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Universidade de São Paulo
Faculdade de Saúde Pública
Exposição a chumbo e comportamento anti-social
em adolescentes
Kelly Polido Kaneshiro Olympio
São Paulo
2009
Tese apresentada ao Programa de Pós-Graduação em
Saúde Pública para obtenção do título de Doutor em
Saúde Pública.
Área de Concentração: Saúde Ambiental
Orientador: Profa. Dra. Wanda Maria Risso Günther
Co-orientador: Prof. Dr. Etelvino José H. Bechara
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Exposição a chumbo e comportamento anti-
social em adolescentes
Kelly Polido Kaneshiro Olympio
São Paulo
2009
Tese apresentada ao Programa de Pós-Graduação
em Saúde Pública para obtenção do título de Doutor
em Saúde Pública.
Área de Concentração: Saúde Ambiental
Orientador: Profa. Dra. Wanda Maria Risso Günther
Co-orientador: Prof. Dr. Etelvino José H. Bechara
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forma impressa como eletrônica. Sua reprodução total ou parcial é permitida
exclusivamente para fins acadêmicos e científicos, desde que na reprodução
figure a identificação do autor, título, instituição e ano da tese.
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DEDICATÓRIA
Ao meu Senhor e Salvador Jesus Cristo
A Ele toda honra e toda glória, agora e para sempre. Amém!
A Nossa Senhora, Maria Santíssima
Dulcíssima e cuidadosa mãe que me envolveu em seu sagrado manto em
todos os momentos de alegrias e de dificuldades.
Ao meu marido, Antonio Carlos
Sem seu auxílio e motivação, absolutamente nada disso teria sido possível.
À minha filha, Maria Clara
Tão pequena e tão compreensiva nos momentos em que estive ausente
para concluir este trabalho.
Aos meus pais, José e Lourdes
Por sempre estarem disponíveis em todos os momentos de necessidade.
Aos meus irmãos, Keith e Marcio
Pelo carinho, amizade e incentivo durante toda a minha vida.
Dedico este trabalho a vocês com todo o amor de meu coração!
.
AGRADECIMENTOS
Aos voluntários desta pesquisa e seus familiares. Sem a participação deles,
nada teria sido possível.
À Creche São José, ao Centro Irmã Adelaide e ao Projeto Girassol por
cederem o espaço físico e facilitarem o contato com seus alunos para a
realização de entrevistas e coletas de amostras de esmalte dentário.
À Comunidade Marianista Católica de Bauru por me indicar possíveis
locais onde eu poderia realizar o trabalho de campo e intermediar os
contatos pessoais necessários.
À CAPES, pela bolsa de estudos a mim concedida durante os quatro anos
de Doutorado.
À FAPESP pelo auxílio financeiro fornecido, através dos processos nº
01/09641-1 e nº 06/56530-4.
Ao CNPq, Projeto do Milênio, Redoxoma, pelo auxílio financeiro.
Aos professores Eurivaldo Sampaio de Almeida, José Carlos Seixas,
Cássia Maria Buchalla, Maria Regina Alves Cardoso, Chéster Luiz
Galvão César, Eliseu Alves Waldman, Oswaldo Yoshimi Tanaka, Sueli
Gandolfi Dallari, Paulo Antonio Fortes. Aos senhores, serei eternamente
grata pelos conhecimentos adquiridos durante as disciplinas que cursei com
tanta satisfação e interesse durante os meu quatro anos de Doutorado. Os
senhores, a cada aula, despertavam-me o orgulho de estudar na Faculdade
de Saúde Pública da USP. Parabéns pela competência e brilhantismo na
exposição de idéias e conteúdos. Muito obrigada!
.
Aos professores Marília Afonso Rabelo Buzalaf (Faculdade de Odontologia
de Bauru, USP) e Manuel Valentim de Pera Garcia (Instituto de Matemática
e Estatística, USP) pela valorosa colaboração metodológica e na discussão
dos resultados obtidos.
Ao Prof. Dr. Antonio Francisco da Silva Marques e sua equipe
(Departamento de Educação, Faculdade de Ciências, UNESP – Bauru) pela
colaboração durante o trabalho de campo, permitindo que eu tivesse acesso
aos dados do censo realizado no bairro Ferradura Mirim, Bauru, SP.
À Dra. Clarice Umbelino de Freitas (Secretaria de Saúde do Estado de São
Paulo, Centro de Vigilância Epidemiológica) pelas importantes sugestões
durante o meu exame de qualificação, resultando no terceiro artigo que
compõe esta tese.
Às professoras Cássia Maria Buchalla e Maria Regina Alves Cardoso
(Departamento de Epidemiologia, Faculdade de Saúde Pública, USP) pela
importante contribuição durante o exame de qualificação, delineamento da
pesquisa e discussão dos resultados.
Ao Prof. Dr. Pedro Vitoriano de Oliveira (Departamento de Química
Analítica, Instituto de Química, USP) pela rotineira disponibilidade em
responder meus questionamentos e pelas análises laboratoriais.
À Profa. Dra. Cleide Lavieri Martins (Departamento de Prática de Saúde
Pública, Faculdade de Saúde Pública, USP) por todos os conhecimentos
transmitidos durante o meu estágio no Programa de Aperfeiçoamento ao
Ensino (PAE), na disciplina Fundamentos de Saúde Pública em Educação
Física (Escola de Educação Física e Esporte da USP). Foi um prazer
participar de cada uma das aulas da disciplina, aprendendo como ensinar
Saúde Pública e despertar o interesse de alunos do 1º ano da graduação
para o assunto. Muito obrigada pela oportunidade.
.
À minha grande amiga, Vanessa Eid da Silva Cardoso, por me apresentar
o Prof. Dr. Etelvino José Henriques Bechara, mentor intelectual do projeto
que resultou nos artigos I e II que compõem esta tese e meu co-orientador,
possibilitando o desenvolvimento deste trabalho. A você, Van, todo meu
carinho, amizade e agradecimento.
.
AGRADECIMENTOS ESPECIAIS
À Profa. Dra. Wanda Maria Risso Günther, por me acolher na Faculdade
de Saúde Pública da Universidade de São Paulo, permitindo a realização de
um sonho - estudar nesta instituição. Pela orientação, respeito e confiança a
mim dedicados durante todo este tempo.
Ao Prof. Dr. Etelvino José Henriques Bechara, pela confiança que sempre
demonstrou ter em mim, o que funcionou como combustível nos muitos
momentos difíceis. Obrigada pela orientação e pelo inestimável auxílio
durante todo o desenvolvimento deste trabalho.
Aos senhores, a minha gratidão e profunda admiração.
.
RESUMO
Olympio KPK. Exposição a chumbo e comportamento anti-social em
adolescentes [Tese de Doutorado]. São Paulo: Faculdade de Saúde Pública
da USP; 2009.
Introdução- A intoxicação por chumbo é um conhecido problema de saúde
pública e o envenenamento por este metal pode causar danos a vários
órgãos, especialmente ao Sistema Nervoso Central de crianças em
desenvolvimento