainly

characterized by the highly exposed neonates (group #1,

with high hair-Hg) in contrast with another group of

infants with mild delay in Gessel scores (group #4).

Discussion

Assuming that hair strands are the best integrator of

past MeHg exposure, the main ﬁnding of this study is

that the marker of ﬁsh consumption (maternal hair-Hg)

signiﬁcantly correlated with fetal hair-Hg; this signiﬁ-

cant association lasted until 6 months of lactation.

Notably, delay in neuromotor developm ent observed in

infants with higher median hair-Hg did not indicate a

dose response relationship: the highest hair-Hg values

were found in the normal infant group (Table 5).

ARTICLE IN PRESS

Table 3. Maternal income, education and tissue Hg concentrations as a function of reported frequency of ﬁsh consumption

Fish

servings/w

N Blood [Hg]

(mgL

À1

)

Hair [Hg]

(mgg

À1

)

Umbilical cord

(mgg

À1

)

Placenta

(mgg

À1

)

Income

(US$)

Education

(y)

0–1 57 0.6 (0.01–10)

3.5 (0.2–28.7) 8.1 (0.53–58.1) 8 (0.7–50.4) 125 (50–1250) 8 (0–18)

X2 43 0.5 (0.01–10) 5.7 (0.16–62.4) 6.5 (0.1–43.7) 7.9 (0.2–56.3) 125 (17–1250) 8 (4–17)

Median (MinÀMax); w ¼ week; y ¼ year.

Table 4. Total Hg concentrations in tissues of mothers and

infants

Minimum Median Maximum

Mothers

Umbilical cord (ng g

À1

) 0.12 7.44 43.74

Placenta (ng g

À1

) 0.37 8.10 56.28

Blood (mgL

À1

) 0.01 0.55 9.97

Hair (mgg

À1

) 0.39 5.40 62.43

Infant’s hair Hg (mgg

À1

)

Fetal 0.05 1.59 19.65

Six months 0.02 1.81 32.95

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–60 55

In Fig. 1, the highest income and educational levels were

clustered in group #2. These ﬁnding suggests the

occurrence of some protective conditions which were

not present in underpriv ileged families. Poorer and less

educated families mo re probably lack mother–fetus

pairs ideal development support.

The mean hair-Hg of these mothers (7.4 mgg

À1

)is

lower than reported values for ribeirinho mothers

(8.3–9.4 mgg

À1

) of the Rio Tapajo

s(Pinheiro et al.,

2005), breastfeeding mothers (14.3 mgg

À1

) of the Rio

Madeira (Barbosa et al., 1997) and the Rio Negro

(Dorea et al., 2003). In the ribei rinho women of the Rio

Negro consuming at least one ﬁsh-based meal a day

median hair-Hg (18.3 mgg

À1

) was much higher than that

of women in the present study (Dorea et al., 2003). In

ribeirinho women of the Rio Madeira, the mean

breastmilk-Hg concentration was 5.8 ng g

À1

and infant

hair-Hg was 9.8 mgg

À1

(Barbosa et al., 1997). There

were indications that MeHg transfer was higher during

pregnancy than during breastfeedi ng. Nevertheless,

there are no reports of clinical signs of neuropathologies

in that population associated with ﬁsh consumption. A

recent study of urban mothers from Paramaribo

(Surinamese Amazon) showed a much lower concentra-

tion (0.8 mgg

À1

) of hair-Hg (Mohan et al., 2005). In

non-traditional urban pregnant-women of Alta

Floresta (southern Amazonia) the mean hair-Hg con-

centrations was also 1.2 mgg

À1

(range, 0.05–8.2 mgg

À1

ARTICLE IN PRESS

Table 5. Frequency distribution of neurodevelopment (Gesell Schedules) of infants at 6 months of age and summary of

corresponding markers of Hg exposure and socioeconomic status

Gesell Scale N

[%]

Infant hair Hg,

0m, (mgg

À1

)

Infant hair Hg,

6m, (mgg

À1

)

Mother hair Hg

(mgg

À1

)

Income

(US$)

Mother

education (y)

Normal 61 [74] 1.4 (0.05–9.3) 1.6 (0.01–33) 4.28 (0.39–62.43) 145.8 (16.7–1250) 8 (4–18)

Motor (M) 1 [1] 0.62 1.7 10.56 375 11

Language (L) 7 [9] 1.59 (0.3–3.5) 2.8 (0.9–26.9) 7.29 (1.30–28.73) 145.8 (50–525) 8 (5–16)

M and L 7 [9] 2.3 (0.6–7.4) 5.6 (1.2–15.1) 10.34 (2.14–12.48) 104.16 (70–125) 8 (4–11)

M, L and A

6 [7] 6.4 (2.3–19.7) 6.9 (0.4–18.1) 5.74 (3.32–20.77) 104.16 (83.3–208.3) 6.5 (6–11)

Overall 82 [100] 1.6 (0.05–19.7) 1.8 (0.02–33) 5.40 (0.39–62.43) 125 (16.7–1250) 8 (0–18)

Median (MinÀMax); m ¼ month; y ¼ year.

A ¼ Adaptive.

1.5

AD:D

If>9

MT:D

LG:D

Nn < 9

AD:L

LG:L

MT:L

$400

$200

$100

WL>5

12ySch

Nn < 6

LG:N

$800

$1200

AD:N

MT:N

18ySch

8ySch

Nn < 3

4ySch

If<9

If<3

If<6

WL<5

Nn>9

1.0

0.5

0.0

-0.5

-1.0

-3.0 -2.5 -2.0 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5

Dimension 1; Ei

envalue: 0.40 (16.06% of inertia)

Dimension 2; Eigenvalue: 0.26 (10.44% of inertia)

Fig. 1. Multivariate analysis (anthropometrics parameter, performance in Gesell schedule and socioeconomics status). MT—Motor,

LG—Language, AD—Adaptive, N—Normal, D—Delay, L—Borderline, If—infant Hg-hair, Nn—newborn Hg-hair, ySch—

mother education in year, $—income, WL—weight-for-length percentile.

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–6056

This population is formed by non-ﬁsh eaters that

migrated from the South of Brazil during the gold rush

(Hacon et al., 2000).

The studied sample of Porto Velho women is not

homogenous, neither culturally or socioeconomically.

Therefore, because there were no severe prenatal insults

that could distort early pattern of neurodevelopment, the

observed delays were within expected rates for Brazilians

(Paine and Pasquali, 1983). Even in homogenous popula-

tions there are variations in neurobehavioral outcomes.

Dodge et al. (1975) have discussed the limitations of the

reliability of a single function as the evaluator of an insult

on normal development. In our case there were only six

cases of retardation of all milestones.

Regarding prenatal Hg exposure, our results (Gesell

Schedules) are consistent with the absence of neurode-

velopmental abnormalities in early childhood rep orted

in Peruvian mothers (Paciﬁc coast) with comparable

mean hair-Hg levels (8.3 mgg

À1

; range 1.2–30 mgg

À1

)

during pregnancy (Marsh et al., 1995). A larger long-

itudinal study with 708 Seychellois infants also reported

that in utero exposure to MeHg (from a maternal ﬁsh

diet) caused no adverse outcomes in neurological and

psychological development at 6 months of age (Myers et

al., 1995). In that study maternal hair-Hg ranged from

0.5 to 26.7 mgg

À1

(Myers et al., 1995). Neurodevelop-

mental and Hg-associated studies of infants and young

children sometimes do not make references to breast-

feeding status. Because of the fundamental role of

breastfeeding and neuromotor development it is im-

portant to emphasize such studies. Only 6% of the

breastfed infants in the present study showed adaptive

behavior delays. Speciﬁc components (PUFA) present in

breast milk, but not in infant formulas, which are

essential for neuronal development and organization

have accounted for cognitive an d developmental scores

in favor of breastfeeding (Agostoni et al., 2001).

Therefore, when breastfeeding is considered in mater-

nal-Hg contamination studies, there are clearly beneﬁts

for the neurodevelopment of the breastfed infants

(Grandjean et al., 1994; Jensen et al., 2005).

Although we collected fetal hair at birth, infant

exposure to maternal Hg (consumed as ﬁsh-MeHg)

during pregnancy is better assessed with maternal hair-

Hg. Indeed, maternal hair-Hg was signiﬁcantly corre-

lated with fetal hair-Hg, which is in agreement with

other Amazonian studies of women with high (Barbosa

and Do

rea, 1998) and low (Mohan et al., 2005) ﬁsh

consumption. Lindow et al. (2003) also found signiﬁcant

correlation betw een maternal hair-Hg and fetal hair-Hg

in a group of mothers predominantly exposed to dental

amalgam-Hg. There are several drawbacks in fetal hair-

Hg as a reliable indicator of intra-uterine exposure. Not

all infants are born with hair; hair starts to grow at the

18–20th week of development at the front al and the

parietal regions and is likely to fall by the time of birth.

Also, oc cipital hair is likely to grow at the time of birth

and to fall after 12 weeks. Furthermore, hair density,

length and texture are dependent upon factors such as

race and sex. Fetal hair-Hg in the present study was

higher (2.4 mgg

À1

) than for newborns (1.6 mgg

À1

)of

urban Surinamese mothers (Mohan et al., 2005).

Studies involving non-occupational maternal Hg

exposure (ﬁsh consumption) during pregnancy and

infant ne uro-motor develop ment are complicate to

design because of the confounding factors. This is

further complicated when mothers are exposed to

neurotoxic substances other than MeHg present in ﬁsh

(Stewart et al., 2003). Nevertheless, maternal ﬁsh intake

during pregnancy was associated with higher develop-

mental scores for language comprehension, and social

activities (Daniels et al., 2004). A recent study by Oken

et al. (2005) in low ﬁsh-eating American mothers

showed that the mean visual recognition memory was

higher at six months in infants born to mothers that ate

42 ﬁsh servings/week but had less than 1.2 of hair-Hg

during pregnancy, clearly suggesting an interaction

between positive effects of ﬁsh eating and negative

effects of Hg contamination. Although the multivariate

model adjusted for breastfeeding duration, Oken et al.

(2005) failed to ad dress the organic Hg effects currently

debated in vaccinated infants.

Fetal Hg exposure is exclusively derived from

maternal contamination. However, during breastfeed-

ing, iatrogenic Hg exposure (vaccines) is frequent but

has not been taken into consideration in infant

neurodevelopmental studies. Redwood et al. (2001)

reviewed Hg exposure due to Thimerosal (Thiomersal,

ethylmercurithiosalicate-TMS), a preservative found in

many infant vaccines. TMS contains 49.6% ethyl

mercury-EtHg (by weight) contributing 25 mg of EtHg

per dose; 12.5–40% depending on the vaccine (Redwood

et al., 2001). During an immunization schedule, infants

may receive vaccines at birth (12.5 mgEtHg), two

(62.5 mgEtHg), four (50 mgEtHg) and six (62.5 mgEtHg)

months that could expose them to a total of

207.5 mgEtHg during the ﬁrst 6 months of life. The

possible associati on of vaccinations (conta ining TMS)

and autism spectrum disorde rs (ASD) is gaining

scientiﬁc attention. Mutter et al. (2004, 2005) discussed

the epidemiology and mechanism of Hg-associated

developmental (behavioral) disorders and suggested that

effects of MeHg found in ﬁsh are less toxic compared to

iatrogenic sources of Hg. They argued that the vaccine

situation resembles the epidemic of acrodynia in the last

century which affected up to 1 of 500 infants. After

removing teething powder, which contained Hg as

calomel (Hg

), acrodynia disappeared. They also

argued that in 1953 immunizations with TMS-contain-

ing vaccines preceded the onset of acrodynia in several

cases. It should be noticed that epidemiologic al studies

relating ASD and vaccine-TMS have not considered

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–60 57

breastfeeding. However, there is one study indicating

that early weaning could contribute to the etiology of

autism (Tanoue and Oda, 1989).

Mercury in the umbilical cord is not frequently

assessed, but one such study (Murata et al., 2004)

reported that maternal hair-Hg concentration was

signiﬁcantly correlated with the MeHg in the umbilical

cord obtained from 49 newborns. This is closely in line

with our present study. Besides easier sampling and

processing, umbilical cord may be collected 1 week after

birth. A retrospective study (babies born between 1950

and 1965) of the Minamata crisis in Japan showed that

the 24 patients diagnosed with Minamata disease had

umbilical cord Hg concentrations of 1.63 mgg

À1

(Akagi

et al., 1998). This is higher than values that we (in the

present study) and others (Daniels et al., 2004) found in

low ﬁsh eating mothers.

Although ﬁsh consumption has been monitored for

riverine populations this is the ﬁrst study reporting ﬁsh

consumption of urban Amazonian mo thers. In Amazo-

nian populations hair-Hg is a surrogate of ﬁsh

consumption of adu lts (Dorea et al., 2005a) and children

(Dorea et al., 2005b). The Brazilian Amazon population

showing elevated hair-Hg exposure is mainly indigenous

and ribeirinho. The impor tance of subsistence ﬁshing for

ribeirinho communities has shown to be proportional to

their distance from urban centers (Alves et al., 2006). In

remote communities of the Rio Madeira subsistence on

ﬁsh is also well characterized by mean hair-Hg

concentrations 416 mgg

À1

. We recently showed that

these communities may consume ﬁsh twice a day (Bastos

et al., 2005). In the present study, ﬁsh consumption of

the Porto Velho mothers is greatly decreased probably

as a result of urbanization and migratory ﬂuxes from

other parts of the country. However, hair-Hg is much

higher than in Paramaribo women (Mohan et al., 2005).

This study also shows that neuro-motor development is

not associated with ﬁsh consumption but could be

placed with health inequalities and social deprivation.

Indeed, Cory-Slechta (2005) discussed low SES itself as

a known risk factor for adverse health outcomes and

behavioral dysfunctions both in adults and children.

Low SES is associated with higher levels of mental

retardation, learning disorders and language and atten-

tion deﬁcits in children. Rice (2005) argued that the

higher incidence of disease and dysfunction accompany-

ing low SES is due to the greater environmental stresses

experienced by such populations: a chronic strain

associated with persistent economic hardships resulting

in protracted elevation of cortisol levels.

Acknowledgements

We are greatly in debt to the mothers for their

participation in the study, to the staff and Directors

(Marineˆ s R. dos Santos Cezar, Tereza Cristina Ramos,

Daniele Brasil, Katia Wendt, and Laura Jane Marques)

of the Hospitals (Hospital de Base Ary Pinheiro,

Hospital Panamericano and Hospital Regina Pacis),

the staff of the UNIR and UFRJ. This work was

supported by UNESCO, Ministe

rio da Sau´ de do Brasil

and The National Research Council of Brazil-CNPq

(PNOPG project-55.0882/01-4) and the RIOMAR

Foundation.

References

Agostoni, C., Marangoni, F., Giovannini, M., Galli, C., Riva,

E., 2001. Prolonged breast-feeding (six months or more)

and milk fat content at six months are associated with

higher developmental scores at one year of age within a

breast-fed population. Adv. Exp. Med. Biol. 501, 137–141.

Akagi, H., Grandjean, P., Takizawa, Y., Weihe, P., 1998.

Methylmercury dose estimation from umbilical cord con-

centrations in patients with Minamata disease. Environ.

Res. 77, 98–103.

Alves, M.F.A., Fraiji, N.A., Barbosa, A.C., Lima, D.S.S.,

Souza, J.R., Do

rea, J.G., Cordeiro, G.W.O., 2006. Fish

consumption, mercury exposure and serum antinuclear

antibody in amazonians. Int. J. Environ. Health 16,

255–262.

Barbosa, A.C., Do

rea, J.G., 1998. Indices of mercury

contamination during breast feeding in the Amazon Basin.

Environ. Toxicol. Pharmacol. 6, 71–79.

Barbosa, A.C., Souza, J.R., Garcia, M.A., 1997. Mercury

contamination in hair of riverine populations of Apiaca

Reserve in the Brazilian Amazon. Water Air Soil Pollut. 97,

1–8.

Barbosa, A.C., Silva, S.R.L., Do

rea, J.G., 1998. Concentra-

tion of mercury in hair of Indigenous mothers and infants

from the Amazon Basin. I. Arch. Environ. Contam.

Toxicol. 34, 100–105.

Barbosa, A.C., Souza, J., Dorea, J.G., Jardim, W.F., Fadini,

P.S., 2003. Mercury biomagniﬁcation in a tropical black

water, Rio Negro, Brazil. Arch. Environ. Contam. Toxicol.

45, 235–246.

Bastos, W.R., Malm, O., Pfeiffer, W.C., Cleary, D., 1998.

Establishment and analytical quality control of laboratories

for Hg determination in biological and geological samples

in the Amazon, Brazil. Cieˆ nc. Cult. 50, 255–260.

Bastos, W.R., Gomes, J.P.O., Oliveira, R.C., Almeida, R.,

Nascimento, E.L., Bernardi, J.V.E., Lacerda, L.D., Sil-

veira, E.G., Pfeiffer, W.C., 2006. Mercury in the environ-

ment and riverside population in the Madeira River Basin,

Amazon, Brazil. Sci. Total Environ. 368, 344–351.

Campos, M.S., Sarkis, J.E.S., Muller, R.C.S., Brabo, E.D.,

Santos, E.D., 2002. Correlation between mercury and

selenium concentrations in Indian hair from Rondonia

State, Amazon region, Brazil. Sci. Total Environ. 287,

155–161.

Castoldi, A.F., Coccini, T., Manzo, L., 2003. Neurotoxic and

molecular effects of methylmercury in humans. Rev.

Environ. Health 18, 19–31.

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–6058

Cordier, S., Garel, M., Mandereau, L., Morcel, H.H.,

Doineau, P., Gosme-Seguret, S., Josse, D., White, A.,

Amiel-Tison, C., 2002. Neurodevelopmental investigations

among methylmercury-exposed children in French Guiana.

Environ. Res. 89, 1–11.

Cory-Slechta, D.A., 2005. Studying toxicants as single

chemicals: does this strategy adequately identify neurotoxic

risk? Neurotoxicology 26, 491–510.

Daniels, J.L., Longnecker, M.P., Rowland, A.S., Golding, J.,

2004. Fish intake during pregnancy and early cognitive

development of offspring. Epidemiology 15, 394–402.

Dodge, P.R., Prensky, A.L., Feigin, R.D., 1975. Nutrition and

the Developing Nervous System. The C.W. Mosby

Company, Saint Louis, pp. 1–538.

Dorea, J.G., 2003. Fish are central in the diet of Amazonian

riparians: should we worry about their mercury concentra-

tions? Environ. Res. 92, 232–244.

Dorea, J.G., 2004. Cassava cyanogens and ﬁsh mercury are

high but safely consumed in the diet of native Amazonians.

Ecotoxicol. Environ. Saf. 57, 248–256.

Dorea, J.G., Moreira, M.B., East, G., Barbosa, A.C., 1998.

Selenium and mercury concentrations in some ﬁsh species

of the Madeira River, Amazon Basin, Brazil. Biol. Trace

Elem. Res. 65, 211–220.

Dorea, J.G., Barbosa, A.C., Ferrari, I., Souza, J.R., 2003.

Mercury in hair and in ﬁsh consumed by riparian women of

the Rio Negro, Amazon, Brazil. Int. J. Environ. Health

Res. 13, 239–248.

Dorea, J.G., Souza, J.R., Rodrigues, P., Ferrari, I., Barbosa,

A.C., 2005a. Hair mercury (signature of ﬁsh consumption)

and cardiovascular risk in Munduruku and Kayabi Indians

of Amazonia. Environ. Res. 97, 208–218.

Dorea, J.G., Barbosa, A.C., Ferrari, I., Souza, J.R., 2005b.

Fish consumption (hair mercury) and nutritional status of

Amazonian Amer-Indian children. Am. J. Hum. Biol. 17,

507–514.

Fadini, P.S., Jardim, W.F., 2001. Is the Negro River Basin

(Amazon) impacted by naturally occurring mercury? Sci.

Total Environ. 275, 71–82.

Gesell, A., 2003. A crianc¸ a de 0 a 5 anos, 6th ed. Martins

Fontes, Sa

o Paulo, pp. 20–300.

Gesell, A., Amatruda, C.S., 2000. Psicologia do Desenvolvi-

mento—do Lactente a

crianc¸ a pequena, bases neuropsico-

gicas e comportamentais, 1st ed. Atheneu, Sa

o Paulo, pp.

30–106.

Grandjean, P., Jorgensen, P.J., Weihe, P., 1994. Human milk

as a source of methylmercury exposure in infants. Environ.

Health Perspect. 102, 74–77.

Grandjean, P., White, R.F., Nielsen, A., Cleary, D., de

Oliveira Santos, E.C., 1999. Methylmercury neurotoxicity

in Amazonian children downstream from gold mining.

Environ. Health Perspect. 107, 587–591.

Hacon, S., Yokoo, E., Valente, J., Campos, R.C., da Silva,

V.A., de Menezes, A.C., de Moraes, L.P., Ignotti, E., 2000.

Exposure to mercury in pregnant women from Alta

Floresta-Amazon basin, Brazil. Environ. Res. 84, 204–210.

Inhamuns, A.J., Franco, M.R.B., 2001. Composition of total,

neutral, and phospholipids in Mapara (Hypophthalmus sp.)

from the Brazilian Amazonian area. J. Agric. Food Chem.

49, 4859–4863.

Jensen, T.K., Grandjean, P., Jorgensen, E.B., White, R.F.,

Debes, F., Weihe, P., 2005. Effects of breast feeding on

neuropsychological development in a community with

methylmercury exposure from seafood. J. Expo. Anal.

Environ. Epidemiol. 15, 423–430.

Kuczmarski, R.J., Ogden, C.L., Grummer-Strawn, L.M.,

Flegal, K.M., Guo, S.S., Wei, R., Mei, Z., Curtin, L.R.,

Roche, A.F., Johnson, C.L., 2000. CDC Growth Charts:

United States. Advance Data from Vital and Health

Statistics, vol. 314. National Center for Health Statistics,

Hyattsville, MD, pp. 1–28.

Lindow, S.W., Knight, R., Batty, J., Haswell, S.J., 2003.

Maternal and neonatal hair mercury concentrations: the

effect of dental amalgam. BJOG 110, 287–291.

Malm, O., Pfeiffer, W.C., Souza, C.M.M., 1989. Utilizac¸ a

odo

acesso

rio de gerac¸ a

o de vapor frio para ana

lise de mercu´ rio

em investigac¸ o

es ambientais por espectrofotometria de

absorc¸ a

o atoˆ mica. Cieˆ nc. Cult. 41, 88–92.

Malm, O., Pfeiffer, W.C., Cleary, D., 1998. Establishment and

analytical quality control of laboratories for Hg determina-

tion in biological and geological samples in the Amazon,

Brazil. Cieˆ nc. Cult. 50, 255–260.

Marsh, D.O., Turner, M.D., Smith, J.C., Allen, P., Richdale,

N., 1995. Fetal methylmercury study in a Peruvian ﬁsh-

eating population. Neurotoxicology 16, 717–726.

Mohan, S., Tiller, M., Van Der Voet, G., Kanhai, H., 2005.

Mercury exposure of mothers and newborns in Surinam: a

pilot study. Clin. Toxicol. (Phila.) 43, 101–104.

Murata, K., Sakamoto, M., Nakai, K., Weihe, P., Dakeishi,

M., Iwata, T., Liu, X., Ohno, T., Kurosawa, T., Kamiya,

K., Satoh, H., 2004. Effects of methylmercury on neuro-

development in Japanese children in relation to the

Madeiran study. Int. Arch. Occup. Environ. Health 77,

571–579.

Mutter, J., Naumann, J., Sadaghiani, C., Schneider, R.,

Walach, H., 2004. Alzheimer disease: mercury as pathoge-

netic factor and apolipoprotein E as a moderator.

Neuroendocrin. Lett. 25, 275–283.

Mutter, J., Naumann, J., Sadaghiani, C., Walach, H., Drasch,

G., 2005. Mercury and autism: Response to the letter of K.

E. v. Muhlendahl [Int. J. Hyg. Environ. Health 208 (2005)

435]. Int. J. Hyg. Environ. Health. 208, 437–438.

Myers, G.J., Davidson, P.W., 1998. Prenatal methylmercury

exposure and children: neurologic, developmental, and

behavioral research. Environ. Health Perspect. 106,

841–847.

Myers, G.J., Marsh, D.O., Davidson, P.W., Cox, C.,

Shamlaye, C.F., Tanner, M.A., Choi, A., Cernichiari, E.,

Choisy, O., Clarkson, T.W., 1995. Main neuro-develop-

mental study of Seychellois children following in utero

exposure to methylmercury from a maternal ﬁsh diet:

outcome at six months. Neurotoxicology 16, 653–664.

Oken, E., Wright, R.O., Kleinman, K.P., Bellinger, D.,

Amarasiriwardena, C.J., Hu, H., Rich-Edwards, J.W.,

Gillman, M.W., 2005. Maternal ﬁsh consumption, hair

mercury, and infant cognition in a US Cohort. Environ.

Health Perspect. 113, 1376–1380.

Paine, P.A., Pasquali, L., 1983. Is motor development really

more advanced in Third World infants? Percept. Mot. Skills

57, 729–730.

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–60 59

Patrick, L., 2002. Mercury toxicity and antioxidants: Part 1:

role of glutathione and alpha-lipoic acid in the treatment of

mercury toxicity. Altern. Med. Rev. 7, 456–471.

Pinheiro, M.C., Muller, R.C., Sarkis, J.E., Vieira, J.L., Oikawa,

T., Gomes, M.S., Guimara

es, G.A., do Nascimento, J.L.,

Silveira, L.C., 2005. Mercury and selenium concentrations

in hair samples of women in fertile age from Amazon

riverside communities. Sci. Total Environ. 349, 284–288.

Redwood, L., Bernard, S., Brown, D., 2001. Predicted mercury

concentrations in hair from infant immunizations: cause for

concern. Neurotoxicology 22, 691–697.

Rice, D.C., 2005. Assessing the effects of environmental

toxicant exposure in developmental epidemiological stu-

dies: issues for risk assessment. Neurotoxicology 26,

483–489.

Stewart, P.W., Reihman, J., Lonky, E.I., Darvill, T.J., Pagano,

J., 2003. Cognitive development in preschool children

prenatally exposed to PCBs and MeHg. Neurotoxicol.

Teratol. 25, 11–22.

Tanoue, Y., Oda, S., 1989. Weaning time of children

with infantile autism. J. Autism Dev. Disord. 19,

425–434.

Tavares, L.M.B., Camara, V.M., Malm, O., Santos, E.C.,

2005. Performance on neurological development tests by

riverine children with moderate mercury exposure in

Amazonia, Brazil. Cad. Saude Publ. 21, 1160–1167.

Vasconcellos, M.B.A., Bode, P., Paletti, G., Catharino,

M.G.M., Ammerlaan, A.K., Saiki, M., Fa

varo, D.I.T.,

Byrne, A.R., Baruzzi, R., Rodrigues, D.A., 2000. Determi-

nation of mercury and selenium in hair samples of Brazilian

Indian populations living in the Amazonic region by NAA.

J. Radioanal. Nucl. Chem. 244, 81–85.

World Health Organization (WHO), 1990. Methylmercury,

vol. 101. WHO, Geneva.

World Health Organization (WHO) Group on the Growth

Reference Protocol, 1998. A growth curve for the 21st

century: the WHO multicentre growth reference study.

WHO, Geneva.

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ.-Health 210 (2007) 51–6060

ORIGINAL PAPER

Hair mercury in breast-fed infants exposed

to thimerosal-preserved vaccines

Rejane C. Marques & José G. Dórea &

Márlon F. Fonseca & Wanderley R. Bastos & Olaf Malm

Received: 10 July 2006 / Revised: 9 October 2006 / Accepted: 28 October 2006 / Published online: 20 January 2007

Springer-Verlag 2007

Abstract Because of uncertainties associated with a possi-

ble rise in neuro-developmental deficits among vaccinated

children, thimeros al-preserved vaccines have not been used

since 2004 in the USA (with the exception of thimerosal-

containing influenza vaccines which are routinely recom-

mended for administration to pregnant women and children),

and the EU but are widely produced and used in other

countries. We investigated the impact of thimerosal on the

total Hg in hair of 82 breast-fed infants during the first

6 months of life. The infants received three doses of the

hepatitis-B vaccine (at birth, 1 a nd 6 mont hs) and three

DTP (diphtheria, tetanus, and pertussis) doses at 2, 4 and

6 months, according to the immunization schedule recom-

mended by the Ministry of Health of Brazil. The thimerosal

in vaccines provided an ethylmercury (EtHg) exposure of

25 μgHg at birth, 30, 60 and 120 days, and 50 μgHg at

180 days. The exposure to vaccine-EtHg represents 80% of

that expected from total breast milk-Hg in the first month

but only 40% of the expected exposure integrated in the

6 months of breastfeeding. However, the Hg exposure

corrected for body weight at the day of immunization was

much higher from thimerosal- EtHg (5.7 to 11.3 μgHg/kg

b.w.) than from breastfeedi ng (0.266 μgHg/kg b.w.). While

mothers showed a relative decreas e (−57%) in total hair-Hg

during the 6 months lactation there was substantial increase

in the infant’s hair-Hg (446%). We speculate that dose and

parenteral mode of thimerosal-EtHg exposure modulated

the relative increase in hair-Hg of breast-fed infan ts at

6 months of age.

Keywords Thiomersal

Ethyl-mercury

Methyl-mercury

Breastfeeding

Immunization

Abbreviations

EtHg ethyl-mercury

MeHg methyl-mercury

DTP difteria tetanus and pertussis

PUFA polyunsatured fatty a cids

DHA docosahexaenoic acid

EPA eicosopentanoic acid

GSH glutathione

Introduction

Great strides in the control of epidemics have been made

possible by the effective production and safe use of vaccines.

The elimination of polio, measles and other scourges are the

hallmark of successful immunization programs. However, in

the earlier days of vaccine production there were several

tragic incidents that caused death in vaccinated children [1].

To reduce the risk of bacterial contamination, many

countries require a preservative in vaccines. The antiseptic

agent of choice to prevent bacterial growth is sodium

ethylmercurithiosalicylate (thiomersal), also known as thi-

merosal (USA). Thimerosal contains 49.6% Hg by weight

and has been used since the 1930s at the preservative level

Eur J Pediatr (2007) 166:935–941

DOI 10.1007/s00431-006-0362-2

R. C. Marques

W. R. Bastos

Fundação Universidade Federal de Rondônia,

Porto Velho, RO, Brazil

R. C. Marques

M. F. Fonseca

O. Malm

Instituto de Biofísica Carlos Chagas Filho,

Universidade Federal do Rio de Janeiro,

Rio de Janeiro, RJ, Brazil

J. G. Dórea (*)

Universidade de Brasília,

C.P. 04322 Brasília, DF, Brazil

e-mail: [email protected]

in vaccines. Some vaccines contain thimerosal at a concen-

tration of 0.01%; thus a vaccine dose of 0.5 ml contains

50 μg of thimerosal or approximately 25 μgofHg[1].

The amount of EtHg received from vaccinations by

infants up to 6 months old is an additional exposure to Hg

frequently found in human milk and formulas [10 ].

However, because the vaccine-Hg dose (extrinsic EtHg),

unlike intrinsic milk-Hg, is not part of a food, it bypasses

the barrier and detoxifying entero-hepatic system, and not

being physiologically processed, it may instead migrate to

the brain . The uncertainties about the safety of thimerosal-

preserved vaccines and controversy about whether EtHg

might be associated with neurodevelopmental disorders

(including autistic spectrum disorder) are debatable issues

that have involved both scientists and society [32]. Because

Hg toxic effects are manifested long after the exposure, it is

necessary to have reliable markers of Hg exposure.

However, depending on the elapsed time, one of the

difficulties in studying neurotoxic effect of vaccine-thimer-

osal is that post-exposure to vaccine-EtHg may not leave

Hg traces to be measured.

The organic Hg form of thimerosal is ethyl-Hg which, like

methyl-Hg (MeHg), can also bind to protein matrices. In vitro

studies showed that the neuronal and glial cell toxicity of

MeHg and EtHg are both mediated by glutathione depletion

[15]. Although chemically and toxicologically related, ethyl-

Hg and methyl-Hg have differences in metabolism. Com-

pared to EtHg, MeHg binds more avidly to cysteine-groups

and is more slowly metabolized. Thus, thimerosal-derived

EtHg half-life in the blood of infant monkeys was 2.1 and

8.6 days after exposure (i.m. injection), significantly shorter

than the 21.5 days elimination half-life of MeHg exposure

by gavage [6]. Additionally, it was observed that the

concentration of inorganic mercury in the brains of

thimerosal-EtHg exposed monkeys was more than double

that found in the MeHg exposed monkeys, and it was

observed that the inorganic mercury in the brains of the

thimerosal-EtHg exposed monkeys showed no significant

decline 120 days after exposure. Pichichero et al. [26]

estimated that half-life of EtHg in human infants is 6 days

compared with 40 to 50 days for MeHg. Furthermore,

concentrations of Hg were low in urine after vaccination,

but were high in stools of thimerosal-exposed infants.

Conversion to inorganic Hg is expected to yield high

urine-Hg; thus, stool Hg elimination is a feature also found

in MeHg metabolism. In such a scenario, the neurotoxic

effects of thimerosal-EtHg evaluated by post-exposure

markers such as hair-Hg can be compromised.

Stajich et al. [31] studied thimerosal-EtHg metabolism in

preterm human infants by measuring total Hg levels in

blood before and after the hepatitis-B vaccination. A

significant increase in Hg was seen in both preterm and

term infants. Additionally, preterm infants had greater than

tenfold higher mean Hg levels at the baseline reading

compared with term infants. Although this difference was

not statistically significant, Stajich et al. [31] speculated

that it might indicate that preterm infants may not be able to

metabolize Hg efficiently. Indeed we have shown that

larger babies, as measured by liver weight, had signifi cantly

higher mean liver Cu concentrations, an indicator of

metallothionein [16], than smaller babies [9]. Either

immaturity of the liver could be responsible for less

metallothionein to bind inorganic Hg or a difference in

body composition of infants with less body mass could

metabolize EtHg at a slower rate [31].

There are no studies that measured hair-Hg in infants at the

time of vaccination, but Redwood et al. [29] developed models

that predicted increases in hair-Hg concentrations after a full

immunization schedule within the first 6 months of life. They

assumed that neonates up to 6 months of age have low Hg

excretion due to immature hepatic function, low bile

production, and insufficient glutathione which binds the Hg.

Substances that accumulate in the body should be looked

at specifically. In the case of Hg, not only the chemical

form but also the exposure route , as well as the modifying

effect of changing metabolism (body weight with accom-

panying blood volume and organ maturity) are critical for

newborns. Therefore, we compared the exposure to injected

EtHg in vaccines with the expected exposure to breast-milk

Hg and examined the hair-Hg of breastfeeding mother-

infant pairs after the recommended immunization schedule

of the first 6 months.

Materials and methods

The research protocol to study the effects of Hg contam-

ination of urban (Porto Velho, Brazil) mothers on neuro-

development of pre-school children was approved by the

Ethics Committee of Studies for Humans of the Universi-

dade Federal de Rondonia and details appeared elsewhere

[21]. In this study we used data from our previous study

which was not collected with the purpose of evaluating the

impact of immunization on hair-Hg, since when the

research project started we were not aware of the EtHg

issue in pediatric vaccines. Briefly, pregnant mothers were

introduced to the study and invited to participate during

their routine visits to the pre-natal clinics of three hospitals

in Porto Velho (Hospital de Base, Hospital Panamericano

and Hospital Regina Pacis). Only Hospital de Base is a

state-run facility that receives mostly poor mothers.

Plain-language information about the study was pre-

sented and a written consent form signed by the volunteer-

ing mother. The written consent stated that participation

was voluntary with assured confidentiality and the right to

withdraw from the study at any time. Potential participants

936 Eur J Pediatr (2007) 166:935–941

were selected among mothers in good health, reporting no

illness or complaints at the time of the study and who were

willing to breast feed and adhere to the post-natal

attendance of the pediatric clinic for the regular immuniza-

tion program. One hundred mothers between the ages of 15

and 45 years were recruited.

At birth the infants were clinically examined with special

attention to vitality, perinatal reflexes, maturity, and

congenital malformations; weight, length, head circumfer-

ence, and Apgar scores were recorded. Ant hropometric data

(weight, length, and head circumference) were recorded and

samples of hair from the mothers and infants (fetal hair)

were collected. Mothers followed the immunization sched-

ule recommended by the Ministry of Health of Brazil and

returned at 30, 60, 120 and 180 days. Only 86 mother-

infan t pairs reported for the programme d clinical and

immunization at 6 months of age when infants were

weighed and measured for length, and hair-samples were

again collected from mothers and infants. Because Hospital

de Base is a publi c health facility, only the babies born at

this hospital (66%) received the hepatitis-B vaccine within

the first day postpartum. Babies born at the Hospital

Panamericano and the Hospital Regina Pacis received the

hepatitis-B vac cine immediately after the mother’s dis-

charge (2–4 days postpartum). At this time the mothers

were taken under our supervision to a state-run clinic where

vaccines are distributed free.

Estimated exposure to Hg from vaccines (injected

thimerosal) and breastfeeding

Differences in infants’ weight at birth and at 6 months were

used to estimate daily weight gain and integrated gain at 30,

60, 120 and 180 days. As stated by manufacturers, vaccines

contained 0.01% Thimerosal; the Hg concentration of the

doses delivered through vaccines was 25 μgHg/0.5 mL for

hepatitis-B (Korea Green Cross Corporation, Kiheung-Eup

Yougin-Goon Kiyunggi-Do, Korea; Euvax B injectable, LG

Life Sciences, Jeonbuk-Do, Korea) and difteria, tuberculo-

sis and pertussis-DTP (Triple Antigen, Serum Institute of

India Ltd., India; Vacina Tríplice, Instituto Butanta, São

Paulo, Brazil).

We used the data of breast milk-Hg concentratio ns

(adapted from Dorea [8]) with the same approach as

Bigham and Copes [4] to estimate Hg exposure during

breastfeeding: infant mean weight × mean daily breast milk

consumption (140 mL/kg) × number of days × mean total

Hg concentrati on in breast milk (1.9 μg/L).

Hair Hg determinations

Hair sample preparation and Hg determination were done

according to routine procedures previ ously established at

the Universidade Federal do Rio de Janeiro [ 3]. We

followed routi ne laboratory procedures after adapting the

analytical protocol used for Hg determination in previous

studies analyzing blood, hair and fish-flesh matrices [3, 19,

20]. Briefly, the hair samples were comminuted with

stainless steel scissor s, weighed an d digested befo re

analysis. Blood samples were digested with concentrated

HNO

(3 mL) and KMnO

(5%, 6 mL) using a microwave

oven system for 35 min (CEM-Corporation, MDS 2000,

Matthews, North Carolina, USA). Placenta and umbilical

cord samples were weighed and digested with HNO

(1:1, 5 mL) and KMnO

(5%, 4 mL) using a

digestion block at 80°C for 1 h (Tecnal Ltd., Piracicaba,

São Paulo, Brazil). Human hair samples were washed with

EDTA 0.01%, dried in an oven at 50°C, weighed and

digested with 5 mL of HNO

:H2SO

(1:1) and 4 mL of 5%

KMnO

using a digestion block at 80°C for 40 min. The

determination of total Hg in the digested samples was done

by cold vapor atomic absorption spectrome try with a flow

injection system-FIMS (CV-AAS, Perkin-Elmer-FIMS 400,

Ueberlingen, Germany). Glassware used in the analytical

protocol was washed clean, rinsed with 5% EDTA and

double distilled, and left to rest in 5% HNO

overnight.

Then it was rinsed again in double distilled water, and dried

at 100°C for 12 h. Precision and ac curacy of Hg

determinations wer e assured by the use of internal stan-

dards, use of triplicate analyses of samples and certified

reference material s (IAEA-085 and 086, Vienna-Austria)

with recoveries of 92%.

Statistical analysis and data summarization (mean, SD)

were performed using SAS software (SAS Institute, Cary,

NC, USA). For the statistical test, p<0.05 was considered

significant.

Results

The anthropometry a nd the hair-Hg concentrations of

infants and mothers are shown in Table 1, while the

immunization schedule and exposure to the injected

thimerosal as well as the expected exposure to total Hg in

breast milk are shown in Table 2.

The mean birth weight was slightly higher for girls, but

there was a relatively higher weight gain for boys at

6 months. Every child received a cumulative dose of

150 μg/Hg (as Thimerosal-EtHg) distributed in five boluses

during the first 6 months of life. An adaptation of world

data on human milk-Hg concentrations [8] showed that the

median mean Hg concentration is 1.9 ngHg/mL (Fig. 1).

The estimated total Hg intake integrated over the first

180 days of breastfeeding showed a cumulati ve exposure of

290 μg of total-Hg (Fig. 2). Comparatively, the integrated

EtHg exposure in the first month was 80% of that from

Eur J Pediatr (2007) 166:935–941 937

breast milk but declined to 40% at 180 days (Fig. 2). At

two months the infants increased body weight by 60% but

received an integrated EtHg challenge threefold higher than

in the perinatal days (Table 2).

Per unit of body weight the amount of EtHg/kg b.w.

given to newborn babies (before hospital discharge) was

higher than at any other time (Fig. 3), even at 6 months

when infants had increased body weight by more than

100%. The corrected exposure to thimerosal (EtHg) and

breast-milk Hg per unit of body weight is shown in Fig. 3.

Considering a milk volume transfer of 140 mL/kg b.w., and

considering the median breast-milk Hg concentration of

1.9 ngHg/mL (Fig. 1), the exposure to breast-milk Hg was

0.266 μgHg/kg b.w. at any time after the first immunization

at 0 days. Excluding the small volumes of milk intake at

birth (day 0 o f first vaccine), this corresponds to a small

percentage (4.7 to 8.1%) o f the EtHg doses injected

between 30 and 180 days.

Because of the variability in hair-Hg concentrations, the

relative changes from 0 days (birth) to 180 days were

calculated and shown in Fig. 4. While mothers had a mean

decrease of 40% and 45%, their girls and boys had,

respectively, increased hair-Hg concentrations by as much

as 398.3% and 487.1%.

Another notable feature is the time of exposure to EtHg

shown in Fig. 5. A distinct pattern is shown as a function of

the immunization policy regarding the dispensation of

vaccines. The infants that received the first vaccine within

the first 24 hours after birth were all from the only public

hospital in Porto Velho (Hospital de Base). Because the

other two hospitals (Panamericano and Regina Pacis) are

privately owned and operated, the infants are vaccinated in

Table 2 Infant immunization schedule, type of vaccine, and Hg

intake during the first 6 months

Age,

days

Vaccine Body

weight, g

Breast

milk

Type

μgHg/

dose

Hg intake,

μg

0 Hp-B 25.0 3,233.17 0

30 Hp-B 25.0 3,862.87 30.83

60 DTP 25.0 4,492.56 44.65

90 – 5,067

120 DTP 25.0 5,751.95 91.80

150 – 6,327

180 DTP+ Hp-B 50.0 7,011.34 111.90

Total

150.0 279.18

Hp-B: Hepatitis B (assumed 0.01%Thimerosal/dose, Korea Green

Cross Corporation, Kiheung-Eup Yougin-Goon Kiyunggi-Do, Korea;

Euvax B injectable, 0.01%Thimerosal, LG Life Sciences, Jeonbuk-

do, Korea); DTP (Serum Institute of India Ltd; Vacina Tríplice,

0.01%Thimerosal/dose; Instituto Butanta, São Paulo, Brazil)

Integrated total Hg intake (estimated from Dorea [8])

Total exposure of the respective Hg chemical forms

Table 1 Mean (and SD) of anthropometry, hair-Hg concentrations

and Gesell scores of infants

Girls Boys

N 38 44

Birth weight, g 3,177.50 (450.8) 3,281.25 (393.4)

Weight at 180 d 7,012.63 (508.7) 7,010.23 (417.3)

Weight gain, g 3,835.13 (434.2) 3,728.98 (382.8)

Weight gain/d 21.31 (2.4) 20.72 (2.1)

Hair [Hg], μg/g

Birth 2.58 (3.7) 2.32 (2.4)

180 days 4.14 (5.9) 3.9 (5.3)

Mean increase at 6 m 1.55 (5.8) 1.27 (5.3)

Relative increase, % 398.28 (541.9) 487.08 (1,515.0)

Maternal hair [Hg] μg/g

Delivery, 0 days 7.14 (6.8) 7.55 (10.2)

180 days 2.97 (3.1) 3.38 (4.4)

Mean decrease at 6 m 4.17 (4.0) 4.16 (6.2)

Relative decrease, % 39.94 (16.2) 45.50 (23.1)

Fig. 1 Distribution of mean total mercury concentrations in studies of

different parts of the world (adapted from Dorea [8])

100

300

500

0 180

0 180 0 180

s of lactation

Girls' mothers

Girls Boys' mothers Boys

Relative change, %

Fig. 2 Estimation of the integrated total-Hg intake during the first

180 days of breastfeeding

938 Eur J Pediatr (2007) 166:935–941

immunization clinics run by the Ministry of Health after the

hospital discharge.

Discussion

Unlike breast-milk intake, there are no body-weight dosing

adjustments for vaccines; therefore, neonates and young

infants (up to 60 days) with less blood volume are exposed

to more injected EtHg than older infants from 120 to

180 days (Table 2). Over the first 6 mont hs, the estimated

accrual of total Hg exposure from breast milk was higher

than the five doses of injected EtHg (Table 2). However,

differing from parenteral EtHg in vaccines, breast-fed

infants were exposed to enteral Hg (intrinsic to the breast

milk matrix) amortized over 6 months. Because the mean

frequency of feeds/day is 5.5 [30], the milk-Hg exposure in

the breastfed infant occurs in several small daily suckling

episodes. This type of exposure amounts to only 4.7%–

8.1% of the vaccine-Hg bolus (per kg/b.w.) taken on the

day of the immunization. Furthermore, the ingested milk-

Hg is hindered by all gastrointestinal barriers, metabolic

and detoxifying mechanisms (albeit immat ure) attendant to

enteral feeding. Coupled with that, another attenuating

factor is the tendency of human milk Hg to decrease during

initial lactation [8], i.e., from day four to 6 weeks after

delivery [5]. Additionally, central to the toxic effects of Hg

during early life is the unique chemical composition of

human milk with its specific neurotoxic attenuating factors.

Neurodevelopment is rapid in the postnatal period and

substantial amounts of long-chain polyunsatured fatty acids

(PUFAs) are critical to neurite growth and proper brain

development. The benefi cial PUFAs are docosahexaenoic

acid (DHA=22:6n-3) and e icosopentanoic a cid (EPA=

20:5n-3). DHA is an essential component of nervous-

system cell-membranes and is transported across the

placenta and across the mammary gland into milk. During

the critical window of early life, significant amounts of

PUFAs are provided via breast milk and then avidly

incorporated into membranes throughout the infant’s

nervous system [22]. Indeed, post-natal DHA is positively

correlated with visual and language development in breast-

fed infants [14].

Another mitigating factor against Hg-induced neurotox-

icity is the intracellular defense provided by glutathione

(GSH). Mercury has a high affinity for thiol (sulfhydryl

groups), the thiol-c ontaining antioxidant of GSH [15].

According to Cai and Sauve [7], thimerosal is a poorly

membrane-permeable hydrophilic-molecule that oxidizes

SH groups with a high affinity. It reduces S–S bonds of

molecules such as GSH. Therefore, the po tential protective

effect of GSH against Hg toxicity can be compromised in

situations limiting cysteine nutrition. Indeed, James et al.

[15] showed that thimerosal-induced cytotoxicity was

associated with depletion of intracellular GSH. Because

brain cells cannot synthesize cysteine, (the rate limiting

amino acid for GSH synthe sis) it is depend ent on the liver

to synthesize and export cysteine to the brain for intracel-

lular glutathione synthesis [15]. Human milk contains a

high am ount of bioavailable cysteine and differences

between breast-fed and formula-fed infants have been

reported with respect to cysteine concentrations and its

metabolites [23].

The safety of thimerosal vaccines is assumed on the

basis of the faster metabolism of EtHg compared to MeHg

[6]. While we do not know if the infants’ hair-Hg in this

study was derived from the vaccine’s thimerosal, it can be

stated that there was an asymmetric change in infants’ hair-

Hg compared to the mothers’ hair in the same period. We

DTwP

180

60 120

Hepatitis B

s of lactation

DTwP +

150

Injected ethylmercury,

g/kg body weight

Estimated breast-milk Hg

exposure,

g/kg body

weight

Hepatitis B

Fig. 3 Infants’ vaccine schedule and injected ethyl-Hg (as Hg), and

estimated total Hg in breast milk (– adapted from Dorea [8]) during

the first 180 days of life

Fig. 4 Relative hair-Hg

changes (%) from birth (0 days)

to 180 days in mothers

according to infant gender

Eur J Pediatr (2007) 166:935–941 939

have reported that fish-eating riverine mothers showed

higher hair-Hg concentrations than their respective breast-

fed infants [2]. In line with these observations it has been

shown that maternal hair-Hg concentration was higher than

fetal hair-Hg [28]. Recently, Lindow et al. [17] reported

higher fetal hair-Hg concentrations in babies of mothers

exposed to dental amalgam restoration either before or

during pregnancy; they showed maternal/fetal Hg ratios>1

in most samples (66%). However, contrary to these studies,

Mohan et al. [24] reported that 80% of newborns had

higher hair-Hg than their mothers. Nevertheless, the mean

increase in hair-Hg concentrations we found at 6 months is

compatible with the levels predicted by Redwood et al.

[29].

Holmes et al. [12] describe that mothers from autistic

children received more mercury during pregnancy than

mothers from healthy controls and also hair-Hg differences

between autistic and control infants at the time of their first

haircut (11–24 months). They speculated that hair excretion

patterns among autistic infants were significantly reduced

relative to controls. Furthermore, they claimed that hair-Hg

in control infants were significantly correlated to Hg

exposure through prenatal maternal dental amalgam

fillings, but that correlations were absent in the autistic

group. Contrary to that, older (4–7 years old) autistic

children showed higher hair-Hg concentrations than age-

matched controls [11]. Hair-Hg association with behavioral

disorders has also been extended to interactions with zinc

deficien cy [13]. However, the assessment of hair-Hg

concentrations after immunizations containing thimerosal

(EtHg) is incomplete without speciation of the Hg forms.

Nevertheless, our total hair-Hg results are in line with

previous work. Barbosa and Dorea [2] estimated that mean

concentration of Hg in breast milk of “riverine” women

(living near rivers, high fish eaters) with higher hair-Hg

(14.3 ppm) would give an exposure to breast-fed infants

(mean hair Hg, 9.8 ppm) of 0.64 μg/kg body weight. In the

present study the urban mothers are low fish eaters and

show much lower mean hair-Hg concentration (7.5 ppm).

The neurotoxicity of administered MeHg is well known

and seems to be higher than EtHg, the Hg metabolite of

thimerosal [18]. There is limited information on human

metabolism of EtHg and even less information is available

for infant metabolism of thimerosal. Because there are no

known studies of intrinsically incorporated EtHg in food

matrices, comparing parenteral vaccine-EtHg to the breast

milk-Hg (intrinsic-Hg) metabolism is challenging. The

decomposition rate of EtHg is higher than MeHg because

of the molecule size. Qvarnstrom et al. [27] reported that

the carbon-Hg bond in C

is less stable than that in

. In addition, the efficiency of the blood-brain

barrier is directly proportional to the size of the organic

radical [18].

Infant Hg exposure in early life can only happen through

milk diets [10]. However, given the variation (24 to 67 h

postpartum) of the onset of lactation [25], for many infants

a high exposure to parenteral EtHg (25 μg) preceded the

much lower (Fig. 2) enteral total-Hg (intrinsic to the human

milk matrix). A second point is the impact caused by

injected EtHg in an immature organism. It has been

speculated that the removal of thimeros al from vaccines

would produce no more than a 50% reduction of Hg

exposure in infancy [4]. This reduction could only happen

with human milk Hg concentrations greater than the median

value (Fig. 1) and more frequently with formulas, which

usually contain higher Hg concentrations [10]. Further-

more, this scenario assumes a debatable equivalency of a

bolus (injected Hg) versus the integration (over 6 months)

of inges ted milk-Hg; it does not contemplate the tenet s of

Hg neurotoxicity: chemical form, dose, route of adminis-

tration, attenuating neurotoxic factors of breast feeding, and

enhancing neurotoxic factors of the perinatal period (birth

weight/prematurity).

The expected metabolic consequences of such extremely

different exposure routes and Hg chemical forms (injected

EtHg versus ingested milk-Hg) as well as Hg dose are

difficult to disentangle; we have the changing physiological

interactions of a self-adjusted daily dose of intrinsic Hg

(inorganic and MeHg) in breastfeeding against the full

impact of an extrinsic dose of EtHg. With regard to

breastfeeding, milk-Hg occurs in a context of proven

benefits in a broad array of neurotoxic mitigating factors.

In this study, we observed a relative increase in hair-Hg.

This finding reinforces a mechanistic connection between

thimerosal-EtHg and hair-Hg increase, supporting the use

of hair-Hg in vaccinated children as a confounder of

neurobehavioral maternal-Hg contamina tion.

Fig. 5 Percent distribution of the time-after-birth interval of the first

vaccine (hepatitis B)

940 Eur J Pediatr (2007) 166:935–941

Acknowledgements We are greatly in debt to the mothers for their

participation in the study, to the staff and directors (Marinês R. dos

Santos Cezar, Tereza Cristina Ramos, Daniele Brasil, Katia Wendt,

Katiane G. Brandão, Laura Jane Marques) of the Hospitals (Hospital

de Base Ary Pinheiro, Hospital Panamericano and Hospital Regina

Pacis), Dr. Cezar Augusto Bezerra B. de Araújo (State Coordinator of

the PNI-MS), the staff of the Fundação Universidade Federal de

Rondônia and the Universidade Federal do Rio de Janeiro.

This work was supported by United Nations Educational, Scientific

and Cultural Organization - UNESCO, Ministério da Saúde do Brasil

(SC27824/2005/914BRA 2000 Decit PRODOC ) and The National

Research Council of Brazil-CNPq (PNOPG project-55.0882/01-4

PPG7, project-556985/2005-2).

References

1. Amanna I, Slifka MK (2005) Public fear of vaccination:

separating fact from fiction. Viral Immunol 18:307–315

2. Barbosa AC, Dórea JG (1998) Indices of mercury contamination

during breast feeding in the Amazon Basin. Environ Toxicol

Pharmacol 6:71–79

3. Bastos WR, Malm O, Pfeiffer WC, Cleary D (1998) Establish-

ment and a nalytical quality control of laboratories for Hg

determination in biological and geological s ample s in th e

Amazon, Brasil. Ciênc Cult 50:255–260

4. Bigham M, Copes R (2005) Thiomersal in vaccines: balancing the

risk of adverse effects with the risk of vaccine-preventable

disease. Drug Saf 28:89–101

5. Bjornberg KA, Vahter M, Berglund B, Niklasson B, Blennow M,

Sandborgh-Englund G (2005) Transport of methylmercury and

inorganic mercury to the fetus and breast-fed infant. Environ

Health Perspect 113:1381–1385

6. Burbacher TM, Shen DD, Liberato N, Grant KS, Cernichiari E,

Clarkson T (2005) Comparison of blood and brain mercury levels

in infant monkeys exposed to methylmercury or vaccines

containing thimerosal. Environ Health Perspect 113:1015–1021

7. Cai S, Sauve R (1997) Effects of thiol-modifying agents on a

KCa

ÀÁ

channel of intermediate conductance in bovine aortic

endothelial cells. J Membr Biol 158:147–158

8. Dorea JG (2004) Mercury and lead during breast-feeding. Br J

Nutr 92:21–40

9. Dorea JG, Brito M, Araujo MO (1987) Concentration of copper

and zinc in liver of fetuses and infants. J Am Coll Nutr 6:491–495

10. Dorea JG, Donangelo CM (2006) Early (in uterus and infant)

exposure to mercury and lead. Clin Nutr 25:369–376

11. Fido A, Al-Saad S (2005) Toxic trace elements in the hair of

children with autism. Autism 3:290–298

12. Holmes AS, Blaxill MF, Haley BE (2003) Reduced levels of

mercury in first baby haircuts of autistic children. Int J Toxicol

22:277–285

13. Hu LW, Bernard J, Che J (2003) Neutron activation analysis of

hair samples for the identification of autism. Trans Am Nucl Soc

89:681–682

14. Innis SM, Gilley J, Werker J (2001) Are human milk long-chain

polyunsaturated fatty acids related to visual and neural develop-

ment in breast-fed term infants? J Pediatr 139:532 –538

15. James SJ, Slikker W 3rd, Melnyk S, New E, Pogribna M, Jernigan

S (2005) Thimerosal neurotoxicity is associated with glutathione

depletion: protection with glutathione precursors. Neurotoxicology

26:1–8

16. Klein D, Scholz P, Drasch GA, Muller-Hocker J, Summer KH

(1991) Metallothionein, copper and zinc in fetal and neonatal

human liver: changes during development. Toxicol Lett 56:61–67

17. Lindow SW, Knight R, Batty J, Haswell SJ (2003) Maternal and

neonatal hair mercury concentrations: the effect of dental

amalgam. BJOG 110:287–291

18. Magos L (2003) Neurotoxic character of thimerosal and the

allometric extrapolation of adult clearance half-time to infants. J

App Toxicol 23:263–269

19. Malm O, Branches FJP, Akagi H, Castro MB, Pfeiffer WC,

Harada M, Bastos WR, Kato H (1995) Mercury and methylmer-

cury in fish and human hair from the Tapajós River Basin, Brazil.

Science Total Environ 175:141–150

20. Malm O, Pfeiffer WR, Souza CMM (1989) Utilização do

acessório de geração de vapor frio para análise de mercúrio em

investig ações ambientais por espectrofotometria de absorção

atômica. Ciênc Cult 41:88–92

21. Marques RC, Dórea JG, Bastos WR, Rebelo MF, Fonseca MF, Malm

O (2006) Maternal mercury exposure and neuro-motor development

in breastfed infants from Porto Velho (Amazon), Brazil. Int J Hyg

Env Health (available online DOI 10.1016/j.ijheh.2006.08.001)

22. Marszalek JR, Lodish HF (2005) Docosahexaenoic acid, fatty

acid-interacting proteins, and neuronal function: breastmilk and

fish are good for you. Annu Rev Cell Dev Biol 21:633–657

23. Minet JC, Bisse E, Aebischer CP, Beil A, Wieland H, Lutschg J

(2000) Assessment of vitamin B-12, folate, and vitamin B-6 status

and relation to sulfur amino acid metabolism in neonates. Am J

Clin Nutr 72:751–757

24. Tiller M, van der Voet G, Kanhai H (2005) Mercury exposure of

mothers and newborns in Surinam: a pilot study. Clin Toxicol

43:101–104

25. Perez-Escamilla R, Chapman DJ (2001) Validity and public health

implications of maternal perception of the onset of lactation: an

international analytical overview. J Nutr 131:3021S–3024S

26. Pichichero ME, Cernichiari E, Lopreiato J, Treanor J (2002)

Mercu ry concentrations and metabolism in infants receiving

vaccines containing thiomersal: a descriptive study. Lancet

360:1737

–1741

27. Qvarnstrom J, Lambertsson L, Havarinasab S, Hultman P, Frech

W (2003) Determination of methylmercury, ethylmercury, and

inorganic mercury in mouse tissues, following administration of

thimerosal, by species-specific isotope dilution GC-inductively

coupled plasma-MS. Anal Chem 75:4120–4124

28. Razagui IB, Haswell SJ (2001) Mercury and selenium concen-

trations in maternal and neonatal scalp hair: relationship to

amalgam-based dental treatment received during pregnancy. Biol

Trace Elem Res 81:1–19

29. Redwood L, Bernard S, Brown D (2001) Predicted mercury

concentrations in hair from infant immunizations: cause for

concern. Neurotoxicology 22:691–697

30. Sievers E, Oldigs HD, Santer R, Schaub J (2002) Feeding patterns

in breast-fed and formula-fed infants. Ann Nutr Metab 46:243–248

31. Stajich GV, Lopez GP, Harry SW, Sexson WR (2000) Iatrogenic

exposure to mercury after hepatitis B vaccination in preterm

infants. J Pediatr 136:679–681

32. Zimmerman RK, Wolfe RM, Fox DE, Fox JR, Nowalk MP, Troy

JA, Sharp LK (2005) Vaccine criticism on the World Wide Web. J

Med Internet Res 7(2):e17

Eur J Pediatr (2007) 166:935–941 941

Acta Pædiatrica ISSN 0803-5253

REGULAR ARTICLE

Time of perinatal immunization, thimerosal exposure and

neurodevelopment at 6 months in breastfed infants

Rejane C Marques

1,2

, Jos´eGD´orea ([email protected])

, Angelo G Manzatto

, Wanderley R Bastos

, Jos´e VE Bernardi

, Olaf Malm

1.Funda¸c˜ao Universidade Federal de Rondˆonia, Porto Velho, RO, Brasil

2.Instituto de Biof´ısica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil

3.Universidade de Bras´ılia, Bras´ılia, DF, Brasil

Keywords

Breastfeeding, Ethylmercury, Neurodevelopment,

Thimerosal, Vaccines

Correspondence

Jos´eG.D´orea, C.P.04322, Universidade de Bras´ılia,

70919-970, Bras´ılia, DF, Brazil.

Email: [email protected]

Received

25 December 2006; revised 17 February 2007;

accepted 26 February 2007

DOI:10.1111/j.1651-2227.2007.00288.x

Abstract

Aim: Brazilian newborns immunized with hepatitis-B (thimerosal containing vaccine, TCV) receive the

ﬁrst dose within 24 h if delivered in public hospitals, but at a later time if born in private hospitals. We

compared neurodevelopment (ND) in infants born in a state hospital (immunized within 24 h) and

in privately run hospitals (immunized 2–4 days postnatally).

Methods: We used the Gesell Developmental Schedules in 82 healthy exclusively breastfed infants at

6 months to assess motor skills, language development, comprehension capacity and social skills.

Results: Compared to the group immunized 2–4 days after hospital discharge, the group immunized

within 24 h showed no signiﬁcant difference in ND delays. Despite the variation in gestational age

(range 36–42 weeks) and TCV-ethylmercury (EtHg) dose (5.7–11.3 ␮gHg/kg b.w.) at birth, time of

exposure to TCV showed no signiﬁcant association with ND. Gesell Developmental Score was not

signiﬁcantly correlated with total parenteral EtHg/unit of body mass neither with the relative increase

in hair-Hg (as an additional challenge to prenatal Hg exposure).

Conclusion: In breastfed infants, differences in early exposure to TCV-EtHg cannot portend clinical

neurodevelopment delays at 6 months. We speculate that breastfeeding remains a signiﬁcant strategy to

improve central nervous system protection of infants facing early exposure challenges.

INTRODUCTION

Despite the universal use of thimerosal-containing vac-

cines (TCV) in immunization of children, our knowledge

of the toxicokinetics (TK) and toxicodynamics (TD) of their

Hg metabolite (ethylmercury—EtHg) is derived mostly from

methylmercury (MeHg) studies. The mechanistic signifi-

cance of thimerosal neurotoxicity through EtHg is unques-

tionable, but its toxicological significance is the result of

direct demonstration in animal and theoretical models. Our

ability to understand the safety of TCV-EtHg is still unsat-

isfactory; although there is no proven direct association of

neurodevelopmental disorders in children immunized with

TCV-EtHg, its plausibility has been shown by neurotoxic dis-

asters caused by organic Hg compounds (1). However, there

are no studies pointing to neurologic disorders being an

unambiguous result of TCV-EtHg; rather, the associative

studies have so far emerged amid intense scientific debate.

Rodier (2) and many others have discussed the vulnera-

bility of the young brain to neurotoxic substances over the

early neonatal period. Compared to other organs, the infant

brain takes an unusually long period to form. Besides the

blood–brain barrier (BBB), which is not fully developed until

the middle of the first year of life, neuron proliferation and

migration also continue in the postnatal period. Addition-

ally, myelin production, along with development of receptors

and transmitter systems, is part of the great postnatal activ-

ity of the central nervous system (CNS). Organic Hg com-

pounds are among the neurotoxic substances with effects

that depend on the neuron structure at time of exposure (2).

Coincidently, during the first 6 months, Brazilian infants are

impacted by a heavy vaccination schedule: six i.m. injections

of TCV (Hepatitis-B at 0, 30 and 180 days; DTP at 60, 120

and 180 days) containing 0.01% of thimerosal as preserva-

tive (25 ␮gHg/dose).

Nursing infants are exposed to Hg from formulas and

breast milk (3); however, the extent of exposure during

breastfeeding depends on the maternal Hg that diffuses into

milk. The amount of Hg in the blood of the nursing mother

enters breast milk at a rate (inorganic Hg more than MeHg)

that depends on its protein-binding capacity. Oskarsson et al.

(4) observed that, compared to organic Hg (MeHg), there

was a more efficient transfer of inorganic Hg from blood to

breast milk. They estimated that infant exposure from breast

milk ranges up to 0.3 ␮gHg/kg/day; approximately 50% was

inorganic Hg. While the breast milk Hg exposure is propor-

tional to milk intake, it is amortized in several sucking events

during the day; furthermore, the newborn is not exposed un-

til lactation is established and during this time breastfeeding

is acting on the CNS priming. On the other hand, TCV are

given immediately after parturition by parenteral route. In

our infants, five vaccines are given in the first 6 months, two

of them given at the same time (180 days) and providing a

double dose of TCV (50 ␮gHg/dose). Indeed, breastfed vac-

cinated infants have shown a substantial increase in hair-Hg

concentrations during the first 6 months (5).

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Marques et al. Early vaccination, thimerosal and neurodevelopment

Guidelines to EtHg use as vaccine preservatives are de-

rived from MeHg toxicity studies. These guidelines from var-

ious countries vary from 0.1 to 0.4 ␮g/kg of MeHg for adults

(1). Harry et al. (6) found that in immature mice, when EtHg

was injected, either as the chloride or as thimerosal, less of

the actual delivered dose reached the brain as compared to

an injection of MeHg. They concluded that MeHg does not

appear to be a good model for EtHg-containing compounds.

Therefore, serum transport of MeHg and EtHg can modulate

toxic differences (in the neonate) by regulating the relative

levels of free forms transported and available to cross the

BBB. Additionally, there are differences in initial weight loss

that are more pronounced in breastfed infants. Macdonald

et al. (7) identified neonatal median time of maximum weight

loss around 2.7 days. Clearly, this can further increase dif-

ferences in TCV-EtHg exposures.

The inability of the neonatal liver to secrete bile, coupled

with an immature renal system, mean that Hg excretion

takes longer than in adults. This is particularly relevant for

the breastfed infant; because maternal milk usually comes in

at between 2–4 days after birth, for breastfed infants this is

the time of the lowest expected bowel output (8). Together

with lower albumin binding capacity, the immaturity of the

transport-protein system in neonates enhances the risk of

mercury toxicity. Perinatal immaturity of the CNS and of

other physiological functions (detoxifying enzymes, renal

and gastrointestinal organs) that interferes with Hg binding

and excretion is well established. Additionally, both birth

weight (9) and gestational age (10) are positively associated

with childhood psychometric intelligence. The TCV-EtHg

impact on CNS remains unstudied; gestational age and birth

weight are modulators of TK and TD of TCV. The parenteral

delivery of TCV-EtHg, albeit in very small quantities, reaches

the neonate circulation very quickly and gains unimpeded

access to the immature BBB and the blood cerebrospinal

fluid barrier (BCSFB). Therefore, the early postnatal days

are important windows of opportunity for CNS insults.

Neurodevelopment delay can occur in infants exposed to

prenatal maternal neurotoxic substances; its etiology is mul-

tifactorial and includes Hg. Given the immaturity of new-

borns, after an acute parenteral exposure of thimerosal, there

is more chance of infants accumulating Hg more readily and

excreting it more tardily. When there are differences in time

of first exposure there might be additional risk factors; there

is, therefore, more likelihood of heightening the risks of

untoward neurological effects. Special population groups,

such as term neonates, have a variety of constitutional mod-

ifying risk factors: (a) parenteral exposure at very early time;

(b) sequence of high magnitude EtHg exposures (first days,

1, 2, 4 and 6 months); (c) differences in maturity at birth (ges-

tational ages 36–42 weeks); (d) differences in body mass at

birth and (e) additivity of TCV-EtHg to prenatal Hg exposure

gradients (especially in fish-eating Amazonian populations).

Because of the complexity of studying neurodevelopment

and ethical issues involving study design with TCV we

took advantage of an existing research project and analysed

recorded data of early and relatively later TCV-EtHg expo-

sure of breastfed infants. Therefore, our primary aim was to

compare the effect of time differences in the first exposure to

TCV on ND at 6 months. Our secondary aim was to identify

modifying factors associated with prenatal Hg exposure.

MATERIALS AND METHODS

The data used in this study were not collected with the pur-

pose of evaluating the impact of immunization on ND. In a

parent publication (11) we described the protocol to assess

growth and development of children exposed to prenatal

background Hg; when the research project started we were

not aware of the TCV issue in paediatric vaccines. The par-

ent publication (11) dealt with prenatal Hg exposure; after

that, when studying the changes in hair Hg concentrations

(5, 11) we realized that because Hospital de Base is a public

health facility, only the babies born at this Hospital (66%)

received the hepatitis-B vaccine within the first day post-

partum. Babies born at the Hospital Panamericano and the

Hospital Regina Pacis received the hepatitis-B vaccine im-

mediately after the mother’s discharge (2–4 days postpar-

tum). This appeared as an opportunity to study differences

in timing of immunization and ND outcomes; additionally,

Hospital de Base as a state-run facility receives mostly poor

mothers.

The previous publications detailed the characteristics of

the population of Porto Velho (West Amazonia); the re-

search protocol was approved by the Ethics Committee

of Studies for Humans of the Universidade Federal de

Rondonia (11). Briefly, pregnant mothers between the ages

of 15 and 45 years were enrolled as volunteers; mothers

were in good health, reporting no illness or complaints and

were willing to breastfeed. They were introduced to plain-

language information about the study, and a written consent

form was presented and signed by the volunteering mother.

Mothers were selected by a nurse during their routine visits

to the Prenatal Clinics (Hospital de Base, Hospital Panamer-

icano and Hospital Regina Pacis). Hospital de Base is a

state run facility while Hospital Panamericano and Hospital

Regina Pacis are not. Excluding factors were occupational

exposure to toxic chemicals and hereditary neurological

illnesses.

After birth, the newborns were clinically examined with

special attention to vitality, perinatal reflexes, maturity and

congenital malformations; weight, length, head circumfer-

ence and Apgar scores were recorded. While in the mater-

nity wards, we collected samples of hair from mothers and

respective infants (fetal hair); the hair sampling was repeated

at 6 months. Hair strands were cut from the occipital region

and placed in plastic bags, with the root end stapled on a

paper sheet. These samples were analysed by routine labo-

ratory procedures described in previous publications (5,11).

The mothers were closely monitored by nurses to guaran-

tee full support for breastfeeding and pre and postnatal care;

immunization scheme followed the Brazilian vaccination

program. The first vaccine (Hepatitis B) was taken either

before hospital discharge (in Hospital de Base) or a few

days latter, and at 30, 60, 120 and 160 days. After hospi-

tal discharge the mothers were taken under our supervision

to a state-run clinic where vaccines are distributed free.

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Early vaccination, thimerosal and neurodevelopment Marques et al.

At 6 months of age, only 82 of the 100 original mother–

infant pairs reported for the programmed clinical and neu-

robehavioural examination when infants were weighed and

measured for length, and hair samples were collected again

(5,11).

The neurodevelopment tests were conducted by trained

professionals using the Gesell Developmental Schedules

(12,13). These tests included all reactions (voluntary, spon-

taneous or learned) and reflexes. We also evaluated postural

reactions, hand pressure, locomotion and coordination,

constructive ability (which is influenced by motor develop-

ment), visible and audible communication, individual reac-

tions regarding people and stimulations (depending mainly

on the temperament of the child and the surroundings). The

results were expressed as developmental scores for the Adap-

tive Ability, Language and Motor deficiencies (gross motor

ability), and Social/Emotional ability.

Estimated exposure to EtHg from vaccines (injected

thimerosal):

Integrated weight gains at 30, 60, 120 and 180 days were

estimated from differences in infants’ weight at birth and

at 6 months. Differences in infants’ weight at birth and at

6 months were used to estimate daily weight gain and in-

tegrated gain at 30, 60, 120 and 180 days. As stated by

manufacturers, vaccines contained 0.01% thimerosal; the Hg

concentration of the doses delivered through vaccines was

25 ␮gHg/0.5 mL for hepatitis-B (Korea Green Cross Cor-

poration, Kiheung-Eup Yougin-Goon Kiyunggi-Do, Korea;

Euvax B injectable, LG Life Sciences, Jeonbuk-Do, Korea)

and diphtheria, tetanus and pertussis-DTP (Triple Antigen,

Serum Institute of India Ltd., India; Vacina Tr

ıplice, Instituto

Butanta, S

ao Paulo, Brazil). The exposure to EtHg derived

from vaccines was based on the current national immuniza-

tion program of the Ministry of Health of Brazil.

Statistical analysis

Statistical analysis for testing differences between groups

was done using the Statistica (StatSoft, Inc., v.6.0) statis-

tical package. One-way analysis of variance was used with

Tukey’s procedure. The Shapiro-Wilk test of normality was

applied and data transformed when required. For the associ-

ation between variables Pearson’s correlation was calculated

and tested for significance; we accepted a value of <0.05 as

statistically significant.

RESULTS

Parameters and outcome differences due to timing of the

first dose of TCV (hepatitis-B) are shown in Table S1; no

significant differences were observed in neurodevelopmen-

tal scores at 6 months. The doses of Hg derived from injected

thimerosal varied greatly as a function of birth weight. Al-

though they may seem comparable between groups, actually

because the estimation was based on birth weight, the infants

that took the vaccine at a later time (mean of 3 days) were

probably underestimated due to obligatory neonatal weight

loss. As a function of body mass, the mean TCV-EtHg peri-

natal dose was equivalent to the two vaccine (third dose

of hepatitis-B and third dose of DTP) doses (50 ␮gHg) at

6 months when infants had double their body weight. De-

spite differences in socio-economic status between women

delivering at the public hospital and at private run facilities,

no significant differences were observed in infant anthro-

pometry (and relative TCV dose) at birth or at 6 months.

Because of variation in body mass gain during the first

6 months there was an asymmetry of TCV-EtHg exposure

at time of immunization; this is illustrated by percentage

distributions in Figure S1 (a, b and c). During this period

(six shots, 150 ␮gHg) there was considerable variation in

individual thimerosal exposure as a function of body mass.

The exposure to TCV-EtHg on the first and last vaccination

day (birth and 180 days) exceeds the recommended tolerable

daily intake of MeHg for adults. The amount of Hg per unit of

integrated body-mass gained over 4 months (Fig. S1c) shows

how high the immunized infant can be exposed to EtHg.

Despite a wide variation in birth weight, the correlation of

first dose of EtHg with Gesell scores was not statistically

significant (Fig. S2a).

Continuous variables were analysed as single linear cor-

relations; Pearson correlations were not statistically signif-

icant and are illustrated in the scatter plots of Figures S2

and S3. The hair-Hg representing antenatal (fetal hair-Hg

at 0 day) and postnatal (hair-Hg of 180 days) Hg exposure

events are plotted as percent change during the study period

(Fig. S3). The percentage increase in hair-Hg concentrations

was not significantly correlated with Gesell scores (Fig. S2a);

it should also be noted that that the change in hair-Hg was

not dependent of changes in body mass (Fig. S2b). Indeed,

Gesell scores at 6 months (Fig. S2c) were independent of

body mass increase.

DISCUSSION

The main finding of this work is that differences in time

of the first exposure to TCV (hepatitis-B) did not pre-

dispose infants to clinical neurodevelopment delays at 6

months. Furthermore, percent change in hair-Hg (prenatal

to 6 months hair-Hg, after the additional load of 150 ␮gHg)

was not significantly correlated with neurodevelopment de-

lays of these breastfed infants. The effect-modifiers of NDD

risk considered in this study [(a) time of exposure of first

EtHg challenge, (b) sensitivity due to immaturity (birth

weight/gestational length) and (c) prenatal Hg exposure]

could have been overruled by breastfeeding.

The maturation of the infant brain is a process that extends

over a long period; during this time, brain fine structure and

functioning are susceptible to neurotoxic substances. Mer-

cury is one of such substance and the earlier the exposure

the higher the risk of adverse effects. We realize that the rel-

atively small amount of thimerosal in vaccines is unlikely

to cause overt clinical neurological alterations (at least in

non-susceptible populations). However, given the time dif-

ferences (0 day versus 3 days), maturation gradients related

to gestational age (36–42 weeks) and attendant birth weight,

these results shed some light on the uncertainties raised

866

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2007 Foundation Acta Pædiatrica/Acta Pædiatrica 2007 96, pp. 864–868

Marques et al. Early vaccination, thimerosal and neurodevelopment

by theoretical models. Furthermore, because TCV-EtHg is

quickly excreted through stools (14) possible differences due

to bile production, bowel movements that could coincide

with differences in time of immunization did not seem to

affect neurological landmarks.

Thimerosal has been used in vaccines since the 1930s

without dispute but since then breastfeeding rates have

fallen considerably. While the adverse effects of TCV-

immunization on children have not been satisfactorily

demonstrated, the absence and/or limited duration of

breastfeeding has been amply demonstrated to negatively

affect neurodevelopment in short- and long-term studies.

It is recognized that the consequences of early exposure

to mercury can be subtle and may be evident after a pro-

longed latency (15–17). Therefore, a causal relationship

between EtHg (from vaccines) exposure and an adverse neu-

rological effect is difficult to establish in the relatively short

time of 6 months, especially when infants had the benefit of

breastfeeding. Nevertheless, the lack of a linear correlation

between neurodevelopment delays with parameters related

to gestational age/birth weight (Fig. S2) indicates that either

infant sensitivity to small doses of injected thimerosal lies

beyond these clinical tests or may manifest itself at a later

time.

The Gesell developmental quotient seems adequate in

gauging neurodevelopmental changes; it was able to detect

differences in mean gross motor development at 1 year of age

between term-infants fed formula with alpha-linolenic acid

(18). The 6-month old breastfed infants in this study were not

susceptible to identifiable effects caused by TCV-EtHg. It is

plausible that estimates of the actual neurotoxic risk from

TCV-EtHg exposure could be overruled by the CNS protec-

tive functions of breastfeeding. It is worth noting that the

small number of observations did not provide the necessary

material for multivariate analysis that could adjust important

confounding factors. Therefore, suboptimal neural develop-

ment due to TCV, if present in the more susceptible individ-

uals, could only be detected in much larger epidemiological

studies. Indeed, subtle NDD which amortized against the

broad spectrum of the Brazilian population could reach sig-

nificant numbers. Indeed, the large number of infants born

annually in Brazil (3.7 million) has national DTP coverage

of 90% in 72% of the Brazilian districts (19).

We can predict outcomes of positive effects of breastfeed-

ing on neuromotor development, but can only indicate the

possibility that NDD may occur as a result of TCV-EtHg.

Furthermore, breastfeeding has been shown to have an im-

munomodulating effect (20) and to significantly increase an-

tibody levels of DTP to a greater degree than in those that are

formula fed (21). Although it is possible that untoward events

in neurodevelopment may not be detected at 6 months, it is

also possible that breastfeeding could act as a modifying-risk

factor. At this young age, few cognitive skills are available

to the infant; the basic neuronal constitution that modu-

lates motor and perception involves time-critical processes.

Therefore, adaptive functions or acquisition of skills that

might be compromised by neurotoxic insults may take much

longer than 6 months, to manifest themselves. Indeed we had

a small number of individuals with a considerable overdose

of thimerosal-EtHg (Fig. S1a). In such individuals, subtle

neurodevelopment may lag behind but impairments could

be detected only in large epidemiological studies.

Although postnatal exposure to toxic metals can occur as

a result of milk feedings (3), exposure to EtHg occurs only

through immunization with TCV. However, contrary to for-

mula feeding, breastfeeding carries an established advantage

of CNS-priming substances and maternal stimuli that aid

infant neurodevelopment; together these neuroprotective ef-

fects of breastfeeding have a lifelong benefit on CNS integrity

and general health status. Studies of perinatal events related

to neurodevelopment and breastfeeding are rare. Radzymin-

ski (22) has noted that intact and functioning CNS is crucial

for breastfeeding behaviour. His study compared breastfeed-

ing behaviours and neurobehaviours at birth and at 24 h of

age in neonates of mothers who received epidural analge-

sia during labor and controls; the higher the infant scored

in relation to neurobehavioural functioning, the higher the

infant scored on breastfeeding behaviours.

The NDD effects of thimerosal depend on the interplay

between TK and TD of EtHg. In infants, these features have

not been sufficiently studied (particularly in neonates with a

wide range of birth weight and maturation—gestational age);

in the neonate, the fast surge of serum thimerosal could be

a factor regulating the relative levels of free forms of EtHg

available to cross the BBB. On the other hand, the TD of

thimerosal have been based on animal (mostly small rodent)

models due to ethical difficulties in conducting such studies

in humans. The lack of even observational studies, like the

present, has made our understanding of the TK and TD of

thimerosal depends on expert opinion (23). Therefore, it is

reassuring to have the opportunity to observe no clinical

neurodevelopment delay as a result of early immunization

timing. However, it is important to emphasize that breast-

feeding could have played a role in overruling subtle effects

that such early impact of EtHg might have caused. Therefore,

it is recommendable that breastfeeding should be considered

as a frontline procedure to minimize uncertainties related to

neurotoxic effects of early TCV-EtHg exposure.

CONCLUSION

This paper draws on information derived from a singular sit-

uation in which breastfed infants, opportunistically divided

into early (<1 day) and late (mean of 3 days) thimerosal-

EtHg exposure groups, did not show untowards effects on

developmental landmarks at 6 months.

ACKNOWLEDGEMENTS

We are greatly in debt to the mothers for their participa-

tion in the study, to the staff and Directors (Marin

es R. S.

Cezar, Tereza C. Ramos, Daniele Brasil, Katia Wendt, Laura

J. Marques) of the Hospitals (Hospital de Base Ary Pinheiro,

Hospital Panamericano and Hospital Regina Pacis), Katiane

G. Brand

ao, Dr. Cezar Augusto B. B. de Ara

ujo (State

Coordinator of the PNI-MS), the staff of the Fundac¸

Universidade Federal de Rond

onia and the Universidade

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2007 Foundation Acta Pædiatrica/Acta Pædiatrica 2007 96, pp. 864–868 867

Early vaccination, thimerosal and neurodevelopment Marques et al.

Federal do Rio de Janeiro. This work was supported

by United Nations Educational, Scientific and Cultural

Organization – UNESCO (SC27824/2005/914-BRA2000-

Decit-PRODOC), Minist

erio da Sa

ude do Brasil and The

National Research Council of Brasil-CNPq (PNOPG

project-55.0882/01–4; PPG7, project-556985/2005–2). The

initial study protocol was planned and conducted by Rejane

Marques; the conception of the present study was by

Jose Dorea and data analysis by Angelo G. Manzatto and

Jos

e Bernardi; Wanderley Bastos and Olaf Malm were re-

sponsible for Hg determinations and analytical quality con-

trol. All the coauthors contributed to the interpretation of

the data and the final version of the manuscript. All the au-

thors vouch for the accuracy and completeness of the re-

ported data and declare no conflict of interest.

References

1. Clarkson TW, Magos L. The toxicology of mercury and its

chemical compounds. Crit Rev Toxicol 2006; 36: 609–62.

2. Rodier PM. Developing brain as a target of toxicity. Environ

Health Perspect 1995; 103(Suppl 6): 73–6.

3. Dorea JG, Donangelo CM. Early (in uterus and infant)

exposure to mercury and lead. Clin Nutr 2006; 25: 369–76.

4. Oskarsson A, Schultz A, Skerfving S, Hallen IP, Ohlin B,

Lagerkvist BJ. Total and inorganic mercury in breast milk in

relation to fish consumption and amalgam in lactating women.

Arch Environ Health 1996; 51: 234–41.

5. Marques RC, D

orea JG, Bastos WR, Fonseca MF, Malm O.

Hair mercury in breast-fed infants exposed to thimerosal-

preserved vaccines. Eur J Pediatr 2007b; in press, doi:10.1007/

s00431-006-0362-2.

6. Harry GJ, Harris MW, Burka LT. Mercury concentrations in

brain and kidney following ethylmercury, methylmercury and

Thimerosal administration to neonatal mice. Toxicol Lett

2004; 154: 183–9.

7. Macdonald PD, Ross SR, Grant L, Young D. Neonatal weight

loss in breast and formula fed infants. Arch Dis Child Fetal

Neonatal Ed 2003; 88: F472–6.

8. Lawrence RA, Lawrence RM. (1999). Breastfeeding: a guide

for the medical profession. 5th ed. St. Louis: Mosby.

9. Lawlor DA, Clark H, Smith GD, Leon DA. Intrauterine

growth and intelligence within sibling pairs: findings from the

Aberdeen children of the 1950s cohort. Pediatrics 2006; 117:

e894–902.

10. Lawlor DA, Bor W, O’Callaghan MJ, Williams GM, Najman

JM. Intrauterine growth and intelligence within sibling pairs:

findings from the Mater-University study of pregnancy and its

outcomes. J Epidemiol Community Health 2005; 59: 279–82.

11. Marques RC, D

orea JG, Bastos WR, Rebelo MF, Fonseca MF,

Malm O. Maternal mercury exposure and neuro-motor

development in breastfed infants from Porto Velho (Amazon),

Brazil. Int J Hyg Env Health 2007a; 210: 51–60.

12. Gesell A, 2003. A crianc¸ade0a5anos. 6rd ed., S

ao Paulo:

Martins Fontes, 20–300.

13. Gesell A, Amatruda CS, 2000. Psicologia do Desenvolvimento

– do Lactente

a crianc¸a pequena, bases neuropsicol

ogicas e

comportamentais. 1st ed. S

ao Paulo: Atheneu, 30–106.

14. Pichichero ME, Cernichiari E, Lopreiato J, Treanor J. Mercury

concentrations and metabolism in infants receiving vaccines

containing thiomersal: a descriptive study. Lancet 2002; 360:

1737–41.

15. Rice DC. Evidence for delayed neurotoxicity produced by

methylmercury. Neurotoxicology 1996; 17: 583–96.

16. Harada M. Minamata disease: methylmercury poisoning in

Japan caused by environmental pollution. Crit Rev Toxicol

1995; 25: 1–24.

17. Amin-Zaki L, Majeed MA, Greenwood MR, Elhassani SB,

Clarkson TW, Doherty RA. Methylmercury poisoning in the

Iraqi suckling infant: a longitudinal study over five years.

J Appl Toxicol 1981; 1: 210–4.

18. Voigt RG, Jensen CL, Fraley JK, Rozelle JC, Brown FR III,

Heird WC. Relationship between omega3 long-chain

polyunsaturated fatty acid status during early infancy and

neurodevelopmental status at 1 year of age. J Hum Nutr Diet

2002; 15: 111–20.

19. WHO. Immunization profile – Brazil. 2005 (Available at

http://www.who.int/countries/bra/en/, accessed Nov/18/

2006).

20. Pabst HF, Spady DW, Pilarski LM, Carson MM, Beeler JA,

Krezolek MP. Differential modulation of the immune response

by breast- or formula-feeding of infants. Acta Paediatr 1997;

86: 1291–7.

21. Hahn-Zoric M, Fulconis F, Minoli I, Moro G, Carlsson B,

Bottiger M, et al. Antibody responses to parenteral and oral

vaccines are impaired by conventional and low protein

formulas as compared to breast-feeding. Acta Paediatr Scand

1990; 79: 1137–42.

22. Radzyminski S. Neurobehavioral functioning and

breastfeeding behavior in the newborn. J Obstet Gynecol

Neonatal Nurs 2005; 34: 335–41.

23. Magos L. Neurotoxic character of thimerosal and the

allometric extrapolation of adult clearance half-time to infants.

J Appl Toxicol 2003; 23: 263–9.

Supplementary material

The following supplementary material is available for this

article:

Table S1 Mean (range) of anthropometry, pre and perinatal

Hg exposure, and Gesell Scores of infants

Figure S1 Percent distribution of Thimerosal-EtHg/ unit of

body weight at birth (a—first dose of hepatite-B, 25 ␮gHg)

and at 180 days (b—third dose of hepatite-B and third dose of

DTP, 50 ␮gHg); (c) percent distribution of Thimerosal-EtHg

(second dose of hepatite-B, first and second dose of DTP,

75 ␮gHg)/weight gain between first and last vaccines.

Figure S2 Plot illustrating correlation between Gesell scores

and (a) dose of injected TCV-EtHg as function of body mass

(r =−0.1724; p = 0.1214), (b) between relative change in

hair-Hg at 6 months (r = 0.0753; p = 0.5037) and (c) relative

weight gain at 6 months (r =−0.1185; p = 0.2892).

Figure S3 Plot illustrating correlation between change in

hair-Hg at 6 months and respective change in body mass

(r =−0.0287; p = 0.7988).

This material is available as part of the online article from:

http://www.blackwell-synergy.com/doi/abs/10.1111/j.1651-

2227.2007.00288.x

(This link will take you to the article abstract).

Please note: Blackwell Publishing is not responsible for the

content or functionality of any supplementary materials sup-

plied by the authors. Any queries (other than missing mate-

rial) should be directed to the corresponding author for the

article.

868



2007 The Author(s)/Journal Compilation



2007 Foundation Acta Pædiatrica/Acta Pædiatrica 2007 96, pp. 864–868

Table S1. Mean (range) of anthropometry, pre- and perinatal Hg exposure, and Gesell

Scores of infants.

Schedule

Within 24 hours

48 to 96 hours

Birth weight, kg

3.2 (2.2-4.4)

3.3 (2.5-4.1)

Gestational age, d

39.4 (36-43)

39.2 (37-41)

Prenatal Hg exposure

Fetal hair [Hg], µg/g

2.1 (0.1-12.9)

3.0 (0.1-19.7)

Maternal hair [Hg] µg/g

7.1 (0.4-62.4)

7.8 (0.6-30.5)

Perinatal Hg exposure

Thimerosal, µgHg/kg

8.0 (5.7-11.3)

7.7 (6.1-10.0)

Outcome at six months

Body weight, kg

7.0 (6.1-8.5)

7.0 (6.4-8.0)

Thimerosal, µgHg/kg

7.2 (5.9-8.2)

7.2 (6.3-8.0)

Gesell Scores, quotient

Motor

76.0 (0-100)

72.5 (0-100)

Language

71.0 (0-100)

62.0 (0-100)

Adaptive ability

81.4 (0-100)

83.2 (20-100)

Social

85.6 (70-100)

87.0 (70-100)

General

78.5 (18-100)

76.2 (23-100)

Mean differences were not statistically significant.

Estimated from birth weight (5,11).

Likely to be underestimated because of obligatory perinatal weight loss

(approximately 10%).

Table S1. Mean (range) of anthropometry, pre- and perinatal Hg exposure, and Gesell

Scores of infants.

Figure S1. Percent distribution of Thimerosal-EtHg/ unit of body weight at birth (a – first

dose of hepatite-B, 25 µgHg) and at 180 days (b - third dose of hepatite-B and third dose of

DTP, 50 µgHg); (c) percent distribution of Thimerosal-EtHg (second dose of hepatite-B,

first and second dose of DTP, 75 µgHg)/weight gain between first and last vaccines.

4 6 8 10 12

100

(a)

Thimerosal, gHg/kg b.w. at birth

Distribution, %

4 6 8 10 12

100

Thimerosal, gHg/kg b.w. at 180 days

Distribution, %

(b)

10 20 30 40

100

Thimerosal, gHg/kg b.w. gained

Distribution, %

(c)

Figure S2. Plot illustrating correlation between Gesell scores and (a) dose of injected TCV-

EtHg as function of body mass (r=-0.1724; P=0.1214), (b) between relative change in hair-

Hg at six months (r=0.0753; P=0.5037) and (c) relative weight gain at six months (r=-

0.1185; P=0.2892).

5.0 7.5 10.0 12.5

100

(a)

Thimerosal, gHg/kg b.w.

Gesell score, %

0 1000 2000 3000 4000

100

150

Hair-Hg increase, %

Gesell score, %

(b)

0 100 200 300

100

150

Weight gain, %

Gesell score, %

(c)

Figure S3. Plot illustrating correlation between change in hair-Hg at six months and

respective change in body mass (r=-0.0287; P=0.7988).

0 1000 2000 3000 4000

100

200

300

Hair-Hg change, %

Body mass change, %

Int. J. Hyg. Environ. Health 211 (2008) 606–614

Principal component analysis and discrimination of variables associated

with pre- and post-natal exposure to mercury

Rejane C. Marques

a,b

, Jose

V.E. Bernardi

, Jose

G. Do

rea

c,Ã

Wanderley R. Bastos

, Olaf Malm

Fundac¸a˜o Universidade Federal de Rondo

nia, Porto Velho, RO, Brazil

Instituto de Biofı

sica Carlos Chagas Filho,Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Department of Nutrition, Universidade de Brası

lia, C.P. 04322, Campus - Asa Norte, 70919-970 Brası

lia, DF, Brazil

Received 28 December 2006; received in revised form 25 April 2007; accepted 12 September 2007

Abstract

The varia nce of variables associated with neurodevelopment at 1 80 days, pre-natal variables (Hg in placenta, blood

and hair) and post-natal Hg exposure (including Thimerosal-containing vaccines, TCV) were exami ned in 82

exclusively breastfed infants using principal component analysis (PCA). This mult ivariate method was applied to

identify hierarchy and sets of interrelated variables. The PCA yielded a two-factor solution, explaining 92% of

variance and summarizing most of the relevant information in the dataset matrix: the ﬁrst component represented birth

weight and vaccine (ﬁrst doses of Hepatitis B and DTP) variability and explained 57% of variance; the second

component represented a gradient of neurodevelopm ent (Gesell scores) and explained 35% of variance. The third

component explained only 3% of the remaining 8% variance. Beside CNS priming by breastfeeding, infant

development (birth weight) and time of immunization with TCV should be considered in epidemiological studies. PCA

can classify sets of variables related to vaccination and neuromotor development schedu les, clearly discriminating

between earlier and later TCV exposures of exclusively breastfed infants. In conclusion, the incommensurable concept

of the chance of toxic risk caused by TCV-EtHg exposure against the proven beneﬁt of immunization is in no way

disputed here. However, infant neurodevelopmental (ND) disorders linked to Thimerosal-Hg stands in need of proof,

but PCA points to the pos sibility of identifying exposure risk variables associated with ND schedules.

Keywords: Thimerosal; Ethylmercury; Methylmercury; Vaccines; Breastfeeding; Neurodevelopment; Infants

Introduction

Mercury’s most widely recognized effects are neuro-

logical; the developing central nervous system (CNS) of

fetus, infants and young children are vulnerable to these

effects. Fetal exposure to methylmercury (MeHg, from

ﬁsh consumption) is thought to lower intelligence and

alter behavior. The Harvard Center for Risk Analysis

panel quantiﬁed the impact of chronic MeHg exposure

on cognitive development (Cohen et al., 2005) and

concluded that pre-natal MeHg exposure sufﬁcient to

increase maternal ha ir-Hg by 1 mg/g at parturition

decreases intelligence quotient by 0.7 points (Cohen

et al., 2005). However, neurological effects of post-natal

ARTICLE IN PRESS

www.elsevier.de/ijheh

doi:10.1016/j.ijheh.2007.09.008

Corresponding author. Tel.: +55 61 3368 3575;

fax: +55 61 3368 5853.

E-mail address: [email protected] (J.G. Do

rea).

Hg exposure are more difﬁcult to evaluate. In infants,

Hg exposure occurs through milk feeding (inorganic Hg

and MeHg) and during immunization with Thimer osal

containing vaccines (TCV), which metabolize into ethyl

mercury (EtHg).

During the 1990s, there were several reports claiming

that immunization with TCV could increase autism and

other neurodevelopmental disorders (NDD). The pre-

ponderance of epidemiologic evidence does not support

an association between TCV and autistic disorders

(Parker et al., 2004), but biological plausibility is well

defended (Mutter et al., 2005). Therefor e, the epidemio-

logical interest in NDD (including autism) and associa-

tion with TCV continues to be studied with conﬂicting

results (Verstraeten et al., 2003; Geier and Geier, 2004,

2006a, b, c; Heron et al., 2004; Andrews et al., 2004).

While risk of pre-natal Hg exposure has prompted

issues of ﬁsh consumption advisories, efforts to decrease

children’s exposure to Hg have included a ban on the

use of TCV by industrialized countries; as a precau-

tionary measure, the USA and EU countries have

shifted from TCV to Thimer osal-free vaccines.

Although the WHO considers TCV safe for use in

underdeveloped countries, uncertainty about the possi-

bility of links between TCV and NDD will continue;

banning immun ization of children with TCV was

recognition by advanced nations that NDD have a

tendency to occur.

There are many studies showing that fetal exposure

to neurotoxicants is overruled by breastfeeding.

Furthermore, the link between NDD and TCV, or

more often, its absence as seen in some epidemiological

studies involves a failure to properly address the

weight of compromising circumstances related to

breastfeeding absence, insufﬁcient quantity or dura-

tion. The immunized infant is exposed to EtHg in

Thimerosal by a parenteral route which is different from

exposure to Hg (organic and inorganic) in human milk.

While Hg exposure in breast milk is buffered by the

enterohepatic barrier, the regulatory mechanisms of

the gastrointestinal tract are bypassed by injected

Thimerosal.

The ﬁrst 6 months of life are marked by signiﬁcant

qualitative (organ maturity, priming of the nervous

system, function of enzymes) and quantitative (tissue

differentiation, organ and body growth) changes.

Therefore, this relatively short period of life is impacted

by a high load of EtHg from immunizations; the smaller

body mass and blood volume of neonates are important

modulators of the toxicokinetics and toxicodynamics of

Thimerosal, especially at times of more vulnerability of

the blood–brain barrier (BBB). Newborns and infants

requiring immunization (in Brazil and many countries)

are still exposed to EtHg through TCV. Although the

use of TCV is considered safe, uncertainties related to

sub-clinical NDD in population studies await statistical

evaluation (Magos and Clarkson, 2006). Indeed, studies

that ignored colinearity or the many confounders

(related to pre- and post-natal exposure and infant

development) have produced rather incongruent results

such as a negative correlation between Thimerosal dose

and risk of neurological disorders (Andrews et al., 2004;

Heron et al., 2004).

PCA is a simple multivariate method useful to extract

relevant information from complex datasets. It is the

objective of the present study (a continuation of part I,

Marques et al., 2007a) to use powerful PCA to

discriminate variables associated with neurodevelop-

ment outcomes, pre- and post-natal Hg exposure and it

is expected that one can learn about the main factors

that inﬂuence variation in neurodevelopment of normal

breastfed ch ildren.

Materials and methods

As part of the study protocol to assess growth and

development of infants exposed to pre-natal back-

ground maternal-Hg load, we took advantage of the

study already published (Marques et al., 2007a) to focus

on variable interactions. During the review process of

the parent publication (Marques et al., 2007a), it was

brought to our attention that the possible post-natal

exposure include the vaccines given to infants which

were the type preserved in Thimerosal. We investigated

the origin of the vaccines and were informed that all

vaccines in the Br azilian immunization program con-

tained Thimerosal within the permitted limits (0.01%).

In order to evaluate metabolic aspect s related to our

main marker of organic Hg exposure (hair Hg

concentrations) in infants, we addressed these issues in

a second publication (Marques et al., 2007b).

The parent publication (Marques et al., 2007a)

dealing with pre-natal Hg exposure described in details

the characteristics of the population of Porto Velho

(State of Rondoˆ nia, West Amazonia). Mothers between

the ages of 15 and 45 years were selected by a nurse

during their routine visits to the Pre-natal Clinics

(Hospital de Base, Hospital Panamericano and Hospital

Regina Pacis), among those in good health, reporting no

illness or complaints. Excluding factors were occupa-

tional exposure to toxic chemicals and hereditary

neurological illnesses. Pregnant mothers who were

willing to breastfeed were introduced to the study’s

plain-language infor mation, and a written consent form

was presented and signed by the volunteer mother.

Conﬁdentiality was assured as well as freedom to

withdraw from the study at any time. The mothers were

closely monitored by nurses to guarantee full support of

breastfeeding and pre- and post-natal care including the

full immunization schedules recommended by the

Brazilian immunization program.

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ. Health 211 (2008) 606–614 607

After birth, the newborns were clinically examined

with special attention to vitality, perinatal reﬂexes,

maturity, and congenital malformations; weight, length,

head circumference, and Apgar scores wer e recorded.

While on the maternity wards, we collected samples of

placenta, blood and hair from mothers and respective

infants (fetal hair); the hair sampling was repeated at 6

months. Hair strands were cut from the occipital region

and placed in plastic bags, with the root end stapled on a

paper sheet. Sample preparation and Hg de terminations

in hair and blood were done according to routine

procedures (Marques et al., 2007a).

After hospital discharge the mothers and the children

were monitored for the programmed post-natal visits

and immunization scheme in accordance with the

Brazilian vaccination program. At 6 months of age,

mother–infant pairs reported for the programmed

clinical and neurobehavioral examination when hair

samples were collected again. Infants were again

weighed and measured for length and hair-samples were

again collected from mothers and infants. Integrated

weight gains at 30, 60 and 120 days were estimated from

differences between infants’ weight measured at birth

and at 6 months. In this study we used only datasets of

82 mother–infant pairs of the initial 100 enrollment; due

to various causes we had drop outs and incomplete data

in 18 pairs (Marques et al., 2007a).

The neurodevelopment tests were conducted by

trained professionals using the Gesell Developmental

Schedules (Gesell, 2003 ; Gesell and Amatruda, 2000).

These tests included all reactions (voluntary, sponta-

neous or learned) and reﬂexes. We also evaluated

postural reactions, hand pressure, locomotion and

coordination, constructive ability (which is inﬂuenced

by motor development), visible and audible commu-

nication, individual reactions regarding people and

stimulations (depending mainly on the temperament of

the child and the surroundings); using a ppropriate

measurements we compare the child developmental

stage with that exp ected for the respective age (qualita-

tive and quantitative). The results (as percentages) were

expressed as developmental quotients (DQ) for the

Adaptive Behavior, Language Development, Gross

Motor Skills and Personal/Social Behaviors.

Estimated exposure to injected Thimerosal-Hg (EtHg)

from vaccines: Mothers followed the immunization

schedule recommended by the Ministry of Health of

Brazil. The ﬁrst dose of Hepatitis B vaccine was taken

by neo nates before hospital discharge and the subse-

quent two doses (at 30 and 180 days) as well as the three

doses of DTP at 60, 120 and 180 days (Table 1). Only

the babies born at the state-run hospital (Hospital de

Base, 66%) received the Hepatitis B vaccine within the

ﬁrst day postpartum. Babies born at the Hospital

Panamericano and the Hospital Regina Pac is received

the Hepatitis B vaccine immediately after the mother’s

discharge (2–4 days postpartum). At this time, the

mothers were taken unde r our supervision to a state-run

clinic where immunizations are done.

The calculated exposure to EtHg was estimated as

Thimerosal-Hg derived from vaccines used in the

current national immunization program of the Ministry

of Health of Brazil. The Hg concentration of the doses

delivered through vaccines (containing 0.01% Thimer-

osal as stated by manufacturers) was 25 mg/0.5 mL for

Hepatitis B (Korea Green Cross Corporation, Kiheung-

Eup Yougin-Goon Kiyunggi-Do, Korea; Euvax B

injectable, LG Life Sciences, Jeonbuk-Do, Korea) and

for DTP (Triple Antigen, Serum Insti tute of India Ltd.,

India; Vacina Trı

plice, Instituto Butanta

,Sa

o Paulo,

Brazil).

Statistical analysis

A principal component analysis (PCA) model was

used for the measured variables to gain information

with fewer varia bles. This mathematical model calcu-

lates new variables (principal components) that account

for the variability in the data and enables the study of

covariances or correlations between variables. Thus, the

principal components are linear combinations of the

original variables. The combination of variables that

explains the greatest amount of variability is the ﬁrst

ARTICLE IN PRESS

Table 1. Infant anthropometry, neurodevelopment schedules

and markers of prenatal Hg exposure

Characteristics Mean7S.D.

Infant anthropometry

Weight at birth (g) 3233.177421.56

Length at birth (cm) 49.7772.43

Head circumference at birth (cm) 33.8872.29

Weight at 6 month (g) 7011.347458.98

Length at 6 month (cm) 67.6072.59

Head circumference at 6 month (cm) 42.5871.08

Pre-natal Hg exposure

Maternal blood Hg (mg/L) 6.03714.07

Placenta Hg (ng/g) 10.7579.82

Umbilical cord Hg (ng/g) 10.5479.93

Maternal hair Hg at delivery (mg/g) 7.3678.73

Infant hair Hg at birth ( mg/g) 2.4473.04

Infant hair Hg at 6 month (mg/g) 3.8475.55

Maternal hair Hg 6 month (mg/g) 3.1973.85

Neurodevelopmental schedules

Motor DQ 74.82731.95

Language DQ 67.87736.23

Adaptive DQ 82.01719.62

Personal social DQ 86.10710.21

Total DQ 77.70722.24

DQ, development quotient.

R.C. Marques et al. / Int. J. Hyg. Environ. Health 211 (2008) 606–614608

principal component. The subsequent ones (second and

third principal component) describe the maximum

amount of remaining variability; they must be indepen-

dent of the ﬁrst principal component. For this multi-

variate model we used the appro ach discussed by others

(Ludwing and Reynolds, 1988; Reyment and Jo

reskog,

1996).

The main steps of the analysis were:

(i) Transformation of the data: PCA standardize the

original matrix assuming means as zero and

standard deviations as one. The purpose of this step

is to derive a small number of linear combinations

(principal components) that retain as much of the

information in the original variables as possible; this

allows many variables to be summarized in a few,

jointly uncorrelated principal components. The

result is considered goo d if we obtain a high

proportion of the total variance explained by a few

principal components. Commensurate with the

analysis done in principal components analysis, we

also treated the variables as active and supplemen-

tary variables. The supplementary variables can be

projected onto the vector subspace generated by the

factors.

We used the STATISTICA package (StatSoft, Inc.,

Data Analysis Software System, version 6, www.

statsoft.com, 2001, Tulsa, OK 74104, USA) on the

following variables: (1) birth weight, (2) length at

birth, (3) head circumference at birth, (4) mate rnal

blood-Hg concentration at delivery, (5) Placenta-Hg

concentration, (6) umbilical cord-Hg concentration,

(7) maternal hair-Hg concentration at delivery,

(8) infant hair-Hg concentration at birth, (9) infant

hair-Hg concentration at 6 months, (10) maternal

hair-Hg concentration at 6 months, (11) infant

weight at 6 months, (12) infant length at 6 months,

(13) head circumference at 6 months, (14) motor DQ

at 6 months, (15) language DQ at 6 months,

(16) adaptative DQ at 6 months, (17) personal and

social DQ at 6 months, (18) total DQ at 6 months,

(19) Thimerosal-Hg (Hepatitis B 1st dose)/kg b.w. at

birth, (20) Thimerosal-Hg (Hepatitis B 2nd dose)/kg

b.w. at 30 days, (21) Thimerosal-Hg (DTP 1st dose)/kg

b.w. at 60 days, (22) Thimerosal-Hg (DTP 2nd

dose)/kg b.w. at 120 da ys, (23) Thimerosal-Hg

(3rd dose of Hepatitis B and DTP)/kg b.w. at 180

days, (24) integrated mean Thimerosal-Hg/kg b.w.

at 180 days; we did not use varimax rotation or any

other additional factor analysis.

(ii) Construction of two-dimensional graph (Fig. 1): This

step organizes the variables on the appropriate

vector basis allowing visual inspection of the data

in a two-dimensional score plot that reveals patterns

hidden in the dataset. To visualize the separation of

groups, Fig. 1 shows score plots of the ﬁrst two PCs

and the values of the PC loadings. Plots of loadings

(PC1 and PC2) on these data allow us to recognize

groups of samples with similar behavior and the

existing association among the original variables.

Results

The summary, as mean and S.D., of the chosen

variables is shown in Tables 1 and 2. These variables

include pre- and post-natal Hg exposure, infant anthro-

pometry and neu rodevelopment scores (Table 1); pre-

natal Hg exposure is represented by hair-Hg of mother

and infants at birth, Hg concentrations in placenta and

umbilical cord. Post-natal Hg exposure is represented by

infant hair-Hg at 6 months and immunization doses of

TCV-Hg (as EtHg) during the ﬁrst 6 months (Table 2).

Higher Thimerosal-Hg dose per unit of body mass is

shown at birth (when newborns had the smallest body

mass) and 6 months (when they were injected with two

doses of TCV – third shot of both Hepatitis B and

DTP).

The main component analysis was used to explore the

data matrix order; this can help determine the weight

(importance) of parameters in the total variability

through vector size and loads and respective percentual

contributions. The result of PCA gives two signiﬁcant

principal components (eigenvalues 41), which explain

92% of the variation in the data (57% and 35%,

respectively). The classiﬁcation of loads (Tables 3–5)

points to the post-natal Hg exposure (immunization

ARTICLE IN PRESS

Active

Supplementary

-1.0 -0.5 0.0 0.5 1.0

-1.0

-0.5

0.0

0.5

1.0

Factor 2 : 34.79%

Fig. 1. Scatter plot of loadings of PCA (principal component

analysis) dataset. The identities of the responses and the

neurodevelopment variables are indicated by the plot labels.

R.C. Marques et al. / Int. J. Hyg. Environ. Health 211 (2008) 606–614 609

schedules of newborns and infants): two vaccine s within

the ﬁrst month (0 and 30 days) and the high Thimerosal-

Hg dose (in the third dos e of Hepatitis B and DTP) at

180 days. The analyzed variables and respective loads

are listed in Table 3; the ﬁrst component (57% of

variance) is accounted for by variables with the highest

loads: (1) birth weight (0.90), (19) Thimerosal-Hg

(Hepatitis B 1st dose)/kg b.w. at birth (À0.90), (20)

Thimerosal-Hg (Hepatitis B 2nd dose)/b.w. at 30 days

ARTICLE IN PRESS

Table 2. Infant immunization schedule, type of vaccine, and Thimerosal-Hg/dose (EtHg) during the ﬁrst 6 months

Age (days) Type

mgHg/dose

Infant weight (kg)

c,d

mgHg/kg

0 Hp-B 25.0 3.23 (0.42) 7.87 (1.09)

30 Hp-B 25.0 3.86 (0.35) 6.53 (0.61)

60 DTP 25.0 4.49 (0.31) 5.59 (0.38)

120 DTP 25.0 5.75 (0.32) 4.37 (0.23)

180 DTP+Hp-B 50.0 7.01 (0.46) 7.16 (0.44)

Total 150.0 6.30 (0.45)

Hp-B: Hepatitis B (Korea Green Cross Corporation, Kiheung-Eup Yougin-Goon Kiyunggi-Do, Korea; Euvax B injectable, LG Life Sciences,

Jeonbuk-do, Korea); DTP (Serum Institute of India Ltd.; Vacina Trı

plice, Instituto Butanta, Sa

o Paulo, Brazil). These vaccines contained 0.01%

Thimerosal/dose as stated by manufactures.

Vaccine dose: 0.5 mL; Hg mass is approximately 50% of the Thimerosal.

Estimated from Marques et al. (2007a).

Mean (and standard deviation).

Table 3. Principal component loads indicating variability percentages of measured parameters (factor-variable correlations (factor

loadings), based on active and supplementary variables

)

Factor 1 Factor 2 Factor 3

1. Weight at birth 0.898249 0.362923 0.064494

14. Index of motor development 0.493417 À0.796391 0.139435

15. Index of language development 0.517388 À0.771637 0.235206

16. Index of adaptative development 0.470206 À0.757382 À0.440593

18. Index of general development 0.534355 À0.841893 0.049363

19. TCV-Hg (Hepatitis B 1st dose, birth)/kg b.w. À0.897585 À0.361978 À0.057133

20. TCV-Hg (Hepatitis B 2nd dose, 30 days)/kg b.w. À0.930730 À0.364520 0.000802

21. TCV-Hg (DTP 1st dose, 60 days)/kg b.w. À0.902208 À0.331506 0.075336

24. Total Thimerosal-Hg/kg b.w. at 180 days À0.917511 À0.336700 0.054712

2. Length at birth

0.319748 0.229549 À0.096108

3. Head circumference at birth

0.484365 0.053901 À0.012174

4. Maternal blood-Hg at delivery

À0.204777 0.163233 0.338478

5. Placenta-Hg

À0.174918 0.030322 À0.219712

6. Umbilical cord-Hg

À0.132638 0.015406 À0.069268

7. Maternal hair-Hg at delivery

À0.001043 0.088459 À0.046659

8. Infant hair-Hg at birth

À0.051069 0.468469 0.092115

9. Infant hair-Hg at 6 months

À0.126927 0.329956 0.120974

10. Maternal hair-Hg at 6 months

À0.001800 0.055173 0.043098

11. Infant weight at 6 months

0.142328 À0.019707 À0.245993

12. Infant length at 6 months

0.537043 0.124353 À0.134842

13. Head circumference at 6 months

0.568066 À0.055300 À0.132429

17. Index of personal and social development

0.372902 À0.650683 0.005994

22. TCV-Hg (DTP 2nd dose-120 days)/kg b.w.

À0.537471 À0.140750 0.209801

23. TCV-Hg (Hepatitis B and DTP – 180 days)/kg b.w.

À0.138998 0.030978 0.242526

TCV: thimerosal-containing vaccines; DTP: diphtheria, tetanus, pertussis.

Supplementary variables are illustrated in Fig. 1 (Eigenvalues o1).

Table 4. Eigenvalues of correlation matrix and related

statistics of active variables only – individual and cumulative

percent contributions

Eigenvalue % Total Cumulative Cumulative

1 5.152354 57.24837 5.152354 57.2484

2 3.130954 34.78837 8.283307 92.0367

3 0.287416 3.19351 8.570724 95.2303

R.C. Marques et al. / Int. J. Hyg. Environ. Health 211 (2008) 606–614610

(À0.93), (21) Thimerosal-Hg (DTP 1st dose)/kg b.w. at

60 days (À0.90), (24) mean Thimerosal-Hg/kg b.w. at

180 days (À0.92). The second component, which

explained 35% of the variation, is accounted for

by the following variables: (14) motor DQ (À0.80),

(15) language DQ (À0.78), (16) total DQ (À0.84),

(18) adaptative DQ (À0.76). This can be interpreted as a

gradient of varia bility among variables; positive signs

mean that variables increase together while a negative

sign means that as one increases the other decreases. It

should also be conceptualized that the sign of the birth-

weight variable is the opposite of those of Thimerosal

doses because these are derived from the former (they

are reciprocals). The third component explained 3% of

the remaining 8% variability. Further details in Table 4

illustrate the percentage of variance explained by

components. Components one and two explain 92% of

total variance, accounting, respectively, for 57% and

35% of the variation. Table 5 summarizes the factor

contribution of the active variables.

Most of the variation is explained by two-dimensional

scattergram (Fig. 1); plotting the values for the ﬁrst two

principal components of the data points gives the

essential features of the multidimensional scatter (PC1

and PC2, whose eigenvalues are 41, represents the main

variables). Fig. 1 illustrates the separation of the

observational variables: the variables associated with

ND sched ules and immunizations (as function of body

weight) are at opposite quadrant.

Discussion

The main ﬁnding of this work is to show that PCA

discriminated variability of early vaccine schedule and

neurodevelopment outcomes from variables that in-

cluded pre- and post-natal Hg exposure; immunizations

given within 60 days were extracted as the ﬁrst principal

component. Multivariate models involving many vari-

ables are often difﬁcult to interpret because plots and

lists of loadings can become too overwhelming to

comprehend. In order to handle a large number of

variables, PCA reorganizes the variables, reducing the

dimensions of the origi nal dataset. In other words,

without a signiﬁcant loss of information, this approach

describes a shorter list of variables. In this dimension,

PCA clearly discriminated the ﬁrst set of vaccines taken

at an earlier time (ﬁrst and second doses of Hepatitis B

at birth and 30 days, and ﬁrst DTP at 60 days).

Although all vaccines contained the same dose of

Thimerosal-Hg, the amount of EtHg/kg b.w. given

varied as a function of body mass change: newborn

babies (within the ﬁrst days) received doses of TCV-

EtHg comparable to those of 6 months old (double the

amount of vaccine-Hg in the two shots given at 6

month). Although these doses of Thimerosal (EtHg) per

unit of body mass represented the highest TCV

exposures, they ended up in different components.

However, it should be kept in mind that a point of

concern can be raised in respect to birth weight (as well

as body mass development) and the given TCV-Hg dose.

Birth weight and weight adjusted Hg produced close co-

linearity (opposite sign factor-loadings but nearly

identical high values – Table 3). For studies intending

to use PCA co-linearity, such as this type, it can bias

regression models. Nevertheless, it points to the

necessity to view the ﬁrst 6 months in its proper

perspective.

Differences in infant growth and maturati on (tissue

differentiation) between a 1-day-old neonate and a

6-month-old infant (who has doubled its original body

mass and already acquired functional abilities) set them

apart in terms of the toxicodynamics of Hg exposure.

Infants of this age range are not just small children,

but a heterog eneous population with a wide range of

physiological, metabolic and anatomical differences. It

is essential to consider the dynamic complexity of early

post-natal development to unde rstand the spectrum of

differentiation that determines the heterogeneity of this

group. At one end we have a neonate (‘‘external fetus’’)

with the highest degree of immaturity and that is deﬁned

as ‘‘born at term and normal’’ only by gestational age,

ARTICLE IN PRESS

Table 5. Discrimination of variables with percent contributions

Factor 1 Factor 2 Factor 3

1. Birth weight 0.156598 0.042068 0.014472

14. Index of motor development 0.047252 0.202571 0.067645

15. Index of language development at 6 months 0.051955 0.190173 0.192479

16. Index of adaptative development 0.042911 0.183212 0.675405

18. Index of general development 0.055419 0.226380 0.008478

19. TCV-Hg (Hepatitis B 1st dose, birth)/kg b.w. 0.156367 0.041849 0.011357

20. TCV-Hg (Hepatitis B 2nd dose, 30 days)/kg b.w. 0.168129 0.042439 0.000002

21. TCV-Hg (DTP 1st dose, 60 days)/kg b.w. 0.157982 0.035100 0.019747

24. Total Thimerosal-Hg/kg b.w. at 180 days 0.163387 0.036208 0.010415

TCV: thimerosal containing vaccines; DTP: diphtheria, tetanus, pertussis.

R.C. Marques et al. / Int. J. Hyg. Environ. Health 211 (2008) 606–614 611

not by markers of immaturity. At the other end,

6-month-old infants are more functional and differen-

tiated (anatomically, biochemically and physiologically).

Therefore, there are implications for Thimerosal-EtHg

during the ﬁrst post-natal 24 weeks in term babies

(deﬁned as born between 36 and 42 weeks) with such a

relatively wide (6 weeks) range of gestational age

difference.

During the ﬁrst 6 months, the fast changes in size,

body composition, and organ function dramatically

affect pharmacokinetics; this is also likely to affect

pharmacodynamics. During this time infants are ex-

posed to Hg in breast milk (Dorea, 2004); such exposure

is amortized against a proportional milk intake

(as a function of body weight). Any form of breast-

milk Hg (inorganic Hg and methyl-Hg, MeHg) is

complexed into the milk–protein matrix and its absorp-

tion and toxicity are attenuated by entero-hepatic

barriers. Concomitantly, CNS is primed by components

naturally present in breast milk. Contrary to the

enteral breast-milk Hg, the parente ral route of TCV

leads to a disproportionate EtHg exposure (per unit of

body mass) at different stages of development (highest

immediately after birth). The parenteral TCV exposure

bypasses the enteral barriers and metabolic defenses,

gaining ready access to the systemic venous system and

thus reachi ng the brain within seconds. Magos et al.

(1989) showed in rat models that circulation time

between Hg absorption site and brain is an important

toxicity factor. Indeed, a peak value of mercury appears

to be the determinant of toxic damage in population

studies (Clarkson and Strain, 2004); depending on post-

natal immaturity, this is likely to occur with injected

Thimerosal (EtHg).

Stajich et al. (2000) speculated that infants with less

body mass could metabolize EtHg at a slower rate than

larger infants and that immaturity of the liver could be

responsible for less metallothionein available to bind

inorganic Hg. However, the reanalysis of their data by

Magos (2003) reached the opposite conclusion: ‘‘pre-

term infants handled ethylmercury load more efﬁciently

than term infants.’’ Regardless of these toxicokinetic

differences, the vulnerability of the developing nervous

system is expecte d to be higher in the ne onatal period

than at 180 days. Both Stajich et al. (2000) and Magos

(2003) referred speciﬁcally to the toxicokinetics of EtHg

and by no means referred to its toxicodynamics.

The decision to eliminate Thimerosal in vaccines

(claimed as a precautionary measure) by some countries

was based on g uidelines for MeHg. Although the

guidelines for MeHg are not equivalent to guidelines

for EtHg (Thimerosal-Hg metabolite), the current

assumption is that the health risks from exposure to

MeHg and EtHg are the same (NIAID, 2005). This is

debatable in the context of the breastfed infant: while

breastfed infants are exposed to MeHg (in lower

amounts than inorganic Hg) only enterally, TCV-EtHg

exposure is a bolus parenteral exposure.

Because we are dealing with exposure to a pure

substance by a parenteral route impacting an immature

organism, there are speciﬁc considerations. The ﬁrst

impact of vaccine-EtHg for some newborns comes

within hours of birth (Marques et al., 2007b). Newborns

show important differences in body weight and tissue

differentiation that affect toxicokinetics. Even in term

infants, the blood–brain barrier (BBB) is not complete

until after 6 months of age (Adinolﬁ, 1985). Therefore,

the pharmacokinetic and pharmacodynamic of Thimer-

osal (EtHg) are modiﬁed by infant development, thus

representing a challenge to infant toxicology. It has been

experimentally shown that both genetic and brain

maturation are critical determinants of post-natal

Thimerosal-related sequelae. Equiva lent EtHg dosing

and timing of exposure caused behavioral disruption in

control mice but not in a strain resistant to autoimmu-

nity (Hornig et al., 2004). However, because only 3% of

neurodevelopmental (ND) disabilities are attributed to

environmental neurotoxic substances (Grandjean and

Landrigan, 2006), a large population study would be

required to detect a post-natal Hg exposure effect

caused by TCV. Furtherm ore, in situations of universal

exposure such as Hg in vaccines, increased risk of ND

disabilities is more complex because of chance of

interactions with pre-natal exposure to MeHg in ﬁsh

eating populations.

It should be stressed that neurodevelopmental vari-

ables in this study were obtained from infants whose

CNS were primed by exclusive breastfeeding. Breast-

feeding has consistently shown efﬁcient CNS priming

that could affect the toxicodynamics of TCV-EtHg. The

counteractive effects of breastfeeding on NDD have

been reported in infants pre-natally exposed to MeHg

(Grandjean et al., 1995; Jensen et al., 2005). Breastfeed-

ing predicts faster CNS maturation rates (Hart et al.,

2003), higher neurodevelopment scores (Vestergaard

et al., 1999), and better intellectual achievements

(Victora et al., 2005).

So far, studies that examined NDD effects of vaccine-

EtHg exposure have not taken breastfeeding into

consideration. One study, however, provides an oppor-

tunity to speculate; in 7-year-old Faroese children,

breastfeeding was associated with marginally better

neuropsychological performance (Jensen et al., 2005).

Hair mercury concentration at age of 1 year (adjusted

confounder), did not change the positive effects of

breastfeeding. These Danish children were born between

1986 and 1987 and probably also received the TCV

(125 mgEtHg, three vaccine doses) still in use at that time

(Hviid et al., 2003).

The full range of options used in epidemiological

studies of infant exposure to TCV (EtHg) and the short-

and long-term effects on neurodevelopment should

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ. Health 211 (2008) 606–614612

consider breastfeeding as an important covariate. Multi-

variate models such as PCA do not establish cause and

effect association between variables, but are useful tools

to sort them. In our study it was possible to line up

vectors that discriminated and classiﬁed the immuniza-

tion schedules (ﬁrst principal component) and neurode-

velopmental scores (second principal component).

Because there were no adverse effects of pre-natal Hg

exposure in this group of breastfed infants (Marques

et al., 2007a), neuro development interaction with early

immunization of infants should also consider birth

weight (or surrogates of fetal development such as

gestational age). This might help devise epidemiological

studies with sufﬁcient reﬁnement to include variables

that can more precisely account for the complexity of

early post-natal development and Hg exposure. There

are no cause–effects relations between early Thimerosal-

Hg exposure and neurodevelopmenta l deﬁcits, but the

challenge of understanding such complex interactions

may beneﬁt from the present ﬁndings.

Conclusion

The chance of harm caused by TCV-EtHg exposure

against the proven beneﬁt of immunization is in no way

argued by these results. Infant NDD linked to Thimer-

osal-Hg stands in need of proof; however, the present

work points to the possibility of identifying modiﬁable

NDD-risk factors associated with infant development

and TCV-EtHg dosing.

Acknowledgments

We are greatly in debt to the mothers for their

participation in the study; Dr. Angelo Gilberto Man-

zatto, Cezar Augusto Bezerra B. de Arau´ jo, MSc, MD

Marineˆ s Rodrigues dos Santos Cezar, MSc, Tereza

Cristina Ram os, Katiane Guedes Branda

o, Rayson

Correa Marques and the staffs of the Fundac¸ a

Universidade Federal de Rondoˆ nia and the Universi-

dade Federal do Rio de Janeiro. This work was

supported by United Nations Educational, Scientiﬁc

and Cultural Organization – UNESCO (SC27824/2005/

914-BRA2000-Decit-P RODOC), Ministe

rio da Sau´ de

do Brasil and The Nation al Research Council of Brasil-

CNPq (PNOPG project-55.0882/ 01-4; PPG7, project-

556985/2005-2).

References

Adinolﬁ, M., 1985. The development of the human blood-

CSF-brain barrier. Dev. Med. Child Neurol. 27, 532–537.

Andrews, N., Miller, E., Grant, A., Stowe, J., Osborne, V.,

Taylor, B., 2004. Thimerosal exposure in infants and

developmental disorders: a retrospective cohort study in

the United Kingdom does not support a causal association.

Pediatrics 114, 584–591.

Clarkson, T.W., Strain, J.J., 2004. Methyl mercury: loaves

versus ﬁshes. Seychelles Med. Dent. J. 7, 61–66.

Cohen, J.T., Bellinger, D.C., Shaywitz, B.A., 2005. A

quantitative analysis of prenatal methyl mercury exposure

and cognitive development. Am. J. Prev. Med. 29, 353–365.

Dorea, J.G., 2004. Mercury and lead during breast-feeding. Br.

J. Nutr. 92, 21–40.

Geier, D., Geier, M.R., 2004. Neurodevelopmental disorders

following thimerosal-containing childhood immunizations:

a follow-up analysis. Int. J. Toxicol. 23, 369–376.

Geier, D.A., Geier, M.R., 2006a. An evaluation of the effects

of thimerosal on neurodevelopmental disorders reported

following DTP and Hib vaccines in comparison to DTPH

vaccine in the United States. J. Toxicol. Environ. Health A

69, 1481–1495.

Geier, D.A., Geier, M.R., 2006b. An assessment of downward

trends in neurodevelopmental disorders in the United

States following removal of thimerosal from childhood

vaccines. Med. Sci. Monit. 12, CR231–CR239.

Geier, D.A., Geier, M.R., 2006c. A meta-analysis epidemio-

logical assessment of neurodevelopmental disorders follow-

ing vaccines administered from 1994 through 2000 in the

United States. Neuro Endocrinol. Lett. 5, 27(4) [Epub

ahead of print].

Gesell, A., 2003. A crianc¸ a de 0 a 5 anos, 6th ed. Martins

Fontes, Sa

o Paulo, pp. 20–300.

Gesell, A., Amatruda, C.S., 2000. Psicologia do Desenvolvi-

mento – do Lactente a

crianc¸ a pequena, bases neuropsico-

gicas e comportamentais, ﬁrst ed. Atheneu, Sa

o Paulo,

pp. 30–106.

Grandjean, P., Weihe, P., White, R.F., 1995. Milestone

development in infants exposed to methylmercury from

human milk. Neurotoxicology 16, 27–33.

Grandjean, P., Landrigan, P.J., 2006. Developmental neuro-

toxicity of industrial chemicals. Lancet 368 (9553),

2167–2178.

Hart, S., Boylan, L.M., Carroll, S., Musick, Y.A., Lampe,

R.M., 2003. Brief report: breast-fed one-week-olds demon-

strate superior neurobehavioral organization. J. Pediatr.

Psychol. 28, 529–534.

Heron, J., Golding, J., ALSPAC Study Team, 2004. Thimer-

osal exposure in infants and developmental disorders: a

prospective cohort study in the United Kingdom does not

support a causal association. Pediatrics 114, 577–583.

Hornig, M., Chian, D., Lipkin, W.I., 2004. Neurotoxic effects

of postnatal thimerosal are mouse strain dependent. Mol.

Psychiatry 9, 833–845.

Hviid, A., Stellfeld, M., Wohlfahrt, J., Melbye, M., 2003.

Association between thimerosal-containing vaccine and

autism. JAMA 290, 1763–1766.

Jensen, T.K., Grandjean, P., Jorgensen, E.B., White, R.F.,

Debes, F., Weihe, P., 2005. Effects of breast feeding on

neuropsychological development in a community with

methylmercury exposure from seafood. J. Expo. Anal.

Environ. Epidemiol. 15, 423–430.

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ. Health 211 (2008) 606–614 613

Ludwing, J.A., Reynolds, J.F., 1988. Statistical Ecology: A

Primer on Methods and Computing. Wiley Interscience

Publication, New York, p. 325.

Magos, L., 2003. Neurotoxic character of thimerosal and the

allometric extrapolation of adult clearance half-time to

infants. J. App. Toxicol. 23, 263–269.

Magos, L., Clarkson, T.W., 2006. Overview of the clinical

toxicity of mercury. Ann. Clin. Biochem. 43, 257–268.

Magos, L., Clarkson, T.W., Hudson, A.R., 1989. The effects

of dose of elemental mercury and ﬁrst-pass circulation time

on exhalation and organ distribution of inorganic mercury

in rats. Biochem. Biophys. Acta 991, 85–89.

Marques, R.C., Do

rea, J.G., Bastos, W.R., Rebelo, M.F.,

Fonseca, M.F., Malm, O., 2007a. Maternal mercury

exposure and neuro-motor development in breastfed

infants from Porto Velho (Amazon), Brazil. Int. J. Hyg.

Environ. Health 210, 51–60.

Marques, R.C., Do

rea, J.G., Fonseca, M.F., Bastos, W.R.,

Malm, O., 2007b. Hair mercury in breast-fed infants

exposed to thimerosal-preserved vaccines. Eur. J. Pediatr.

[Epub ahead of print, doi:10.1007/s00431-006-0362-2 ].

Mutter, J., Naumann, J., Schneider, R., Walach, H., Haley, B.,

2005. Mercury and autism: accelerating evidence? Neuro

Endocrinol. Lett. 26, 439–446.

NIAID. 2005. National Institute of Allergy and Infectious

Diseases Supported Studies on Mercury. Thimerosal and

Vaccine Safety (http://www.niaid.nih.gov/factsheets/

thimerosalqa.htm; available on line April 11, 2006).

Parker, S.K., Schwartz, B., Todd, J., Pickering, L.K., 2004.

Thimerosal-containing vaccines and autistic spectrum

disorder: a critical review of published original data.

Pediatrics 114, 793–804.

Reyment, R.A., Jo

reskog, K.G., 1996. Applied Factor

Analysis in the Natural Sciences. Cambridge University

Press, p. 383.

Stajich, G.V., Lopez, G.P., Harry, S.W., Sexson, W.R.,

2000. Iatrogenic exposure to mercury after hepatitis

B vaccination in preterm infants. J. Pediatr. 136,

679–681.

Vestergaard, M., Obel, C., Henriksen, T.B., Sorensen, H.T.,

Skajaa, E., Ostergaard, J., 1999. Duration of breastfeeding

and developmental milestones during the latter half of

infancy. Acta Paediatr. 88, 1327–1332.

Verstraeten, T., Davis, R.L., DeStefano, F., Lieu, T.A.,

Rhodes, P.H., Black, S.B., Shineﬁeld, H., Chen, R.T.,

Vaccine Safety Datalink Team, 2003. Safety of thimerosal-

containing vaccines: a two-phased study of computerized

health maintenance organization databases. Pediatrics 112,

1039–1048.

Victora, C.G., Barros, F.C., Horta, B.L., Lima, R.C., 2005.

Breastfeeding and school achievement in Brazilian adoles-

cents. Acta Paediatr. 94, 1656–1660.

ARTICLE IN PRESS

R.C. Marques et al. / Int. J. Hyg. Environ. Health 211 (2008) 606–614614

Changes in children hair-Hg concentrations during the ﬁrst 5

years: Maternal, environmental and iatrogenic modifying factors

Rejane C. Marques

a,b

, Jose

G. Do

rea

, Wanderley R. Bastos

, Olaf Malm

Fundac¸a

o Universidade Federal de Rondo

nia, Porto Velho, RO, Brazil

Instituto de Biofı

sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil

Universidade de Brası

lia, C.P. 04322, 70919-970 Brası

lia, DF, Brazil

Received 21 February 2007

Available online 10 May 2007

Abstract

Children are exposed to Hg from mothers (via placenta and lactation), environment (food), and in many parts of the world by thi-

merosal-containing vaccines (TCV) during immunization. Neurodevelopment studies based on infant hair-Hg (HHg) have been designed

without explicit attention to the factors associated with changes in infant physiology and Hg sources of exposure. A longitudinal study of

changes in HHg concentrations from birth to 5 years was done in a sample of children from Porto Velho (Rondonia), Brazilian Amazo-

nia. The study extracted information from the asymmetry associated with maternal and infant HHg changes at speciﬁed sampling: birth

(fetal exposure), 6 months of exclusive breastfeeding, 36 months (weaning) and 60 months (pre-school). The distribution of HHg in

breastfed infants followed a pattern diﬀerent from their mothers. While mothers had the highest HHg concentrations at childbirth,

infants showed the highest HHg values at 6 months after the recommended full schedule (six shots) of immunization with TCV; after

that, the downward trend in HHg shown by children coincided with both weaning and less frequent vaccination period (5 years).

Extended lactation (up to 36 months) was not signiﬁcantly associated with HHg of infants or mothers; however, signiﬁcant association

(Spearman’s r) between maternal and infant HHg concentration was seen at birth (r = 0.3534; P = 0.001), 6 months (r = 0.4793;

P < 0.0001), 3 years (r = 0.0122; P = 0.012) and 5 years (r = 0.0357; P = 0.005). Maternal postpartum metabolic changes, infant devel-

opment and transitional diets and possibly Hg from TCV contribute to the asymmetry of HHg changes between mothers and children.

Ó 2007 Published by Elsevier Inc.

Keywords: Vaccines; Thimerosal; Hair-Hg; Breastfeeding; Fish consumption; Amazon; Neuro-motor development

1. Introduction

Current studies associating Hg exposure and neurode-

velopmental disorders (NDD) in children rely on the metal

concentrations in hair. Hair is composed of a pro tein com-

plex formed from amino acids that avidly binds to methyl-

Hg (MeHg). The assumption is that the occurrence of

NDD results from consuming ﬁsh contaminated with

MeHg, which avidly binds to hair (Cernichiari et al.,

2007). Indeed, epidemiologic studies dealing with MeHg

exposure from ﬁsh consumption have used HHg concen-

trations as a reliable indicator. However, during the ﬁrst

years of life, infants can be exposed to Hg in their changing

food sources: from breast milk (or formulas) to weaning

foods, and at a latter age, from other dietary sources (that

might include ﬁsh) when eating adult food. Young children

can also be exposed to other forms of organic Hg (ethyl-

mercury—EtHg) when immunized with thimerosal (Mar-

ques et al., 2007a).

Children of ﬁsh-eating populations of the Amazon

Basin are exposed to maternal MeHg via placental transfer

and breast milk (Barbosa et al., 1998; Barbosa and Do

rea,

1998; Marques et al., in press). In this context, the increase

in the risk of NDD has been strongly suggested to be asso-

ciated with MeHg naturally present in ﬁsh from the Ama-

zonian rivers (Grandj ean et al., 1999). The possibility of

0273-2300/$ - see front matter Ó 2007 Published by Elsevier Inc.

doi:10.1016/j.yrtph.2007.05.001

Corresponding author. Fax: +55 61 368 5853.

E-mail address: [email protected] (J.G. Do

rea).

www.elsevier.com/locate/yrtph

Available online at www.sciencedirect.com

Regulatory Toxicology and Pharmacology 49 (2007) 17–24

NDD raised by these studies has relied on HHg concentra-

tions (biomarkers of ﬁsh consumption). Indeed, variability

in HHg of riverine and Amerindian populations has been

correctly assumed to be a function of ﬁsh consumption.

Barbosa et al. (1995) have shown important diﬀerences in

urine and HHg concentrations due to occupational expo-

sure (gold miners) and ﬁsh consumption for native Amazo-

nians. However, we have recently raised the possibility

that, at least for the ﬁrst 6 months of life, HHg concentra-

tions may be inﬂuenced by the heavy immunization (with

TCV) schedule of urban populations ( Marques et al.,

2007a). Indeed, during the infant’s ﬁrst years, susceptibility

to modifying factors remains unknown, but physiolo gical

and dietary changes (breast milk, weaning and habitual

family diets) are likely to inﬂuence HHg changes.

Body retention of mercury and its hair uptake depend

on dietary and physiological factors. In children, there

are drastic physiological changes during the ﬁrst years

that aﬀect the toxic okinetics of Hg. Mercury is taken

up from the bloodstream during hair formation. New-

born hair essentially represents Hg exposure during fetal

development. During the ﬁrst 6 months, at least in

breastfed infants, the origi n of Hg is still maternal, but

it is not through the fetal circulation but enteral through

breast milk. Enteral exposure to Hg from weaning food

has not been explored for ﬁsh-eating populations, but

it is expected to be diﬀerent from an adult’s diet. How-

ever, for children with access to health services, another

source of Hg exposure has been unaccounted for in ﬁsh-

eating populations, that is, immunization with TCV

(Marques et al., in press).

Easy collection and preservation make the minimally

invasive HHg sampling a method of choice in evaluating

MeHg exposure from ﬁsh consumption. Indeed, HHg has

made it possible to estimate ﬁsh consumption more reliably

than with conventional dietary recall methods (Richardson

and Currie, 1993; Gosselin et al., 2006); it has been conve-

niently applied in diﬀerentiating occupational and environ-

mental (ﬁsh intake) exposures (Barbosa et al., 1995) and to

overcome diﬃculties of communication involving cross-

cultural studies of Amazonian Amer-Indians (Dorea

et al., 2005) and isolated co mmunities of riverine popula-

tions (Alves et al., 2006).

Ponce et al. (1998) compared Hg exposure estimates

based on self-reported ﬁsh intake and measured ﬁsh-Hg

concentrations and HHg concentrations; they showed bias

and random error as components of uncertainty regarding

HHg exposure estimates. Indeed, Canuel et al. (2006) dem-

onstrated signiﬁcant regional diﬀerences in HHg kinetics;

therefore, generalization of HHg models needs to take into

account not only the organic form of Hg and source of

exposure but also physiological diﬀerences and genetic

diversities of individuals within populations.

Both immunization (primary prevention) and breast-

feeding (secondary prevention) share desirable health out-

comes: prevention of infectious diseases (vaccines) and

long-term health CNS and general health indicators war-

ranted by breastfeeding. However, such health-related pro-

cedures are also the initial way infants and young children

are exposed to Hg (Marques et al., in press). Infant routes

of Hg exposure entail a varie ty of sources and a corre-

sponding chemical species: placenta (MeHg), breast mil k

(MeHg and inorganic Hg), TCV (EtHg) and weaning foods

(MeHg and inorganic Hg); each source of exposure is part

of a unique set of events at speciﬁc time which inﬂuences

substantially the toxicokinetics of Hg.

The underlying metabolic pathways attendant on the

changing anatomical and physiological functions of early

infancy, as well as the interactions among sources (Hg

forms, dos e and duration) make young children a heter-

ogeneous population in respect to the toxicokinetics of

Hg. Additionally, during this time there are special cir-

cumstances of intens e iatrogenic exposure during the

immunization with TCV that still prevails in some coun-

tries; these interactions are likely determinants of Hg

retention in children’ hair. The data collected for this

study were conceived for evaluating the neurodevelop-

ment of children exposed to pre-natal exposure Hg from

maternal ﬁsh consumption (Marques et al., 2007a).

When the research project started we wer e not aware

of the TCV issue in pediatric vaccines; this was brought

up during the revision process of the parent publication

(Marques et al., 2007a). Because we had all records of

immunization we could conceptualize the assessment of

thimerosal-Hg in the young children. Therefore, we took

advantage of our cohort to study longitudinal changes in

HHg of mothers and respective children from birth to

key ages of 6, 36 and 60 months.

2. Materials and methods

2.1. Sample description

The city of Porto Velho, capital of the state of Rondonia (West

Amazonia), has experienced signiﬁcant demographic changes with people

coming from many other Brazilian regions. During the last 30 years, it has

changed from a traditional Amazonian city to one under the impact of

heavy migration brought by agricultural projects in the south-west region

and especially the inﬂux of prospectors seeking alluvial gold along the

banks of the Rio Madeira basin. The present population has both tradi-

tional families that base their diets on ﬁsh and starchy foods and city

dwellers with more cosmopolitan food habits. In this changing environ-

ment, we investigated the health status of breastfed infants with special

reference to food habits and possible Hg exposure due to ﬁsh

consumption.

The research protocol was approved by the Ethics Committee for

Human Studies of the Universidade Federal de Rondonia and details have

already been published (Marques et al., 2007a,b).

2.2. Study protocol

Mothers were introduced to the study and invited to participate by a

nurse during their routine visits to the Pre-natal Clinics of three hospitals

in Porto Velho: Hospital de Base, Hospital Panamericano and Hospital

Regina Pacis. One-hundred and sixty potential participants received

plain-language information about the study and a written consent form

was presented and signed by the 155 that agreed to participate—115 were

eligible; the written consent stated that participation was voluntary, their

18 R.C. Marques et al. / Regulatory Toxicology and Pharmacology 49 (2007) 17–24

conﬁdentiality was assured and that they could withdraw from the study

at any time. Mothers (between the ages of 15 and 45 years) were selected

among those in good health, reporting no illness or complaints at the time

of the study and who were willing to breastfeed up to 6 months of age.

Excluding factors were occupational exposure to toxic chemicals and

hereditary neurological illnesses. While at the maternity wards, we col-

lected samples of hair from mothers and respective infants (fetal hair).

Hair strands were cut from the occipital region and placed in plastic bags,

with the root end stapled on a paper sheet; newborn hair was sampled in

suﬃcient amount. Hair samples were again taken at the follow-up visits at

6, 36 and 60 months.

2.3. Hair-Hg analysis

Sample preparation and analytical procedures were done according

to our standardized protocol for Hg determination in hair as described

elsewhere (Marques et al., 2007a). Hair analysis was done on the ﬁrst

centimeter closest to root end. Sample preparation and Hg determina-

tion were done according to routine procedures previously established

at the Universidade Federal do Rio de Janeiro (Bastos et al., 1998).

We followed routine procedures after adaptation of analytical protocol

used for Hg determination in previous studies. Brieﬂy, the hair samples

were comminuted with stainless steel scissors, weighed, and digested

before analysis. Human hair samples were washed with EDTA 0.01%,

dried in an oven at 50 °C, weighed and digested with 5 mL of

HNO

:H2SO

(1:1) and 4 mL of 5% KMnO

using a digestion block

at 80 °C for 40 min. The determination of total Hg in the digested sam-

ples was done by cold vapor atomic absorption spectrometry with a

ﬂow injection system-FIMS (CV-AAS, Perkin-Elmer—FIMS 400,

Ueberlingen, Germany).

All glassware used in the analytical protocol was washed clean,

rinsed with 5% EDTA and double distilled, and left to rest in 5%

HNO

overnight. Then it was rinsed again in double distilled water,

and dried at 100 °C for 12 h. Precision and accuracy of Hg determina-

tions were assured by the use of internal standards, use of triplicate

analyses of samples and certiﬁed reference materials (IAEA-085 and

086, Vienna, Austria) with recoveries of 92%. The limit of detection

for the procedure was determined at <0.01 lg/g and there was hair-

Hg determination below the limits.

2.4. Immunization vaccines and schedule

Infants up to 6 months of age received the full immunization scheme

recommended by the Brazilian immunization program. After that time

mothers were oriented about the importance of immunization and

followed recommendations of available pediatric services. Among the

vaccines taken by infants during the 5 years of the study some were pre-

served with 0.01% thimerosal (which metabolizes into EtHg). The Hg con-

centration of the doses delivered through vaccines was 25 lg Hg/0.5 mL.

The immunization schedule with TCV and respective intake of Hg as sta-

ted by manufacturers is summarized in Table 1. These were: hepatitis-B

(Korea Green Cross Corporation, Kiheung-Eup Yougin-Goon

Kiyunggi-Do, Korea; Euvax B injectable, LG Life Sciences, Jeonbuk-

Do, Korea), diphtheria, tetanus and pertussis-DTP (Triple Antigen,

Serum Institute of India Ltd, India; Vacina Trı

plice, Instituto Butanta,

o Paulo, Brazil), Hib: Haemophilus inﬂuenzae type b (HibTITER

Lederle-Praxis), Flu: inﬂuenza (VAXIGRIP, Pasteur Me

rieux Connaught,

Sao Paulo, Brazil). Children that were immunized against Hib within the

ﬁrst year (two doses) received the booster dose 6–12 months after the last

dose, otherwise received only one dose in the second year of life. The rec-

ommendation for the Flu vaccine is to be taken between the ages of 6 and

35 months in two doses of 0.25 mL (1 month apart) and one single dose

(0.5 mL) the following year; although the vaccines were taken the ages

given represent approximations of the recommended date.

The exposure to EtHg derived from vaccines was based on the current

national immunization program of the Ministry of Health of Brazil (Table

1). After 6 months only 20 of the 82 infants were given a fourth dose of

DTP between 9 and 12 months. We estimated breast-milk Hg exposure

from weight gain up to 6 months, when the infants were exclusively breast-

fed, and discussed it elsewhere (Marques et al., 2007a).

2.5. Statistical analysis

Of the 100 original enrolled mothers, only complete data of 82 mother–

infant pairs could be obtained at the end of the study (Marques et al., in

press). The statistical packages contained in Excel and Prism were used for

data summarization (means, standard deviation, changes in mean Hg con-

centrations) and correlation analysis. Repeated measurements analysis

was run to test sampling eﬀect on HHg on infant and maternal HHg con-

centrations; this statistical analysis was performed using SAS release 9.1.3

(SAS Institute Inc., Cary, North Carolina). We accepted a value of <0.05

as statistically signiﬁcant.

3. Results

A summary of the estimation of Hg forms in each of

the exposure media (TCV and breast milk) that can con-

tribute to HHg is shown in Table 1. During the ﬁrst 6

months, infants received up to six shots of TCV (and were

exposed to 150 lg Hg from thimerosal); the TCV corre-

sponded to Hepatitis B and DTP; subsequently the immu-

nization with TCV were for inﬂuenza and Hib with a

coverage ranging from 2.5 to 25% of the children. Age

of children corresponds to recommended vaccination

schedules, but only 6-month-olds had the full schedule

of immunization and the corres ponding EtHg exposure.

It is important to note that the exposure to TCV-Hg from

the ﬁrst vaccine taken at birth (0 day) is the highest and

the most challenging dose; at this time the small body

mass of neonates takes an impact equivalent to the double

doses (of DTP and hepatitis B) at 6 and 12 months. The

estimation of total Hg exposure from breast milk was

possible only up to 6 months when mothers were closely

monitored and psychologically supported to keep on with

exclusive breastfeeding; indeed, most mothers (66%)

breastfed for 12 months and some reported to breastfeed

up to 60 months (Fig. 1). However, the eﬀect of lactation

on HHg concentrations of mothers (excreting Hg for 60

months) and children (absorbing Hg) over the 36 months

is illustrated in Fig. 1: there was no statistically signiﬁcant

association between HHg and length of lactation, either

for mothers or for children.

Table 2 summarizes HHg concentrations of children and

mothers during the study period. The higher mean HHg at

6 mo nths coincided with both Hg exposure from breast

milk and the heavy vaccine schedule. Indeed, the distribu-

tion of HHg in infants follo wed a diﬀerent pattern of their

mothers (Fig. 2). After 6 months, exclusively breastfed

infants receiving a full load of immunization (ﬁve types

of vaccine, 150 lg Hg) showed an expansion of HHg con-

centrations greater than HHg at birth and at 3 and 5 years

of age. HHg in their respective mothers showed the highest

concentrations at childbirth, but a similar pattern at 6

months and 3 years later. Fr equency of ﬁsh consumption

(0–1 servings/week vs. compared to 2–7 servings/week)

was presented and discussed previously (Marques et al.,

R.C. Marques et al. / Regulatory Toxicology and Pharmacology 49 (2007) 17–24 19

2007a); in these urban mothers ﬁsh consumption depended

on the species available at market (season and price are

strong determinants).

Fig. 3 can be interpreted as diﬀerence in Hg transfer

rates between placenta and mammary gland. The proﬁle

of ratios of mother:infant HHg clearly indica te that moth-

ers’ decreases while infants’ increases. However, the associ-

ation between maternal and infant HHg (Spearman’s r)

illustrated in Fig. 4 was signiﬁcant at all times: statistical

signiﬁcant association between maternal and infant HHg

concentration was seen at birth (r = 0.3534; P = 0.001), 6

months (r = 0.4793; P < 0.0001); Spearman’s r values at 3

years (r = 0.0122; P = 0.012) and 5 years (r = 0.0357;

P = 0.005) although signiﬁcant were low and indicative

of less perfect correlations. Indeed, sampling time was sta-

Table 1

Children immunization schedule, type of vaccines, and corresponding EtHg intake (as Hg) during the ﬁrst 60 months

Age (months)

Vaccine Breast milk

% coverage Type

lg Hg/dose lg Hg/day

0 100 Hp-B 25.0 0

1 100 Hp-B 25.0 30.83

2 100 DTP 25.0 44.65

3 100 — — —

4 100 DTP 25.0 91.80

5 100 — — —

6 100 DTP + Hp-B 50.0 111.90

Total Hg injected 150.0

8 2.4 Hib 25.0 NE

10 8.5 Hib 25.0 NE

12 8.5 Hib + Flu 50.0 NE

15 25.5 DTP 25 NE

24 10 Flu 12.5 NE

36 5.0 Flu 12.5 NE

48 2.4 Flu 25 NE

NE, not estimated.

All 82 infants had a full immunization schedule during the ﬁrst 6 months; the fourth dose of DTP was taken after the ﬁrst year only by 25.5%. The Hib

and inﬂuenza vaccines taken after 6 months were at the recommended age by the speciﬁed percentage of the children; the age of immunization after 6

months are approximations based on current recommendations.

Hp-B: Hepatitis B (0.01% thimerosal/dose, Korea Green Cross Corporation, Kiheung-Eup Yougin-Goon Kiyunggi-Do, Korea; Euvax B injectable,

0.01% thimerosal [LG Life Sciences, Jeonbuk-do, Korea]); DTP (Serum Institute of India Ltd; Vacina Trı

plice, 0.01% thimerosal/dose [Instituto Butanta,

o Paulo, Brazil]); Hib: Haemophilus inﬂuenzae type b (HibTITER

, 0.01% thimerosal/dose, Lederle-Praxis); Flu: inﬂuenza (VAXIGRIP 0.01%

thimerosal/dose, Pasteur Me

rieux Connaught, Sao Paulo, Brazil). Children that were immunized against Haemophilus inﬂuenzae type b (Hib) within the

ﬁrst year (two doses) received the booster dose 6–12 months after the last dose, otherwise received only one dose in the second year of life. The

recommendation for the anti-ﬂu vaccine is to be taken between the ages of 6 and 35 months in two doses (1 month apart) of 0.25 mL and one single dose

(0.5 mL) the following year; although the vaccines were taken the ages given represent approximations of the recommended date.

Integrated total Hg intake adapted from Marques et al. (2007a); we used the data of breast milk-Hg concentrations (adapted from Dorea (2004))to

estimate Hg exposure during breastfeeding: infant mean weight · mean daily breast milk consumption (140 mL/kg) · number of days · mean total Hg

concentration in breast milk (1.9 lg/L).

0 10 20 30 40 50 60

Length of lactation, months

Maternal hair [Hg] μg/g

0 10 20 30

Length of lactation, months

Infant hair [Hg] μg/g

0 10 20 30 40 50 60

100

Length of lactation, months

Distribuiton, %

Fig. 1. Length of lactation and its impact on hair-Hg concentrations of mothers and infants.

Table 2

Mean hair-Hg concentrations (SD) in children and respective mothers

during the ﬁrst 5 years

Children Mothers

N 82 82

Hair [Hg] lg/g, birth 2.4 (3.0) 7.4 (8.7)

Hair [Hg] lg/g, 6 months 3.8 (5.5) 3.2 (3.8)

Hair [Hg] lg/g, 36 months 2.6 (3.7) 2.8 (3.2)

Hair [Hg] lg/g, 60 months 2.6 (3.5) 2.6 (3.0)

20 R.C. Marques et al. / Regulatory Toxicology and Pharmacology 49 (2007) 17–24

tistically signiﬁcant for mothers (<0.0001) but not for chil-

dren (0.0987).

The distribution of HHg in infants followed a pattern

diﬀerent from their mothers. While mothers had the highest

concentrations of HHg at birth, breastfed children experi-

ence the highest HHg concentrations after the heaviest

schedule (six shots) of immunization with TCV. Extended

lactation (>36 months) was not signiﬁcantly associated

with changes in HHg of infants or mothers; both maternal

and children HHg followed a downward trend.

4. Discussion

The proﬁles of distribution of maternal and infant HHg

concentrations during the ﬁrst 5 years revealed diﬀerences

in type of Hg exposure. The chronology of infant hair sam-

pling matched events associated with diet (that included

breastfeeding) and immunization schedules. We found a

clear trend in infant HHg coinciding with vaccine schedule

but cannot prove a cause–eﬀect relationship. The intercur-

rence of TCV-EtHg and the transitional weaning diets may

have co ntributed to the pattern of infant HHg changes.

Diﬀerent cumulative distribution of HHg (mother:infant)

ratios extracted information in response to short-term

changes in source and dose of Hg exposure: the eﬀects of

the short-term but heavy schedule of immunization with

TCV were likely modifying facto rs.

Based on HHg concentrations, the urban mothers in this

study had a much lower consumption of ﬁsh than Amazo-

nian ‘‘ribeirinho’’ women; we reported before that 57/82 of

these mothers ate at most one ﬁsh meal a week showed a

lower mean hair-Hg concentration than mothers that con-

sumed more than 2 times a week (Marques et al., in press).

HHg concentrations were lower than those observed for

0 5 10 15 20 25 30

100

Birth

36m

60m

Infant hair [Hg], μg/g

Distribution, %

0 5 10 15 20 25 30

100

Birth *

36m

60m

Maternal hair [Hg], μg/g

Distribution, %

Fig. 2. Frequency distribution of maternal and infant hair-Hg concentrations at speciﬁed sampling times; maternal outlier:

=62lg Hg/g.

0 10 20 30

100

Birth

Six months

Ratios of maternal:infant hair-Hg

Distribution, %

a a'

b b'

Fig. 3. Frequency distribution of maternal:infant hair-Hg ratios and

outliers: a = 52, a

= 158; b = 140, b

= 151.

0 5 10 15 20 25 30

Maternal hair [Hg] μg/g Maternal hair [Hg] μg/g

Maternal hair [Hg] μg/g

(Birth)

Infant hair [Hg] μg/gInfant hair [Hg] μg/g

0 10 20 30

(Six months)

0 10 20 30

(36 months)

0 10 20 30

(60 months)

Fig. 4. Scatter plot of correlation of infant and maternal hair-Hg concentrations at speciﬁed sampling.

R.C. Marques et al. / Regulatory Toxicology and Pharmacology 49 (2007) 17–24 21

‘‘ribeirinho’’ women of the Rio Madeira in 1991 by Barbosa

et al. (1998). Mean HHg concentrations of studies in ﬁsh-eat-

ing Amazonians have been reported to vary between 6.5 and

34.2 ppm (Barbosa et al., 2001); however, adjusting for

inherent HHg variability or long-term studies of HHg

changes (within individuals) are rare. Because of HHg vari-

ability, the eﬀects of pregnancy and lactation may not impact

the mean HHg deposition in Amazonian high ﬁsh eaters

(Barbosa et al., 2001) but the relative changes (percent

adjusted) within mothers show that it decreases during preg-

nancy (Barbosa et al., 1998); thus, in agreement with the

present ﬁndings. The percent decline in maternal HHg con-

centrations observed in this and previous study (Barbosa

et al., 1998) may reﬂect the metabolic changes associated

with lactation which includes transfer of Hg to milk.

Events related to pregnancy and lactation accelerate

maternal-Hg metabolism ( Greenwood et al., 1978), but Hg

transfer rates in utero (pregnancy) and ex utero (breast-feed-

ing) diﬀer depending on the organic (MeHg) or inorganic Hg

form (Dorea, 2004). As discussed elsewhere (Dorea, 2004)

both Hg forms are equally transferred through milk but

MeHg is more readily transferred across the placent a than

inorganic Hg. Indeed, inorganic Hg absorption through

milk is not a signiﬁcant source of Hg exposure to breastfed

infants (Sandborgh-Englund et al., 2001). But, compared

to inorganic Hg, Bjornberg et al. (2005) reported that MeHg

seems to contribute more to infant exposure via breast milk;

additionally, total Hg in breast milk decreased signiﬁcantly

from day 4 to 6 weeks, remaining unchanged thereafter

(Bjornberg et al., 2005 ). Studies of lactating mothers (after

the accidental poisoning in Iraq) showed that the milk-Hg

was 5% of blood-Hg but the organic fraction of milk-Hg

was only 3% of blood-MeHg (Bakir et al., 1973); this indi-

cates that the placenta plays a greater role in Hg transfer than

the mammary gland (Bjornberg et al., 2005). The HHg ratios

clearly showed diﬀerences in Hg transfer rates between preg-

nancy and lactation (Fig. 3).

In the present study, the length of breastfeeding does not

seem to be a signiﬁcant determ inant of HHg concentrations

of mother s or infants, thus showing that the mammary gland

is a more eﬀective barrier for Hg transfer than the placenta.

Indeed, we had showed that during lactation mothers

increased HHg to pre-pregnancy levels (Barbosa et al.,

1998). Studies relating length of breastfeeding with HHg

concentrations of mothers and infants are rare and results

of correlation analysis are not always concurrent. There

are diﬀerences between populations: infants’ HHg and dura-

tion of breastfeeding were signiﬁcant correlated in the Amer-

Indians (of eastern Amazonia) but not in the ‘‘ribeirinhos’’ of

Rio Madeira studied by Barbosa et al. (1998).

The Hg found in hair of breastfed infants comes from

both exogenous (breast milk) and endogenous (metabolic

turnover of fetal tissues) sources; because of postnatal

hair change and growth, the contribution of residual fetal

tissue may become negligible at a certain age: nonetheless,

HHg in breastfed infants (if immunized with thimerosal-

free vaccines) originates from maternal sources. This close

association between maternal and children HHg remains

longer after breastfeeding: at weaning and at 5 years

(Fig. 2). Irresp ective of the level of HHg the mother–

infant dyad remains in very close association; this indi-

cates that dietary characteristics (type of ﬁsh or frequency

of consumption) are strong within families (Fig. 2). These

observations are in agreement with results obtained with

high ﬁsh-eating population living at the banks of the

Rio Madeira. Barbosa et al. (1998) reported a statistically

signiﬁcant correlation between ‘‘ribeirinho’’ mothers and

breastfed infants’ HHg of older age. This signiﬁcant cor-

relation was reported by some (in Madrid—Gonzalez

et al., 1985) but not by others (Fujita and Takabatake,

1977). Nevertheless, our ﬁnding of signiﬁcant association

between maternal and fetal hair is in agreement with stud-

ies in other parts of Amazonia (Mohan et al., 2005)and

in low ﬁsh-eating mothers of other countries (

Sikorski

et al., 1986; Lindow et al., 2003).

The mean HHg values of our breastfed children are

lower than the means reported for 1-year-olds of several

Eastern Amazonian communities of the Rio Tapajo

s(Pin-

heiro et al., 2007 ). Although there are no studies of infant

HHg (due to breastfeeding and TCV) our mean values

(Table 2) are within HHg concentrations predicted from

Redwood et al. (2001) TCVl-exposure model. All infants

in our study received a substantial load of parenteral EtHg

concentrated in the ﬁrst 6 months; only a small percentage

(2.5–25%) of children received an equivalent load

(175 lg Hg) distributed over 42 months at a much older

age (12–48 months) and larger body weight. The proﬁle

of the mother:infant HHg ratios indicates that the breast-

fed infants’ hair retains more Hg than the fetus (Fig. 4 );

however, since we do not have speciation of Hg chemical

forms (EtHg or MeHg), it can not be solely attributed to

TCV. Our ﬁndings suggest that the signiﬁcant association

between mothers’ and infants’ HHg at all sampling times

reﬂect the family environmental exposure through ﬁsh-Hg.

Neonate metabolic pathways and attendant excretion

rates of Hg vary according to stage of development

mainly because of diﬀerences in gut development and bile

secretion as well as source of Hg exposure. The wider

distribution (greater variability) of infant HHg at 6

months can be attributed to the fact that in addition

to Hg from breast milk, HHg values can be inﬂuenced

by the EtHg breakdown from TCV, thus increasing

organic Hg exposure. Predictive models of hair-Hg con-

centrations in infants have assumed low or no Hg excre-

tion up to 6 months (Redwood et al., 2001). Therefore,

positive Hg balance in infants contrasting with negative

balance of mothers can be interpreted from Fig. 4. Nev-

ertheless, additional research is necessary to understand

the relationship between infant early development

(weight gain and tissue diﬀerentiation), and diﬀerences

in the metabolism of Hg from such distinct sources—

Hg intrinsic to breast milk and parenteral EtHg from

TCV. The physiological barriers that breast-milk Hg

has to cross are bypassed by parenteral-administered

22 R.C. Marques et al. / Regulatory Toxicology and Pharmacology 49 (2007) 17–24

thimerosal-EtHg. This particular aspect of HHg change

was discussed in the parent publication (Marques et al.,

2007a).

After exclusive (up 6 months) and extended breastfeeding

(36 months), infant mean HHg values declined toward the

maternal values; this reﬂects both, decreased Hg exposure

from weaning foods and a greatly reduced frequency of

TCV-EtHg exposure (Table 1). Overall, the decrease in

infant HHg during weaning was relatively less compared to

maternal HHg; but the mean HHg of infants at 5 years is

equivalent to maternal HHg, thus possibly reﬂecting expo-

sure to the same dietary Hg sources . Since infants were

exposed to higher doses of EtHg during the ﬁrst 6 months,

infant’s increase in HHg may result from higher transfer

rates of EtHg-to-hair compared to subsequent sampling

periods. HHg speciation that could diﬀerentiate MeHg from

EtHg would be necessary to ascertain the origin of total

HHg.

Fluctuation in maternal HHg concentrations has been an

unveriﬁed event that might occur as a result of altered metab-

olism during pregnancy. Because the epidemiological evi-

dence suggests a causal link between exposure to ﬁsh-Hg

and NDD, avoidance of ﬁsh consumption during pregnancy

and lactation has been warranted for NDD prevention

(Ronchetti et al., 2006). The Harvard Center for Risk

Analysis panel used maternal HHg to quantify the impact

of pre-natal MeHg chronic exposure on infant cognitive

development; the panel concluded that MeHg exposure

suﬃcient to increase maternal HHg by 1 lg/g decreases

intelligence quotient by 0.7 points (Cohen et al., 2005).

Therefore, ﬂuctuation in maternal HHg is an important issue

with implications for public he alth policies.

The strengths of this study include its prospective design

and the high rate of retention of mother–infant pairs at key

chronological sampling: birth (fetal exposure), 6 months of

exclusive breastfeeding, 36 months (weaning) and 60 months

(pre-school); additionally we identiﬁed input of other

organic Hg sources. However, our limitation in analytical

capability to determine the TCV-EtHg in hair is an impor-

tant constraint in interpreting these aspects of the results.

A common problem faced by Hg toxic eﬀects or susceptibil-

ity at sub-clinical levels is the diﬃculty of accurately recon -

structing responses to sources after the exposure event.

This is especially the case with young infants and accompa-

nying changes in diets intertwined with attendant sources

of Hg (which might include extrinsic EtHg) such as in the

present study; additionally, there are ethical considerations

related to breastfeeding and immunization that limit group

(without such life-saving features) comparisons, thus mak-

ing exposure studies uniquely diﬃcult.

5. Conclusion

Unveriﬁed changes in maternal and infant HHg are

important issues relating this marker to studies of expo-

sure, eﬀects and susceptibility. Maternal postpartum meta-

bolic changes, infant development and transitional diets

and possibly Hg from TCV contribute to the asymmetry

of HHg changes between mothers and children.

Acknowledgments

We are greatly in debt to the mothers for their participa-

tion in the study, to Prof. Edina Myazaki (for helping with

statistical an alysis), to the staﬀ and Directors (Marine

sR.

dos Santos Cezar, Tereza Cristina Ramos, Daniele Brasil,

Katia Wendt, Katiane G. Branda

o, Laura Jane Marques)

of the Hospitals (Hospital de Base Ary Pinheiro, Hospital

Panamericano and Hospital Regina Pacis), Dr. Cezar

Augusto Bezerra B. de Arau

jo (State Coordinator of the

PNI-MS), the staﬀ of the Fundac¸a

o Universidade Federal

de Rondo

nia and the Universidade Federal do Rio de Ja-

neiro. This work was supported by United Nations Educa-

tional, Scientiﬁc and Cultural Organization—UNESCO,

Ministe

rio da Sau

de do Brasil (SC27824/2005/

914BRA2000 Decit PRODOC) and The National Re-

search Council of Brazil-CNPq (PNOPG project-55.0882/

01-4; PPG7, project-556985/2 005-2).

References

Alves, M.F.A., Fraiji, N.A., Barbosa, A.C., Lima, D.S.S., Souza, J.R.,

rea, J.G., Cordeiro, G.W.O., 2006. Fish consumption, mercury

exposure and serum antinuclear antibody in amazonians. Int. J.

Environ. Health 16, 255–262.

Bakir, F., Damluji, S., Amin-Zair, H., et al., 1973. Methylmercury

poisoning in Iraq. An interuniversity report. Science 181, 230–

240.

Barbosa, A.C., Do

rea, J.G., 1998. Indices of mercury contamination

during breast feeding in the Amazon Basin. Environ. Toxicol.

Pharmacol. 6, 71–79.

Barbosa, A.C., Boischio, A.A., Ponce, G.E., Ferrari, I., Gonc¸alves, A.S.,

Silva, P.R.M., Cruz, T.M.E., 1995. Mercury contamination in the

Brazilian Amazon. Environmental and occupational aspects. Water

Air Soil Pollut. 80, 109–121.

Barbosa, A.C., Silva, S.R., Dorea, J.G., 1998. Concentration of mercury

in hair of indigenous mothers and infants from the Amazon basin.

Arch. Environ. Contam. Toxicol. 34, 100–105.

Barbosa, A.C., Jardim, W., Dorea, J.G., Fosberg, B., Souza, J., 2001. Hair

mercury speciation as a function of gender, age, and body mass index

in inhabitants of the Negro River Basin, Amazon, Brazil. Arch.

Environ. Contam. Toxicol. 40, 439–444.

Bastos, W.R., Malm, O., Pfeiﬀer, W.C., Cleary, D., 1998. Establishment

and analytical quality control of laboratories for Hg determination in

biological and geological samples in the Amazon, Brasil. Cie

nc. Cult.

50, 255–260.

Bjornberg, K.A., Vahter, M., Berglund, B., Niklasson, B., Blennow, M.,

Sandborgh-Englund, G., 2005. Transport of methylmercury and

inorganic mercury to the fetus and breast-fed infant. Environ. Health

Perspect. 113, 1381–1385.

Canuel, R., de Grosbois, S.B., Atikesse, L., Lucotte, M., Arp, P.,

Ritchie, C., Mergler, D., Chan, H.M., Amyot, M., Anderson, R.,

2006. New evidence on variations of human body burden of

methylmercury from ﬁsh consumption. Environ. Health Perspect.

114, 302–306.

Cernichiari, E., Myers, G.J., Ballatori, N., Zareba, G., Vyas, J., Clarkson,

T., 2007. The biological monitoring of prenatal exposure to methylmer-

cury. Neurotoxicology. doi:10.1016/j.neuro.2007.02.009, Epub ahead of

print.

R.C. Marques et al. / Regulatory Toxicology and Pharmacology 49 (2007) 17–24 23

Cohen, J.T., Bellinger, D.C., Shaywitz, B.A., 2005. A quantitative analysis

of prenatal methyl mercury exposure and cognitive development. Am.

J. Prev. Med. 29, 353–365.

Dorea, J.G., 2004. Mercury and lead during breast-feeding. Br. J. Nutr.

92, 21–40.

Dorea, J.G., Barbosa, A.C., Ferrari, I., De Souza, J.R., 2005. Fish

consumption (hair mercury) and nutritional status of Amazonian

Amer-Indian children. Am. J. Hum. Biol. 17, 507–514.

Fujita, M., Takabatake, E., 1977. Mercury levels in human maternal and

neonatal blood, hair and milk. Bull. Environ. Contam. Toxicol. 18,

205–209.

Gonzalez, M.J., Rico, M.C., Hernandez, L.M., Baluja, G., 1985. Mercury

in human hair: a study of residents in Madrid, Spain. Arch. Environ.

Health 40, 225–228.

Gosselin, N.H., Brunet, R.C., Carrier, G., Bouchard, M., Feeley, M., 2006.

Reconstruction of methylmercury intakes in indigenous populations

from biomarker data. J. Exp. Sci. Environ. Epidemiol. 16, 19–29.

Grandjean, P., White, R.F., Nielsen, A., Cleary, D., de Oliveira Santos,

E.C., 1999. Methylmercury neurotoxicity in Amazonian children

downstream from gold mining. Environ. Health Perspect. 107,

587–591.

Greenwood, M.R., Clarkson, T.W., Doherty, R.A., Gates, A.M., Amin-

Zaki, L., Elhassani, S., Majeed, M.A., 1978. Blood clearance half-

times in lactating and nonlactating members of a population exposed

to methylmercury. Environ. Res. 16, 48–54.

Lindow, S.W., Knight, R., Batty, J., Haswell, S.J., 2003. Maternal and

neonatal hair mercury concentrations: the eﬀect of dental amalgam.

BJOG 110, 287–291.

Marques, R.C., Do

rea, J.G., Bastos, W.R., Rebelo, M.F., Fonseca, M.F.,

Malm, O., 2007a. Maternal mercury exposure and neuro-motor

development in breastfed infants from Porto Velho (Amazon), Brazil.

Int. J. Hyg. Environ. Health 210, 51–60.

Marques, R.C., Do

rea, J.G., Bastos, W.R., Fonseca, M.F., Malm, O., in

press. Hair mercury in breast-fed infants exposed to thimerosal-

preserved vaccines. Eur. J. Pediatr., (doi: 10.1007/s00431-006-0362-2).

Mohan, S., Tiller, M., van der Voet, G., Kanhai, H., 2005. Mercury

exposure of mothers and newborns in Surinam: a pilot study. Clin.

Toxicol. 43, 101–104.

Pinheiro, M.C., Crespo-Lopez, M.E., Vieira, J.L., Oikawa, T., Guimara

es,

G.A., Arau

jo, C.C., Amoras, W.W., Ribeiro, D.R., Herculano, A.M.,

do Nascimento, J.L., Silveira, L.C., 2007. Mercury pollution and

childhood in Amazon riverside villages. Environ. Int. 33, 56–61.

Ponce, R.A., Bartell, S.M., Kavanagh, T.J., Woods, J.S., Griﬃth, W.C.,

Lee, R.C., Takaro, T.K., Faustman, E.M., 1998. Uncertainty analysis

methods for comparing predictive models and biomarkers: a case study

of dietary methyl mercury exposure. Regul. Toxicol. Pharmacol. 28,

96–105.

Redwood, L., Bernard, S., Brown, D., 2001. Predicted mercury concen-

trations in hair from infant immunizations: cause for concern.

Neurotoxicology 22, 691–697.

Richardson, G.M., Currie, D.J., 1993. Estimating ﬁsh consumption rates

for Ontario Amerindians. J. Expo. Anal. Environ. Epidemiol. 3, 23–38.

Ronchetti, R., Zuurbier, M., Jesenak, M., Koppe, J.G., Farah Ahmed, U.,

Ceccatelli, S., Pia Villa, M., 2006. Children’s health and mercury

exposure. Acta Paediatr. 95, 36–44.

Sandborgh-Englund, G., Ask, K., Belfrage, E., Ekstrand, J., 2001.

Mercury exposure in utero and during infancy. J. Toxicol. Environ.

Health 63, 317–320.

Sikorski, R., Pszkowski, T., Szprengier-Juszkewicz, T., 1986. Mercury in

neonatal hair. Sci. Total Environ. 57, 105–110.

24 R.C. Marques et al. / Regulatory Toxicology and Pharmacology 49 (2007) 17–24

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Maternal fish consumption in the nutrition transition of

the Amazon Basin: Growth of exclusively breastfed

infants during the first 5 years

Rejane C. Marques

; José Garrofe Dórea

; José V. E. Bernardi

; Wanderley R.

Bastos

; Olaf Malm

Universidade Federal de Rond nia, Porto Velho, Brazil

University of Brasilia, Nutrition, Faculdade de Ciencias da Sa de, Brasilia,

70919-970 Brazil

First Published: July 2008

To cite this Article: Marques, Rejane C., Dórea, José Garrofe, Bernardi, José V.

E., Bastos, Wanderley R. and Malm, Olaf (2008) 'Maternal fish consumption in the nutrition transition of the Amazon

Basin: Growth of exclusively breastfed infants during the first 5 years', Annals of Human Biology, 35:4, 363 — 377

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Annals of Human Biology, July–August 2008; 35(4): 363–377

ORIGINAL ARTICLE

Maternal fish consumption in the nutrition transition of the

Amazon Basin: Growth of exclusively breastfed infants

during the first 5 years

REJANE C. MARQUES

, JOSE

GARROFE DO

REA

JOSE

V. E. BERNARDI

, WANDERLEY R. BASTOS

, & OLAF MALM

Universidade Federal de Rondo

nia, Nursing, Porto Velho, Porto Velho, Brazil, and

University of Brasilia, Nutrition, Faculdade de Ciencias da Sau

de, C.P. 04 322, Brasilia,

70919-970 Brazil

(Received 31 January 2008; revised 18 March 2008; accepted 31 March 2008)

Abstract

Background: Changes in fish-eating habits due to rapid urbanization in Western Amazon was used as

model to investigate whether maternal fish-intake rate impacts on children’s weight and height during

the first 5 years.

Aim: The study examined the growth of 82 breastfed children, and maternal fish consumption

(hair mercury concentrations, HHg) during pregnancy and lactation.

Subjects and methods: Fish consumption in mothers and children was estimated through HHg.

The children were measured and weighed at birth and at 6 (exclusive breastfeeding), 36 and

60 months.

Results: Fish consumption rate (HHg) had no significant impact on children’s growth at the specified

ages (p ¼ 0.35). After 6 months of exclusive breastfeeding, children had the highest proportion of

Z-scores <À1 SD; however, weaning (with extended breastfeeding) had a substantial impact in moving

up the attained growth at 3 years. The duration of breastfeeding was significantly correlated with

attained Z-scores for weight-for-age (r ¼ 0.26; p ¼ 0.02) and weight-for-height (r ¼ 0.22; p ¼ 0.04) but

not for height-for-age. At 3 years most children had improved Z-scores (>À1 SD) for height-for-age

(70/82), weight-for-age (74/82) and weight-for-height (74/82). At 5 years, all but one child attained

Z-scores >À1.

Conclusion: The apparently good nutritional status of subjects is more likely due to a well balanced diet

composition than to only one dietary protein source – fish.

Keywords: Nutrition transition, Amazon, growth, breastfeeding, fish

Correspondence: Jos Garrofe Do

rea, University of Brasilia, Nutrition, Faculdade de Ciencias da Sau

de, C.P. 04 322, Brasilia,

70919-970, Brazil. E-mail: [email protected]

ISSN 0301–4460 print/ISSN 1464–5033 online ß 2008 Informa UK Ltd.

DOI: 10.1080/03014460802102495

Downloaded By: [Dórea, José Garrofe] At: 14:40 24 July 2008

Introduction

The population that inhabits the Amazon Basin shows a wide diversity: Currently, native

peoples living traditional lifestyles (in dense forests) contrast with a new urbanization profile.

Piperata (2007) has recently described differences between traditional lifestyles and those found

among the new Amazonian populations. The more urbanized environment caused by recent

Amazonian growth is exemplified in the city of Porto Velho. Gradually replacing riverside

communities, the new inhabitants no longer depend on the traditional dominance of fish as the

source of animal protein, but instead use markets for their food supply. This fast urbanization

has changed the traditional food supply chain and, as a consequence, the typical diet has shifted

away from high fish consumption (Dorea 2004) to dependency on industrial or mass processed

foods (Piperata 2007). The key challenge in studies of nutrition transition is the ability to

characterize which dietary item has substantially changed and its impact on health.

Fish is an abundant natural resource in Amazonian Rivers that is well utilized by riverside

populations (Dufour 1991); in traditional communities its consumption is high and this

tends to increase the farther these communities are from urban centres (Alves et al. 2006). In

the context of traditional riverine diet, the high protein content of fish balances high starchy

food consumption (Dorea 2004). Fish are a good source of sulphur amino acids and

bioavailable iodine, both crucial to counterbalance cassava goitrogens and low iodine foods

produced in iodine-depleted soils of tropical rain forests (Dorea 2004). Indeed, dietary fish

enhances absorption of zinc (Garcia-Arias et al. 1993) and iron from plant foods (Layrisse

et al. 1990; Gibson and Hotz 2001); dietary fish has been positively associated with women’s

iodine status (Zollner et al. 2001) and children’s ferritin stores (Michaelsen et al. 1995;

Gunnarsson et al. 2007). Additionally, Amazonian fish is a good source of selenium (Dorea

et al. 1998), known to counteract the toxic effects of Hg. Other essential nutrients, such as

zinc, are significantly higher in protein foods like fish when compared to food with low

protein content (Terres et al. 2001).

As a specific source of omega-3 polyunsaturated fatty acids (Inhamuns and Bueno Franco

2001), fish is nutritionally important for people living on cassava-dominant diets, especially

during breastfeeding (Rocquelin et al. 1998). Tanzanian women with high intakes of

freshwater fish (as the only animal lipid source) had milk arachidonic acid (AA) and

docosahexaenoic acid (DHA) contents that were well above present recommendations for

infant formulae (Muskiet et al. 2006); such findings inspired Muskiet et al. to hypothesize

that the ‘optimal homeostasis’ of AA and DHA is lacking in Western diets and is causing

subtle signs of unbalanced maternal glucose homeostasis. Indeed, breast-milk DHA of

women from the marine region was higher compared to other regions and twice as high as

any reported previously; it was comparable to the amounts found in the milk of women fed

fish oil (Ruan et al. 1995).

Fish protein has good biological value (Sikka et al. 1979) that is well utilized by children

(Hofvander 1973); porridge containing dried fish has been successfully used to treat

undernourished children (Greco et al. 2006). It is not surprising that per se, addition of fish

powder supported growth just as well as a nutritional supplement fortified with vitamins and

minerals (Lartey et al. 1999). The effects of maternal fish consumption on infant growth may

start early during pregnancy; Olsen et al. (1993) noticed that the weight and length of the

newborn increased with the frequency of seafood meals consumed in pregnancy. After

reporting that infant size at birth increased with fish consumption, Thorsdottir et al. (2004)

hypothesized that constituents of fish and fish oil might affect birth size. Indeed, increasing

fish intake during pregnancy might increase foetal growth rate (Rogers et al. 2004) and

cognitive functions (Hibbeln et al. 2007) of British infants.

364 R. C. Marques et al.

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Fish are complex nutrient-dense foods with exceptional functional characteristics:

Increasing maternal fish intake during pregnancy from once a week to 2.5 times per week

decreased the risk of eczema at age 1 year by 37%, and the risk of positive skin prick test at

age 6 years by 35% (Romieu et al. 2007). Furthermore, regular fish consumption in infancy

(before age 1) is associated with a reduced risk of allergic disease and sensitization to food

and inhalant allergens during the first 4 years of life (Kull et al. 2006).

One underappreciated problem in dietary surveys is error in recalling meal size. This has

been documented in several settings where portions and cultural standards could be

favourable; Nelson et al. (1996) showed that small portion sizes tended to be overestimated,

and large portion sizes underestimated. That is the reason many studies use intake biomarkers

to evaluate the extent of protein misreporting (Kipnis et al. 2002; Subar et al. 2003). Using

hair mercury (HHg) we accomplish reduction of intraindividual and interindividual variability;

this is particularly important in longitudinal studies subjected to seasonal variations in urban

socio-economically limited populations. These variations are amply documented in the

epidemiologic literature (Wu et al. 1986). The methylmercury (MeHg) consumed in fish is

easily absorbed, binds to protein sulphydril groups, and accumulates preferentially in human

hair; the proportion of blood to HHg is estimated at 250 times (Clarkson 2002). This

particular characteristic of hair, coupled with its easy collection and preservation, makes HHg

a first-choice marker of fish consumption in Amazonian populations (Barbosa et al. 1997,

1998; Dorea et al. 2003); the attendant changes in fish consumption rates for native

Amer-Indians have been successfully assessed by HHg determination (Dorea et al. 2005a,b).

Mean daily fish consumption rates of subsistence communities from the Rio Tapajo

s have

shown a variation of 115–171 g day

À1

(Passos et al. 2007); this is in close agreement with

170 g day

À1

derived from HHg concentrations of Rio Negro mothers (Dorea et al. 2003).

It should be noticed that that carnivorous fishes are more likely to have higher mercury content

than non-carnivorous fishes (Dorea 2003).

The importance of adequately supplying protein during pregnancy and lactation to ensure

full reproductive success is well recognized. Traditionally Amazonians have relied heavily on

fish to complement their starch-based diet (Giugliano et al. 1978). Population studies

dealing with nutrition transition in the Amazon are rare, and the most recent one has

focused on nutritional outcomes (Piperata 2007). In a previous publication we reported

mothers as moderate or high fish consumers (Marques et al. 2007); however, our present

construct is not typical of a nutrition survey. This study was created to test a selected aspect

(shift of fish consumption) in the nutrition transition in the Amazon Basin and its impact on

growth of breastfed children.

Materials and methods

Background

The nutrition transition of Porto Velho is typical of many conurbations along the most

remote Brazilian frontier. Agricultural projects and a gold rush have changed the face of this

Rio Madeira city in the last 30 years, attracting people from different parts of the country.

Incomers brought different food habits, and rapid urbanization accompanied social and

economic trends that impacted on fish consumption. During the last 30 years the city of

Porto Velho, capital of the state of Rondonia (Western Amazonia), has experienced

significant demographic changes; as a result of gold prospecting and agricultural

development it has changed its traditional Amazonian characteristics. With people coming

Nutrition transition and growth of children 365

Downloaded By: [Dórea, José Garrofe] At: 14:40 24 July 2008

from many other Brazilian regions, the present population has both traditional families that

base their diets on fish and starchy foods and city dwellers with more cosmopolitan food

habits. In this changing environment, we investigated the growth of a sample of urban

breastfed infants with special reference to family fish consumption.

The study protocol was approved by the Ethics Committee of Studies for Humans of the

Universidade Federal de Rondonia. During routine visits to the pre-natal clinics of three

hospitals in Porto Velho pregnant mothers between the ages of 15 and 45 years were initially

recruited; the mothers were selected among those in good health, reporting no illness or

complaints at the time of the study and who were willing to breastfeed. Excluding factors

were occupational exposure to toxic chemicals and hereditary neurological illnesses. Written

consent (stating that participation was voluntary and confidentiality was assured) was signed

by the participant mother, who could withdraw from the study at any time.

This is part of an ongoing cohort study conceived to study (a) fish Hg exposure of urban

Amazonian mothers; (b) to associate maternal tissue Hg with factors relevant to neuro-

motor development of breastfed infants; and (c) to examine the association between

generated dimensions of infant neurodevelopment and maternal socio-economic and Hg

exposure features. A component of the study focusing on the 6-month-old infants has

already been published (Marques et al. 2007). Briefly, recruitment of 100 mothers began in

2000; detailed information concerning diet and anthropometry (and infant growth and

development) was completed for 82 mother–infant pairs. For each mother a complete

clinical evaluation was obtained from medical records. The newborns were clinically

examined with special attention to vitality, perinatal reflexes, maturity, and congenital

malformations; weight, length, head circumference, and Apgar scores were recorded.

Data collection

We recorded the maternal diet only during the first post-selection visit; this constitutes an

assessment of the principal fish consumption characteristics at the time of the study.

Participants were interviewed about diet; a questionnaire that could identify frequency and

preference of fish was completed. Eighty-two women carried out their intention of

breastfeeding exclusively for 6 months, and some extended lactation to 36 months.

Exclusive breastfeeding was supported throughout the first 6 months and defined according

to the WHO: ‘the infant received breast milk only and allowed supplementation of drops and

syrups such as vitamins, minerals, and medicines’.

At birth and at ages of 6, 36 and 60 months anthropometric measurements of mothers and

infants were taken; weight and height were measured by trained nurses and monitored

regularly according to standard procedures. Weight (kg) and height (cm) of mothers were

measured at each observation and were used to calculate body mass index (BMI, weight/

height

). Length of newborn babies and of 6-month-old infants was measured in recumbent

position with a 0.1 cm stadiometer. At 36 months and 60 months the children were measured

and weighed barefoot and dressed in underwear only; standing height was measured to the

nearest 0.1 cm and weight was measured to the nearest 0.1 kg with an electronic scale.

Both recumbent length (at birth and 6 months) and standing height (at 36 months and 60

months) were measured for all children. At the same time as recording anthropometry, data

were collected on feeding practices, child morbidity, perinatal factors, and socio-economic,

demographic and environmental characteristics. Z-scores for attained weight-for-age,

length-for-age, BMI-for-age and weight-for-length were based on the WHO

Child Growth Standards (WHO Multicentre Growth Reference Study Group 2006).

366 R. C. Marques et al.

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Therefore, the weight-for-height Z-scores (WHZ) were calculated using EPI-INFO (version

4.1; Centers for Disease Control and Prevention, Atlanta) and the WHO recommended

growth curves. Infant growth rate up to 6 months was estimated as weight gain percentage

(IWG%) after Xiong et al. (2007): (infant weight – birth weight)/birth weight Â 100. At the

specified age, we also collected samples of hair from mothers and respective children during

the anthropometric measurements.

Hair Hg determination

Hair sample preparation and analytical procedures were carried out according to

our standardized laboratory procedure for Hg determination (Marques et al. 2007).

Briefly, the hair samples were comminuted with stainless steel scissors, weighed, and

digested before analysis. Human hair samples were washed with EDTA 0.01%, dried in an

oven at 50



C and weighed. Samples were then digested using a digestion block at 80



C for

40 min with concentrated HNO

(3 mL) and KMnO

(5%; 6 mL) in a microwave oven

system for 35 min (CEM-Coorporation, MDS 2000, Matthews, NC, USA). The

determination of total Hg in the digested samples was done by cold vapour atomic

absorption spectrometry with a flow injection system FIMS (CV-AAS, Perkin-Elmer, FIMS

400, Ueberlingen, Germany).

All glassware used in the analytical protocol was washed clean, rinsed with 5% EDTA and

double distilled, and left to rest in 5% HNO

overnight. Then it was rinsed again in double

distilled water, and dried at 100



C for 12 h. Precision and accuracy of Hg determinations

were assured by the use of internal standards, use of triplicate analyses of samples and

certified reference materials (IAEA-085 and 086, Vienna, Austria) with recoveries of 92%.

The limit of detection for the procedure was determined at <0.01 mgg

À1

and there was no

HHg determination below the limits.

Statistical analysis

The statistical packages, contained in Excel and Prism, were used for data summarization

(means, standard deviation, changes in variables) and correlation analysis. The Shapiro–

Wilk test of normality was applied and data transformed when required. Statistical analysis

to test children’s attained growth at 0, 6, 36 and 60 m was carried out using the Statistica

(StatSoft, Inc., v.6.0) statistical package. During data analysis, children were classified into

two groups on the basis of reported maternal fish consumption (Marques et al. 2007). The

General Linear Model of Statistica used a factorial design with repeated measured factors.

Pearson’s (p) correlation was also used to examine the strength of association between the

fish consumption marker (HHg) and anthropometric measures. A value of <0.05 was

accepted as statistically significant.

Results

A previous publication has already partially discussed results of maternal fish consumption

and infant neurodevelopment at 6 months (Marques et al. 2007); the influence of maternal

fish consumption rates on mean HHg concentrations was not statistically significant (Table I).

These contemporary urban mothers purchased fish from open markets and food stores.

Nutrition transition and growth of children 367

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Only 34 of them showed explicit fish species preference: Pacu (Mylossoma spp.; 58%),

Tambaqui (Colossoma spp.; 35%), Jaraqui (Semaprochilodus spp.) and Jatuarana (Brycon spp.)

(33%), Dourado (Brachyplatystoma flavicans; 30%), Tucunare

(Cichla spp.) and Curimata˜

(Prochilodus nigricans) (28%); other species <30% (Filhote – Brachyplatystoma filamentosum;

Piranha - Serrasalmus spp., and Surubim – Pseudoplatystoma spp.).

Both infant HHg at birth and at 6 months represent Hg transferred through placenta

and breast milk, whereas in children at 5 years, HHg concentrations are the result

of exposure to fish in the family diet; only during the weaning period or extended lactation

are children exposed to both sources, breast milk and family diet. Only at birth and

6 months did HHg concentrations substantially increase in both mothers and infants.

Attained Z-scores summarized in Table II showed no statistically significant (p ¼ 0.36)

effect of fish consumption rate (repeated measurement analysis). Also, no statistically

significant correlation was noticed between HHg and respective attained Z-scores

(Figure 1).

Infant characteristics at birth as a function of frequency of maternal fish consumption rates

shown in Table III were adapted from a previous publication (Marques et al. 2007). Mean

birth weights (3.21 vs 3.29 kg), as well as other variables, were nearly identical. The

summary of attained growth and length/height (Z-scores) as a function of fish consumption

Table II. Summary (means) of attained growth and length/height (Z-scores) as a function of

maternal fish consumption; no statistically significant difference between groups (p ¼ 0.36).

Fish servings, groups

0–1 per week (n ¼ 49) !2 per week (n ¼ 33)

Mean SD Mean SD

Weight-for-age

Birth

À0.23673 0.93 À0.02576 0.93

Weight-for-age

6 months

À0.87061 0.69 À0.61364 0.62

Weight-for-age

36 months

0.50041 0.98 0.64061 0.94

Weight-for-age

60 months

0.35653 0.46 0.27636 0.59

Length-for-age

Birth

0.20755 0.91 0.20758 1.08

Height-for-age

6 months

0.31224 1.30 0.46909 1.37

Height-for-age

36 months

0.10612 0.97 0.27606 0.91

Height-for-age

60 months

À0.69245 0.65 À0.52788 0.62

Weight-for-length

Birth

À0.57 1.15 À0.23 1.15

Weight-for-height

6 months

À1.31 1.17 À1.07 1.31

Weight-for-height

36 months

0.63 1.24 0.70 0.95

Weight-for-height

60 months

1.15 0.74 0.87 0.76

Table I. Maternal and infant HHg (mean Æ SD; values in parentheses are SD) concentrations (mgg

À1

)asa

frequency of fish consumption of mothers.

Fish servings

Birth 6 months 3 years 5 years

per week n Mother Infant Mother Infant Mother Infant Mother Infant

0–1 49 6.02 1.97 2.61 3.09 2.39 2.53 2.23 2.50

(5.54) (2.31) (2.48) (4.56) (2.59) (4.16) (2.43) (3.81)

!2 33 9.35 3.14 4.06 4.96 3.39 2.63 3.10 2.84

(11.8) (3.80) (5.09) (6.67) (3.92) (2.96) (3.79) (3.12)

p 0.09 0.08 0.09 0.13 0.21 0.91 0.21 0.66

368 R. C. Marques et al.

Downloaded By: [Dórea, José Garrofe] At: 14:40 24 July 2008

is shown in Table II and the profiles of attained Z-scores are shown in Figure 2. The

cumulative differences between linear and ponderal Z-score outcomes are clear. At birth,

most children showed adequate Z-scores (>À1) for length-for-age (74/82), weight-for-age

(68/82) and weight-for-length (56/82). However, attained growth at 6 months showed

remarkable differences; the infants grew much more in height than in weight. After 6 months

of exclusive breastfeeding the proportion of infants with attained growth (Z-scores <À1) was

14/82, 36/82 and 52/82, respectively, for length-for-age, weight-for-age, and weight-for-

length. Most children showed Z-scores (>À1) for height-for-age (70/82), weight-for-age

(74/82) and weight-for-height (74/82). However, it should be noted that only one child had a

Z-score <À2 for length-for-age during breastfeeding, but five showed stunting at 36 months.

Malnutrition (weight-for-age <À2) was seen in only two breastfed infants. All but one child

attained Z-scores <À1 at 60 months. Part of the nutritional outcome due to the quality of the

weaning diets was due to extended lactation.

0 5 10 15 20 25 30

−5.0

−2.5

0.0

2.5

Infant hair [Hg] µg/g

(Birth)

Weight-for-height Z-score

0 5 10 15 20 25 30

−5.0

−2.5

0.0

2.5

(Six months)

Infant hair [Hg] µg/g

Weight-for-height Z-score

0 10 20 30

−2.5

0.0

2.5

5.0

(36 months)

Infant hair [Hg] µg/g

Weight-for-height Z-score

0 10 20 30

−2

−1

(60 months)

Infant hair [Hg] µg/g

Weight-for-height Z-score

Figure 1. Association between infant HHg and Z-scores (weight-for-height) at 6, 36 and 60 months.

Table III. Maternal and infant characteristics (mean Æ SD) as a function of reported frequency of fish consumption

(adapted from Marques et al. 2007).

Fish servings

per week n

Gestation

(weeks)

Length at

birth (cm)

Infant weight

at birth (kg)

Infant weight

at 6 months (kg) IWG%*

0–1 49 39.3 Æ 1.3 50.0 Æ 1.7 3.21 Æ 0.4 6.97 Æ 0.5 121 Æ 3

!2 33 39.4 Æ 1.4 49.9 Æ 1.9 3.29 Æ 0.4 7.07 Æ 0.5 119 Æ 4

p 0.76 0.76 0.41 0.32 0.82

*IWG%: Infant weight gain at 6 months divided by birth weight.

Nutrition transition and growth of children 369

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Exclusive breastfeeding for 6 months was accomplished by all mothers. After that, many

mothers extended breastfeeding throughout the first year (Figure 3); 10 extended even

further and there was one child still breastfeeding at 5 years. The duration of breastfeeding

did seem to interact with the dietary patterns of weaning; there was significant correlation

with attained Z-scores at 36 months for weight-for-age (r ¼ 0.26; p ¼ 0.02) and weight-for-

length (r ¼ 0.22; p ¼ 0.04) but not for length-for-age (Figure 3). There was a wide spread in

birth weight (range: 2.2–4.3 kg); however, exclusive breastfeeding substantially reduced (by

50%) the range of body mass at 6 months (6.1–8.5 kg). Indeed, the IWG% was inversely

proportional to birth weight in a very high and significant correlation (r ¼À0.88; p ¼ 0.00)

illustrated in Figure 4.

−4 −3 −2 −1 0 1 2 3 4

100

Birth

6 meses

3 years

5 years

(a)

Height-for-age Z-score

Distribution, %Distribution, %Distribution, %

−4 −3 −2 −1 0 1 2 3 4

100

Birth

6 month

3 years

5 years

Birth

6 months

3 years

5 years

(b)

Weight-for-age Z-score

−4 −3 −2 −1 0 1 2 3 4

100

(c)

Weight-for-height Z-score

Figure 2. Cumulative frequency of Z-scores of attained growth during sampling times.

370 R. C. Marques et al.

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The possible taxing of mothers caused by milk demanded to sustain increased infant

growth is illustrated in Figure 5; maternal BMI showed no significant association with

breastfeeding duration. Mothers with higher BMI at the end of pregnancy had returned to

pre-pregnancy levels at 6 months (exclusive breastfeeding) and 3 years (extended

breastfeeding). Figure 5 illustrates that the BMI profile is indistinguishable between

pre-pregnancy, 6 and 36 months post-natal.

Discussion

Quality of the diet for pregnant and lactating women is a key issue in public health nutrition,

especially in poor communities with economic constraints. The mothers of Porto Velho,

0 20 40 60

−2.5

0.0

2.5

5.0

Length of lactation, months

(a)

Weight-for height Z-score

0 20 40 6

−2.5

0.0

2.5

5.0

Length of lactation, months

BMI/age Z-score

(c)

0 20 40 60

−2

−1

(b)

Len

th of lactation, months

Weight-for-age Z-scores

0 20 40 60

−3

−2

−1

(d)

Len

th of lactation, months

Height-for-age Z-scores

Figure 3. Length of lactation and attained growth.

2 3 4 5

100

200

300

Birth wei

ht, k

Relative IWG, %

(6 months)

0 1 2 3 4 5

−2

−1

Birth wei

ht, k

W/A Z-scores

(5 years)

Figure 4. Relative infant weight gain (IWG% – infant weight gain divided by birth weight) at

6 months and weight-for-age Z-score (W/A) as a function of birth weight.

Nutrition transition and growth of children 371

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with respect to levels of dietary fish consumption, showed no significant impact on the

attained growth of their young children; this indicates that changes from traditional

fish-dominated diet to other animal or vegetal-source food have occurred without

consequences for infant development. By examining the longitudinal growth of their

children it was revealed that mothers’ milk, irrespective of level of fish consumption, could

compensate for low birth-weights; furthermore, the length of lactation (beyond 6 months)

was an important nutritional supplement measurable at 3 years (Figure 2).

Fish is a differentiator of the survival strategy of some Amazonian villages (Dorea et al.

2005b); it is consumed more in isolated communities (Alves et al. 2006). Early dietary

studies of poor Amazonian families showed that they can consume 151 g fish per day

comprising 37% of their dietary protein (Giugliano et al. 1978). HHg is a specific and

reliable indicator of fish consumption that has been used to study Amazonian populations

and successfully employed to quantify the amount of freshwater fish consumed (Dorea et al.

2003). Our study showed that in urban population, no longer following traditional food

habits, HHg was higher (although not significantly) in the group reporting higher fish

consumption rate. Therefore, HHg can be valuable to understand fish-MeHg exposure as

well as to trace beneficial outcomes of fish consumption in Amazonians (Dorea et al.

2005a,b). However, because of concerns for neurodevelopmental disorders due to

intrauterine exposure to MeHg, recommendations to reduce fish intake have been made

(Cohen et al. 2005). For economically stressed families, especially in regions accustomed to

high frequency of fish consumption, such recommendations may not take into account the

effect that reducing fish consumption will have on the nutritional status of vulnerable infants

and young children. Indeed, Arnold et al. (2005) have questioned whether it is worth

curtailing fish Hg exposure because this can tax the consumption of an important source of

nutrients; beneficial effects on foetal neurological outcome have been shown as a result of

maternal fish consumption (Hibbeln et al. 2007). Additionally, animal-source foods have

been less appreciated in studies of economically strained urban families in developing

nations. We, therefore, took into consideration the changing habits arising from fast

urbanization of an Amazonian city to assess the role of HHg (marker of fish consumption)

and outcomes of exclusively breastfed-infant development.

We speculate that dominance of fish consumption, although important for some specific

nutrients, was successfully replaced by other animal-food sources. The lack of significant

association between HHg and WHZ (especially at 5 years) indicates that difference in

protein quality (fish vs other sources) was not a determinant of attained growth in these

children. This is in agreement with our studies in Amer-Indian children inhabiting the banks

0 10 20 30 40

Length of lactation, months

MaternalBMI

(b)

0 10 20 30 40

100

Birth

Pre-pregnancy

6 months

36 months

(a)

Maternal BMI

Distribution, %

Figure 5. Maternal BMI (body mass index); (a) profile changes during the study; (b) scatter plot as a

function of length of lactation.

372 R. C. Marques et al.

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of the Xingu-basin Rivers in Eastern Amazonia (Dorea et al. 2005a); in these subsistence-

level communities of indigenous people a clear change in dominance of fish consumption

between the tribes (Kayabi and Munduruku) was not associated with infant growth; other

factors like infectious diseases, may influence the health of subjects.

Infant weight at birth (intrauterine growth rate) is directly influenced by maternal

constitutional (size) and environmental factors (nutrition). Weight and linear growth of

breastfed infants are influenced by human-milk output and nutritional profile (Fornes and

Dorea 1995); although genetic factors may be significant, infant postnatal growth is also

greatly influenced by illnesses. In the case of exclusive breastfeeding, weight gain is indirectly

a result of breastfeeding protection against the nutritional consequences of illness; by

decreasing the number of days with gastrointestinal and respiratory tract illnesses (Launer

et al. 1990), infant food efficiency is optimized with growth maximization.

Infant growth is the key to assessing nutritional status; while cross-sectional growth data

are useful to identify infants whose weight and length outcomes are considered substandard

they are limited in assessing the dynamics of breastfeeding in support of the growing child.

In this context monitoring infant growth to understand true growth potential is better when

based on longitudinal data (Xiong et al. 2007). Several papers have drawn attention to the

supposition that infants born with low birth weight may remain lighter and shorter (Xiong

et al. 2007); however, in our study, breastfeeding showed a growth rate inversely

proportional to birth weight. Overall, our results showed an accelerated rate of length

gain in the first few months. These results are in agreement with others (Cole et al. 2002),

showing that prolonged and exclusive breastfeeding accelerated weight and length gain in

early weeks. Infants in our study showed an apparent increase in thinness at 3 years but no

detectable deficit at 5 years.

The infants with lower birth weight – or small size – reflected poor intrauterine growth

(or shorter gestational age); however, in the present study, they grew at a faster rate during

exclusive breastfeeding. At 5 years, it was not the birth weight but the rate of change in body

mass that modulated the attained growth. The very high linear correlation (Figure 4)

between birth weight and IWG% indicates a catch-up process. We do not yet understand

how the compensatory mechanism operates in breastfeeding to promote the faster move-up

of centiles in the smaller babies. Considering that the breast-milk nutrients originated from

the (same) maternal sources, higher rates of growth indicate more efficient transfer of

nutrients (mother) or more efficient utilization (infant) or, most probably, both.

Differences in placental function (birth weight) and milk supply (postnatal growth) with

regard to maternal nutrient transfer are not yet understood. Weaver (2006) has

differentiated rapid weight-gain catch-up (after intrauterine restriction) and accelerated

growth as a result of excessive energy intake in formula-fed infants. In our study, however,

the self regulating (breastfeeding) milk supply does not seem to lead to an excessive energy

intake; but the same mother, who apparently produced a smaller foetus, is capable of

compensating post-natal infant growth through breast milk. After birth, the (active) infant is

apparently more efficient in nutrient acquisition (sucking) and utilization than the (passive)

foetus. Indeed, Karaolis-Danckert et al. (2006) recently reported that relatively smaller and

lighter babies grow faster than their larger-at-birth counterparts; they appeared to diverge in

growth patterns as early as 6 months. In their study only 30% of fast growers were breastfed

for more than 4 months.

Rich in nutrients, hormones and complex non-nutritional factors, human milk is better

suited to meet specific requirements for infant development; such a complexity of bioactive

substances and neuro-stimulation factors modulates neonatal adaptation and provides

protection and immunity; exclusive breastfeeding is also finely tuned to regulate infant

Nutrition transition and growth of children 373

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growth (Weaver 2006). In certain circumstances, infant growth is associated with breast-

milk fat content (Fornes and Dorea 1995); however, in regard to birth weight, breast-milk’s

intrinsic nutritional-attributes are not sufficient to explain the inverse relationship of infant

growth and birth weight.

In epidemiological studies the differences in attained growth between bottle- and breast-

fed infants have been attributed to reverse causality (feeding choice influenced by child

health/weight). Cole et al. (2002) discussed reverse causality in the context of feeding-mode

differences: ‘slow-growing infants who are ‘falling off’ their growth curve trajectories may be

deliberately supplemented or weaned in an effort to reverse those trends’. Apparently, this is

not the case in our study; our infants showed an inverse correlation (birth weight vs IWG%,

Figure 4) and a direct association of extended breastfeeding (beyond 6 m) and attained

Z-scores at 3 years (Figure 4). It is amply reported that in the developing world, absence of

or insufficient breastfeeding or early weaning are all frequently associated with diarrhoea and

other infections that can tax growth rate.

In poor urban environments in underdeveloped countries breastfeeding is intertwined

with maternal capability to lactate and infant growth rate and health. Regardless of whether

the mother lactates or not, postpartum weight and body fat changes are maternal

physiological occurrences. We (Fornes and Dorea 1995) have shown that poor urban

mothers (in Goiania, Brazil) can sustain a successful exclusive breastfeeding of 3 months

with BMI values comparable to the present study. The socio-economically disadvantaged

urban mothers of that study, taxed by milk supply demanded by the growing infant, did not

show significant BMI changes (from 15 to 90 days) but showed significant differences in

subcutaneous fat change (Fornes and Dorea 1995). Indeed our study did not show

significant association between maternal BMI (at 36 months) and attained infant weight.

Collectively, there is a distinct pattern of postpartum changes in body weight and adiposity

between affluent and socially disadvantaged mothers (in developing countries): Lactating

mothers in less developed countries lose subcutaneous fat when measured singly (triceps,

more sensitive) or as a sum of several skin-fold measurements. Comparing different studies

of different countries it was shown that these effects tend to occur mainly during the second

trimester of lactation, but will occur in the first semester of lactation in circumstances of

relative maternal malnutrition (Dorea 1997). This is in agreement with the changes observed

in the BMI of the mothers in the present study: The profile between pre-pregnancy and at 6

months and 3 years are indistinguishable.

Fish is important in the diets and health of many poor people suffering from vitamin and

mineral deficiencies, and yet we lack studies of its consumption by populations most

vulnerable to nutrient deficiencies (Roos et al. 2007). The unique feature of this study is to

be able to evaluate the impact of a dietary item (fish consumption) on critical periods of

infant development (gestation, breastfeeding) which is sensitive to the maternal effects of

nutritional factors. Although HHg – the signature of fish consumption – was not significantly

associated with infant growth, the study revealed that extended breastfeeding had a

statistically significant impact on profiles of attained height and weight at 3 years thus

indicating that breastfeeding can be an important modifying factor of weaning.

Conclusion

Fish consumption rates can be traced through HHg; this biomarker showed that nutrient

intakes in tandem with maternal fish consumption rate had no impact on outcomes of growth

374 R. C. Marques et al.

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and development of breastfed children. In the food transition scenario of Porto Velho,

regardless of the animal-source food, the habitual plane of nutrition of these urban mothers

was sufficient to sustain satisfactory Z-scores for weight and height at 5 years.

Acknowledgements

We are greatly in debt to the mothers for their participation in the study, to the staff

and Directors (Marine

s R. S. Cezar, Tereza C. Ramos, Daniele Brasil, Katia Wendt,

Laura J. Marques) of the Hospitals (Hospital de Base Ary Pinheiro, Hospital Panamericano

and Hospital Regina Pacis), Katiane G. Branda

o and the staff of the Fundac¸a

o Universidade

Federal de Rondo

nia. This work was supported by United Nations Educational, Scientific

and Cultural Organization – UNESCO (SC27824/2005/914-BRA2000-Decit-PRODOC),

Ministe

rio da Sau

de do Brasil and The National Research Council of Brasil – CNPq

(PNOPG project-55.0882/01-4; PPG7, project-556 985/2005-2).

References

Alves MFA, Fraiji NA, Barbosa AC, Lima DSS, Souza JR, Dorea JG, Cordeiro GWO. 2006. Fish consumption,

mercury exposure and serum antinuclear antibody in Amazonians. Int J Environ Health Res 16:255–262.

Arnold SM, Lynn TV, Verbrugge LA, Middaugh JP. 2005. Human biomonitoring to optimize fish consumption

advice: Reducing uncertainty when evaluating benefits and risks. Am J Public Health 95:393–397.

Barbosa AC, Silva SRL, Dorea JG. 1998. Concentration of mercury in hair of indigenous mothers and infants from

the Amazon Basin. I. Arch Environ Contam Toxicol 34:100–105.

Barbosa AC, Souza JR, Garcia MA. 1997. Mercury contamination in hair of riverine populations of Apiaca’s

Reserve in the Brazilian Amazon. Water Air Soil Pollut 97:1–8.

Clarkson TW. 2002. The three modern faces of mercury. Environ Health Perspect 110:(Suppl 1)11–23.

Cohen JT, Bellinger DC, Shaywitz BA. 2005. A quantitative analysis of prenatal methyl mercury exposure and

cognitive development. Am J Prev Med 29:353–365.

Cole TJ, Paul AA, Whitehead RG. 2002. Weight reference charts for British long-term breastfed infants. Acta

Paediatr 91:1296–1300.

Dorea JG. 1997. Changes in body weight and adiposity during lactation. Nutr Res 17:379–389.

Dorea JG. 2003. Fish are central in the diet of Amazonian riparians: Should we worry about their mercury

concentrations?. Environ Res 92:232–244.

Dorea JG. 2004. Cassava cyanogens and fish mercury are high but safely consumed in the diet of native

Amazonians. Ecotoxicol Environ Saf 57:248–256.

Dorea JG, Moreira MB, East G, Barbosa AC. 1998. Selenium and mercury concentrations in some fish species of

the Madeira River, Amazon Basin, Brazil. Biol Trace Elem Res 65:211–220.

Dorea JG, Barbosa AC, Ferrari I, Souza JR. 2003. Mercury in hair and in fish consumed by riparian women of the

Rio Negro, Amazon, Brazil. Int J Environ Health Res 13:239–248.

Dorea JG, Barbosa AC, Ferrari I, Souza JR. 2005a. Fish consumption (hair mercury) and nutritional status of

Amazonian Amer-Indian children. Am J Hum Biol 17:507–514.

Dorea JG, Souza JR, Rodrigues P, Ferrari I, Barbosa AC. 2005b. Hair mercury (signature of fish consumption) and

cardiovascular risk in Munduruku and Kayabi Indians of Amazonia. Environ Res 97:208–218.

Dufour DL. 1991. Diet and nutritional status of Ameridians: A review of the literature. Cad Saude Publica

7:481–502.

Fornes NS, Dorea JG. 1995. Subcutaneous fat changes in low-income lactating mothers and growth of breast-fed

infants. J Am Coll Nutr 14:61–65.

Garcia-Arias MT, Castrillon AM, Navarro MP. 1993. Bioavailability of zinc in rats fed on tuna as a protein source

in the diet. J Tr Elem Electrol Health Dis 7:29–36.

Gibson RS, Hotz C. 2001. Dietary diversification/modification strategies to enhance micronutrient content and

bioavailability of diets in developing countries. Br J Nutr 85:S159–S166.

Nutrition transition and growth of children 375

Downloaded By: [Dórea, José Garrofe] At: 14:40 24 July 2008

Giugliano R, Shrimpton R, Arkcoll DB, Giugliano LG, Petrere M Jr. 1978. Diagno

stico da realidade alimentar e

nutricional do Estado do Amazonas 1978. Acta Amaz 8:(Suppl. 2)5–54.

Greco L, Balungi J, Amono K, Iriso R, Corrado B. 2006. Effect of a low-cost food on the recovery and death rate of

malnourished children. J Pediatr Gastroenterol Nutr 43:512–517.

Gunnarsson BS, Thorsdottir I, Palsson G. 2007. Associations of iron status with dietary and other factors in 6-year-

old children. Eur J Clin Nutr 61:398–403.

Hibbeln JR, Davis JM, Steer C, Emmett P, Rogers I, Williams C, Golding J. 2007. Maternal seafood consumption

in pregnancy and neurodevelopmental outcomes in childhood (ALSPAC study): An observational cohort

study. Lancet 369:(9561)578–585.

Hofvander Y. 1973. Growth of infants fed a formula containing fish protein concentrate (FPC). Upsala J Med Sci

78:60–64.

Inhamuns AJ, Bueno Franco MR. 2001. Composition of total, neutral, and phospholipids in Mapara

(Hypophthalmus sp.) from the Brazilian Amazonian area. J Agric Food Chem 49:4859–4863.

Kipnis V, Midthune D, Freedman L, Bingham S, Day NE, Riboli E, Ferrari P, Carroll RJ. 2002. Bias in dietary-

report instruments and its implications for nutritional epidemiology. Public Health Nutr 5:915–923.

Kull I, Bergstrom A, Lilja G, Pershagen G, Wickman M. 2006. Fish consumption during the first year of life and

development of allergic diseases during childhood. Allergy 61:1009–1015.

Lartey A, Manu A, Brown KH, Peerson JM, Dewey KG. 1999. A randomized, community-based trial of the effects

of improved, centrally processed complementary foods on growth and micronutrient status of Ghanaian

infants from 6 to 12 mo of age. Am J Clin Nutr 70:391–404.

Layrisse M, Martı

nez-Torres C, Me

ndez-Castellano H, Taylor P, Fossi M, Lo

pez de Blanco M,

Landaeta-Jime

nez M, Jaffe W, Leets I, Tropper E, et al. 1990. Relationship between iron bioavailability

from diets and prevalence of iron deficiency. Food Nutr Bull 12:301–309.

Launer LJ, Habicht JP, Kardjati S. 1990. Breast feeding protects infants in Indonesia against illness and weight loss

due to illness. Am J Epidemiol 131:322–331.

Karaolis-Danckert N, Buyken AE, Bolzenius K, Perim de Faria C, Lentze MJ, Kroke A. 2006. Rapid growth

among term children whose birth weight was appropriate for gestational age has a longer lasting effect on body

fat percentage than on body mass index. Am J Clin Nutr 84:1449–1455.

Marques RC, Do

rea JG, Bastos WR, Rebelo MF, Fonseca MF, Malm O. 2007. Maternal mercury exposure and

neuro-motor development in breastfed infants from Porto Velho (Amazon), Brazil. Int J Hyg Env Health

210:51–60.

Michaelsen KF, Milman N, Samuelson G. 1995. A longitudinal study of iron status in healthy Danish infants:

Effects of early iron status, growth velocity and dietary factors. Acta Paediatr 84:1035–1044.

Muskiet FA, van Goor SA, Kuipers RS, Velzing-Aarts FV, Smit EN, Bouwstra H, Dijck-Brouwer DA,

Boersma ER, Adders-Algra M. 2006. Long-chain polyunsaturated fatty acids in maternal and infant nutrition.

Prostaglandins Leukot Essent Fatty Acids 75:135–144.

Nelson M, Atkinson M, Darbyshire S. 1996. Food photography II: Use of food photographs for estimating portion

size and the nutrient content of meals. Br J Nutr 76:31–49.

Olsen SF, Grandjean P, Weihe P, Videro T. 1993. Frequency of seafood intake in pregnancy as a determinant of

birth weight: Evidence for a dose dependent relationship. J Epidemiol Community Health 47:436–440.

Passos CJ, Mergler D, Fillion M, Lemire M, Mertens F, Guimaraes JR, Philibert A. 2007. Epidemiologic

confirmation that fruit consumption influences mercury exposure in riparian communities in the Brazilian

Amazon. Environ Res 105:183–193.

Piperata BA. 2007. Nutritional status of Ribeirinhos in Brazil and the nutrition transition. Am J Phys Anthropol

133:868–878.

Rocquelin G, Tapsoba S, Dop MC, Mbemba F, Traissac P, Martin-Prevel Y. 1998. Lipid content and essential

fatty acid (EFA) composition of mature Congolese breast milk are influenced by mothers’ nutritional status:

Impact on infants’ EFA supply. Eur J Clin Nutr 52:164–171.

Rogers I, Emmett P, Ness A, Golding J. 2004. Maternal fish intake in late pregnancy and the frequency of low birth

weight and intrauterine growth retardation in a cohort of British infants. J Epidemiol Community Health

58:486–492.

Romieu I, Torrent M, Garcia-Esteban R, Ferrer C, Ribas-Fito N, Anto JM, Sunyer J. 2007. Maternal fish intake

during pregnancy and atopy and asthma in infancy. Clin Exp Allergy 37:518–525.

Roos N, Wahab MA, Chamnan C, Thilsted SH. 2007. The role of fish in food-based strategies to combat vitamin A

and mineral deficiencies in developing countries. J Nutr 137:1106–1109.

Ruan C, Liu X, Man H, Ma X, Lu G, Duan G, De Francesco CA, Connor WE. 1995. Milk composition in women

from five different regions of China: The great diversity of milk fatty acids. J Nutr 125:2993–2998.

376 R. C. Marques et al.

Downloaded By: [Dórea, José Garrofe] At: 14:40 24 July 2008

Sikka KC, Singh R, Gupta DP, Duggal SK. 1979. Comparative nutritive value of fish protein concentrate (FPC)

from different species of fishes. J Agric Food Chem 27:946–949.

Subar AF, Kipnis V, Troiano RP, Midthune D, Schoeller DA, Bingham S, Sharbaugh CO, Trabulsi J, Runswick S,

Ballard-Barbash R, et al. 2003. Using intake biomarkers to evaluate the extent of dietary misreporting in a

large sample of adults: The OPEN study. Am J Epidemiol 158:1–13.

Terres C, Navarro M, Martin-Lagos F, Gimenez R, Lopez H, Lopez MC. 2001. Zinc levels in foods from

southeastern Spain: Relationship to daily dietary intake. Food Addit Contamin 18:687–695.

Thorsdottir I, Birgisdottir BE, Halldorsdottir S, Geirsson RT. 2004. Association of fish and fish liver oil intake in

pregnancy with infant size at birth among women of normal weight before pregnancy in a fishing community.

Am J Epidemiol 160:460–465.

Weaver LT. 2006. Rapid growth in infancy: Balancing the interests of the child. J Pediatr Gastroenterol Nutr

43:428–432.

WHO Multicentre Growth Reference Study Group. 2006. WHO Child Growth Standards based on length/height,

weight and age. Acta Paediatr Suppl 450:76–85.

Wu ML, Whittemore AS, Jung DL. 1986. Errors in reported dietary intakes. I. Short-term recall. Am J Epidemiol

124:826–835.

Xiong X, Wightkin J, Magnus JH, Pridjian G, Acuna JM, Buekens P. 2007. Birth weight and infant growth:

Optimal infant weight gain versus optimal infant weight. Mat Child Health J 11:57–63.

Zollner H, Below H, Franke G, Piek M, Kramer A. 2001. Present alimentary supply of iodine in Western

Pomerania. Deut Lebens-Runds 97:376–380.

Nutrition transition and growth of children 377

149

(Anexo 7)

TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO

Projeto: Avaliação da Exposição ao Mercúrio e Seus Compostos Sobre o

Desenvolvimento Neuropsicomotor em Crianças de Porto Velho – RO.

Estou sendo orientado (a) quanto ao estudo Avaliação da Exposição ao Mercúrio e

Seus Compostos Sobre o Desenvolvimento Neuropsicomotor em Crianças de Porto

Velho – RO, cujos dados serão colhidos nas pacientes do Centro Obstétrico do

Hospital de Base Ary Pinheiro, Maternidade Regina Pacis e Hospital Panamericano.

Este estudo tem por objetivo estudar a exposição por mercúrio nos recém-nascidos

da comunidade. É importante que se estude a mãe e o filho, pois a exposição é

passada de mãe para filho e continuada por alimentos contaminados, entre eles o

peixe. Esta exposição pode levar a problemas de saúde em crianças e adultos. O

estudo será feito com perguntas de importância para o estudo da exposição e

análise dos seguintes materiais: cortes de pedaços de cabelo da mãe e recém-

nascido, amostra de sangue e leite materno, sangue e pedaço de cordão umbilical e

placenta. Os participantes devem ser moradores da cidade de Porto Velho. A

pesquisa será realizada sem riscos para quem participa, uma vez que o exame

clínico não machuca, o cabelo cresce de novo e será utilizado material descartável

para coleta de sangue. Os materiais acima citados serão estudados na busca da

quantidade de mercúrio no corpo. Os pesquisadores comprometem-se a utilizar os

materiais e dados coletados exclusivamente para os fins previstos no protocolo e a

publicar os resultados, sejam eles favoráveis ou não.

Rejane Corrêa Marques

Endereço: Rua 27, casa 14, condomínio Fabiane Asfuri,

Bairro: Jardim das Mangueiras II, Porto Velho – RO.

Telefone: 225-5436/216-8550.

Estou ciente de que não sou obrigado (a) a participar ou deixar nenhum familiar

participar, e que posso desistir a qualquer momento da pesquisa. Concordo com o

que foi dito, permitindo que meu filho (a) e eu participemos da pesquisa. E estou

recebendo cópia deste papel assinado por mim e pela pesquisadora.

Nome da mãe e/ou responsável pela criança: .____________________________

Porto Velho, ________de __________________de 2000.

150

(Anexo 8)

Questionário mães e recém-nascidos

UNIVERSIDADE FEDERAL DE RONDÔNIA – UNIR

Projeto: Avaliação da Exposição ao Mercúrio e Seus Compostos Sobre o

Desenvolvimento Neuropsicomotor em Crianças de Porto Velho – RO.

Área de atuação: Porto Velho (RO)

Ficha n.º :................. Data....../......./........

MÃE

Nome:.....................................................................................................................................

Data de nascimento:....../...../......... Idade:.......... Cor: branca( ) parda( ) negra( ) índia ( )

Lugar de nascimento:.............................................................................................................

Endereço Atual:...............................................................................FONE:............................

Pré-natal: sim( ) não( ) Início:........................ Local:........................................................

Início dos movimentos fetais:............................. Intensidade:...............................................

Intercorrências/Terapêutica:..................................................................................................

Trabalho durante a gestação:................................................................................................

Tipo de parto: ( )normal ( )fórceps ( )cesárea: Indicação:....................................................

DUM: ....../....../......... IG: ........semanas DPP:............ Informante:...........................

RECÉM-NASCIDO:

Sexo: ( ) feminino ( ) masculino Peso:................g Estatura:...........cm PC:........cm

Condições do RN ao nascer: Apgar 1º minuto.............5º minuto................

Circular de cordão: ( )não ( )sim Reanimado: ( )não ( )sim: ( )O

nasal ( )ambu ( )entubação

Amniorrexe prematura: ( ) não ( )sim quanto tempo:..............horas

Uso de antibiótico pré-parto: ( )não ( )sim tipo:......................................................................

Dose:........................................... Tempo de uso:.................................................................

Eliminou mecônio antes da expulsão: ( )não ( )sim

Maturidade: ( )a termo ( )prematuro ( )pós-maduro IG pelo Índice de capurro:.....sem e....dias

Classificação quanto ao peso: ( )AIG ( ) PIG ( ) GIG

Medicação na sala de parto: ( )não ( )sim; especificar a medicação:.................................

Internação no berçário: ( ) não ( ) sim: período: ..............................................................

Intercorrências no berçário:...................................................................................................

Malformação: ( )não ( )sim qual: ...........................................................................................

Observações:.........................................................................................................................

Condições de alta do RN: ( )com a mãe ( )do berçário para o ambulatório ( )TFD

ANTECEDENTES FAMILIARES MATERNOS:

Tipagem sangüínea: ( )O ( )A ( )B ( )AB Fator Rh: ( )Positivo ( )Negativo

Pais consangüíneos: ( ) sim ( ) não

Gesta:............... Para.............. Abortos......... (espontâneos:............... provocados:.......)

Tipo de parto: ( ) normais ( )cesarianas ( ) complicações: ....................................................

( )Natimortos ( )Prematuro ( )malformados tipo de malformações:...............................

Antecedentes patológicos:

( )malária ( )leishmaniose ( )chagas ( )amebíase ( )esquistossomose ( )diabetes ( )sífilis

( )hepatite ( )toxoplasmose ( )rubéola ( )citomegalovírus ( )HIV ( )caxumba ( )tuberculose

151

Outras:...................................................................................................................................

Educação Materna (anos): ..................... Renda mensal familiar: R$.................................

Quanto tempo mora no endereço atual:.........................

Trabalho em garimpo: ( )não ( )sim período:...........................

Quantas refeições faz por dia:.......... Come peixe: ( )não ( )sim, quantidade/dia:................

Freqüência: ........... Tipo:.............................................................. Procedência:.......................

Usa medicamentos: ( )não ( )sim, qual:.............................. Quanto tempo:.......... Dose/dia:........

Contato com substâncias praguicidas ou semelhantes: ( )não ( )sim Quais:............................

Uso de drogas narcóticas: ( )não ( )sim, Qual:........................................ tempo de uso:..........

Usa xampu: ( )não ( )sim, marca:............. Tinge os cabelos: ( )não ( ) sim, periodicidade:......

Ingere bebida alcoólica: não ( ) sim( ) Freqüência: .............. Quantidade/dia:.................

Tipo de bebida.......................................................................................................................

Viagens ao exterior: ( )não ( )sim : país..........................................período:..........................

ANTECEDENTES FAMILIARES PATERNOS:

Saúde: ( )normal ( )com problemas; quais:............................................................................

Educação Paterna (anos): ................. Trabalho em garimpo: ( )não ( )sim período:..............

Quantas refeições/dia:.......... Come peixe: ( )não ( )sim quantidade/dia:............................

Qual tipo:............................................................................ Procedência:............................

Usa medicamentos: ( )não ( )sim qual:....................... Quanto tempo:.......... dose/dia:............

Ingere bebida alcoólica: não ( ) sim( ) Freqüência: .................... Quantidade/dia:................

Tipo de bebida.......................................................................................................................

Uso de drogas narcóticas: ( )não ( )sim tipo de droga:........................tempo de uso:...........

IRMÃOS DO RECÉM-NASCIDO: Número total:........... Sexo e idade em ordem decrescente:

Idade

Sexo

Saúde dos irmãos: ( )boa ( )com problemas, quais:....................................................

CONDIÇÕES DE MORADIA DA FAMÍLIA:

Situação da residência: ( )própria ( )alugada ( )de parentes ( )cedida pelo empregador

( )madeira ( )alvenaria ( )mista ( )enchimento ( )em terreno alagado

No de moradores:............................ Entra luz em todos os cômodos: ( ) sim ( ) não

Presença de insetos e roedores: ( )não ( )sim, qual:.........................................................

Destino das fezes: ( )fossa seca ( )enterrado ( )céu aberto ( )rio ( )fossa negra

Abastecimento de água: ( )rio ( )encanada ( )Poço ( )torneira pública ( ) outra:...................

NÍVEIS DE MERCÚRIO ENCONTRADOS:

No cabelo da mãe:...........................................

No cabelo do recém nascido:...........................

No sangue da mãe:...........................................

No sangue umbilical:...........................................

No cordão umbilical:........................................

Na placenta:......................................................

No leite materno: ...............................................

Avaliador:........................................................................

152

(ANEXO 9)

Questionário Para Crianças 6 meses

UNIVERSIDADE FEDERAL DE RONDÔNIA – UNIR

Projeto: Avaliação da Exposição ao Mercúrio e Seus Compostos Sobre o

Desenvolvimento Neuropsicomotor em Crianças de Porto Velho – RO.

Ficha n.º _______ Data ___/___/____

IDENTIFICAÇÃO

Nome: __________________________________________________________________

Data de nascimento: ___/___/____ Idade:_____ Sexo: ( )Masc ( )Fem Cor:_________

Endereço Atual:___________________________________________________________

Informante/Responsável:________________________ Telefone para Contato:_________

Nome da mãe: ______________________________________ Profissão: ____________

Nome do Pai: _________________________________Profissão:___________________

SAÚDE

História Vacinal

Apresentou cartão de vacinação: ( )sim ( )não

Vacinas

Datas

BCG

Anti-Hepatite B

DPT

Outras

Antecedentes patológicos:__________________________________________________

HISTÓRIA ALIMENTAR

Tempo de amamentação:_________________ n.º de refeições/dia:_________________

Alimentos mais freqüentes:__________________________________________________

Come Peixe: ( )não ( )sim, tipo de peixe:_______________________________________

Freqüência:______Quantidade:______ Procedência/peixe:_________________________

EDUCAÇÃO:

Freqüenta creche: ( )sim ( )não Tempo de permanência na creche:_________horas

Educação Paterna (anos):__________ Educação Materna (anos):____________

Renda Familiar:__________________________

Irmãos (total):.....................

Idade

Sexo

153

Saúde dos irmãos: ( )boa ( )com problemas, quais:..................................................

CONDIÇÕES DE MORADIA DA FAMÍLIA:

Situação da residência: ( )própria ( )alugada ( )de parente/amigos ( )cedida pelo empregador

( )madeira ( )alvenaria ( )enchimento ( )em terreno alagado

Presença de insetos e roedores:( )não ( )sim, qual:_______________________________

Quantos moram na casa:____________ Entra luz em todos os cômodos: ( )sim ( )não

Destino das fezes: ( )fossa seca ( )enterrado ( )céu aberto ( )rio ( ) fossa negra

Abastecimento de água: ( )rio ( )encanada ( )Poço ( )torneira pública ( )outra:__________

EXAME FÍSICO

Mãe:

Peso:___________ Altura:_______ Paridade:_________

Criança:

Peso:___________ Altura:_______ PC:_______

Permanece sentado: ( )sim ( )não Idade que começou a sentar:_____________

Engatinha: ( )sim ( )não Idade que começou a engatinhar:_________________

Outros achados:__________________________________________________________

Impressão diagnóstica:_____________________________________________________

DESENVOLVIMENTO NEUROPSICOMOTOR:

Idade cronológica:___________

CONDUTA

Idade (meses)

MOTORA

ADAPTATIVA

LINGUAGEM

compreensiva

expressiva

PESSOAL-SOCIAL

Níveis de Hg no cabelo da criança:__________________________________________

Níveis de Hg no cabelo da mãe:_____________________________________________

Avaliador:___________________________

154

(Anexo 10)

UNIVERSIDADE FEDERAL DE RONDÔNIA – UNIR

UNIVERSIDADE DE BRASÍLIA – UnB

UNIVERSIDADE FEDERAL DO RIO DE JANEIRO – UFRJ

Projeto: Avaliação da exposição ao mercúrio e suas repercussões sobre o

desenvolvimento neuropsicomotor em crianças de Porto Velho

TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO

Estou sendo orientada (o) quanto ao estudo ―Avaliação da Exposição ao Mercúrio e

suas Repercussões Sobre o Desenvolvimento Neuropsicomotor em Crianças de

Porto Velho – RO‖, cujos dados serão colhidos com o objetivo estudar a exposição

por mercúrio nas crianças da comunidade. É importante que se estude as crianças,

pois a exposição pode ser passada de mãe para filho e continuada por alimentos

contaminados, entre eles o peixe. Esta exposição pode levar a problemas de saúde

em crianças e adultos. O estudo será feito com exame físico completo, perguntas de

importância para o estudo da exposição e amostras de cabelo da criança. A

pesquisa será realizada sem riscos para os participantes, uma vez que o exame

clínico não machuca e o cabelo cresce de novo. O material acima citado será

estudado na busca da quantidade de mercúrio no corpo. A pesquisadora

compromete-se a utilizar o material e dados coletados exclusivamente para os fins

previstos no protocolo e a publicar os resultados, sejam eles favoráveis ou não.

Rejane Corrêa Marques

Endereço: Laboratório de Biogeoquímica

Br 364, km 9,5, Campus UNIR.

Telefone: (69) 2182-2122

Estou ciente de que não sou obrigada (o) a participar ou deixar nenhum familiar

participar, e que posso desistir a qualquer momento da pesquisa, tendo sido

informada (o) dos endereços para contato com a pesquisadora. Concordo com o que

foi dito, permitindo que meu filho (a) e eu participemos da pesquisa, e estou

recebendo cópia deste papel assinado por mim e pela pesquisadora.

Nome da mãe e/ou responsável pela criança______________________________

Porto Velho,_____ de ____________ de 200__.

155

(ANEXO 11)

Questionário Para Crianças de 3 a 5 anos

UNIVERSIDADE FEDERAL DE RONDÔNIA – UNIR

UNIVERSIDADE DE BRASÍLIA – UnB

UNIVERSIDADE FEDERAL DO RIO DE JANEIRO – UFRJ

Projeto: Avaliação da exposição ao mercúrio e suas repercussões sobre o

desenvolvimento neuropsicomotor em crianças de Porto Velho

Ficha n.º _______ Data ___/___/____

IDENTIFICAÇÃO

Nome: ______________________________________________________________

Data de nascimento: ___/___/____ Idade:_____ Sexo: ( )Masc ( )Fem

Lugar de nascimento: __________________________________________________

Endereço Atual:_______________________________________________________

Nome da mãe: ___________________________________ Profissão: ___________

Nome do Pai: ____________________________________ Profissão:____________

FONE:_______________Informante:______________________________________

SAÚDE

História Vacinal: Apresentou carteira: ( )sim ( )não

Vacinas

Datas

BCG

Anti-Hepatite B

DPT + Hib

Anti-polio

Anti-Sarampo

Anti-Amarílica

Triplice viral s

Outras

Teve alguma reação não esperada após receber alguma vacina? ( ) sim ( )não

Se sim, qual (s)_____________________________________________________

Usou algum tipo de remédio: ( )sim ( )não) Qual:_________________________

DOENÇAS:

( )parasitoses ( )catapora ( )dengue ( )hepatite ( ) pneumonia ( ) malária

outras:______________________________________________________________

HISTÓRIA ALIMENTAR

Tempo de amamentação (meses):_________________N.º de refeições/dia:_______

Alimentos mais freqüentes:______________________________________________

Come Peixe: ( )não ( )sim, tipo de peixe:___________________________________

Freqüência do consumo de peixe:________ Quantidade/dia:____________

156

Consome enlatado: ( )sim ( )não Quais:__________________________________

Come ovos de aves domésticas: ( )sim ( ) não tipo de ave:_______________

EDUCAÇÃO

Educação Paterna (anos):_________ Educação Materna (anos):_______________

Renda Familiar:_____________________________________

Freqüenta creche/escola: ( )sim ( )não Tempo de permanência no local:______horas

Qual o meio de informação que a criança dispõe:____________________________

Irmãos: número total:..................... Sexo e idade em ordem decrescente:

Idade

Sexo

Saúde dos irmãos: ( )boa ( )com problemas, Quais:_______________________

CONDIÇÕES DE MORADIA DA FAMÍLIA:

Situação da residência: ( )própria ( )alugada ( )de parente/amigos ( )cedida pelo

empregador ( )madeira ( )alvenaria ( )enchimento ( )em terreno alagado

Presença de insetos e roedores:( )não ( )sim, qual:_________________________

Quantos moram na casa:_____ Entra luz em todos os cômodos: ( )sim ( )não

Destino das fezes: ( )fossa seca ( )enterrado ( )céu aberto ( )rio ( ) fossa negra

Abastecimento de água: ( )rio ( )encanada ( )Poço ( )torneira pública ( ) outra:______

EXAME FÍSICO:

Peso:___________ Altura:_______ PC:_______

Idade que começou a andar:_______ Idade que começou a falar:____________

Outros achados:____________________________________________________

___________________________________________________________________

DESENVOLVIMENTO NEUROPSICOMOTOR:

Idade Cronológica: ______

CONDUTA

Idade (meses)

MOTORA

ADAPTATIVA

LINGUAGEM

compreensiva

expressiva

PESSOAL-SOCIAL

Quociente de Desenvolvimento (QD) = ____________________

Impressão diagnóstica:_______________________________________________

Avaliador:_______________________________________

157

(ANEXO 12)

Etapas do desenvolvimento infantil segundo Gesell

Idade

Adaptativo

Motor Grosseiro

Motor Fino

Linguagem

Pesssoal-Social

semanas

Seguimento restrito do

olhar

Reflexo tônico-cervical

Punhos fechados

Olha para a face do

examinador

semanas

Olha para chocalho na

mão

Postura simétrica

Mãos abertas;

arranha e agarra

Sorri; vocaliza

socialmente

Brinca com as mãos;

reconhece a mamadeira

semanas

Transfere o cubo de mão

Senta; projeta-se para frente

apoiando nas mãos;

sustenta peso com MMII

Agarra o cubo com

as mãos; tenta pegar

objetos pequenos

Vocaliza para

brinquedos; emite

sons consonantais

Brinca com os pés; Toca

na imagem no espelho

semanas

Segura mamadeira;

segura objetos pequenos

Senta-se sem ajuda;

engatinha; fica de pé

Solta objetos; aponta

com o dedo em

direção a objeto

Palavra-frase; imita

sons da fala

Brinca de jogos simples de

berçário; come biscoito

com as mãos

semanas

Coloca cubo dentro do

copo; tenta torre com 2

cubos

Anda com ajuda; dá alguns

passos sem apoio

Preensão em pinça

Fala 2-3 palavras;

reconhece objeto pelo

nome

Coopera no vestir-se;

brinca com a bola

18 meses

Retira pequeno objeto do

copo; rabisca

espontaneamente

Anda com equilíbrio;

agacha-se

Torre de 3 cubos;

folheia 2-3 páginas

de livro de uma só

vez

Jargões; reconhece

figuras

Usa a colher derramando;

puxa o brinquedo andando

24 meses

Constrói torre com 6

cubos; imita círculo com

lápis

Corre com equilíbrio; chuta

bola

Torre com 6 cubos;

folheia páginas 1 por

vez

Usa frases;

compreende ordens

simples diretas

Coloca partes simples da

vestimenta; brinca com

bonecos

36 meses

Imita ponte com 3 cubos;

copia círculo

Fica num pé só; pula

Torre com 10 cubos;

segura o lápis de

forma adequada

Fala formando

sentenças; responde a

questões simples

Usa a colher

adequadamente; coloca

sapatos; aguarda a vez

48 meses

Imita portão com 5 cubos;

copia linhas cruzadas

Pula num pé só; pulo amplo

Traços entre linhas

Usa conjunções;

compreende

preposições

Lava e enxuga a face;

brinca de forma interativa

60 meses

Conta 10 objetos; copia

triângulo

Pula no pé só

alternadamente

Fala sem dislalias;

pergunta ―por que?‖

Veste-se sem auxílio;

pergunta o significado das

palavras

158

APÊNDICE

Calendário Básico de Vacinação da Criança 2007

IDADE

VACINAS

DOSES

DOENÇAS EVITADAS

Ao nascer

BCG - ID

dose única

Formas graves de tuberculose

Vacina contra hepatite B (1)

1ª dose

Hepatite B

1 mês

Vacina contra hepatite B

2ª dose

Hepatite B

2 meses

Vacina tetravalente (DTP + Hib)

(2)

1ª dose

Difteria, tétano, coqueluche, meningite e outras

infecções causadas pelo Haemophilus influenzae

tipo b

VOP (vacina oral contra pólio)

1ª dose

Poliomielite (paralisia infantil)

VORH (Vacina Oral de

Rotavírus Humano) (3)

1ª dose

Diarréia por Rotavírus

Vacina tetravalente (DTP + Hib)

2ª dose

Difteria, tétano, coqueluche, meningite e outras

infecções causadas pelo Haemophilus influenzae

tipo b

4 meses

VOP (vacina oral contra pólio)

2ª dose

Poliomielite (paralisia infantil)

VORH (Vacina Oral de

Rotavírus Humano) (4)

2ª dose

Diarréia por Rotavírus

6 meses

Vacina tetravalente (DTP + Hib)

3ª dose

Difteria, tétano, coqueluche, meningite e outras

infecções causadas pelo Haemophilus influenzae

tipo b

VOP (vacina oral contra pólio)

3ª dose

Poliomielite (paralisia infantil)

Vacina contra hepatite B

3ª dose

Hepatite B

9 meses

Vacina contra febre amarela (5)

dose inicial

Febre amarela

12 meses

SRC (tríplice viral)

dose única

Sarampo, rubéola e caxumba

15 meses

VOP (vacina oral contra pólio)

reforço

Poliomielite (paralisia infantil)

DTP (tríplice bacteriana)

1º reforço

Difteria, tétano e coqueluche

4 - 6 anos

DTP (tríplice bacteriana

2º reforço

Difteria, tétano e coqueluche

SRC (tríplice viral)

reforço

Sarampo, rubéola e caxumba

10 anos

Vacina contra febre amarela

reforço

Febre amarela

(1) A primeira dose da vacina contra a hepatite B deve ser administrada na maternidade, nas primeiras 12 horas de vida do

recém-nascido. O esquema básico se constitui de 03 (três) doses, com intervalos de 30 dias da primeira para a segunda dose e

180 dias da primeira para a terceira dose.

(2) O esquema de vacinação atual é feito aos 2, 4 e 6 meses de idade com a vacina Tetravalente e dois reforços com a Tríplice

Bacteriana (DTP). O primeiro reforço aos 15 meses e o segundo entre 4 e 6 anos.

(3) É possível administar a primeira dose da Vacina Oral de Rotavírus Humano a partir de 1 mês e 15 dias a 3 meses e 7 dias

de idade (6 a 14 semanas de vida).

(4) É possível administrar a segunda dose da Vacina Oral de Rotavírus Humano a partir de 3 meses e 7 dias a 5 meses e 15

dias de idade (14 a 24 semanas de vida). O intervalo mínimo preconizado entre a primeira e a segunda dose é de 4 semanas.

(5) A vacina contra febre amarela está indicada para crianças a partir dos 09 meses de idade, que residam ou que irão viajar

para área endêmica (estados: AP, TO, MA MT, MS, RO, AC, RR, AM, PA, GO e DF), área de transição (alguns municípios

dos estados: PI, BA, MG, SP, PR, SC e RS) e área de risco potencial (alguns municípios dos estados BA, ES e MG). Se

viajar para áreas de risco, vacinar contra Febre Amarela 10 (dez) dias antes da viagem.

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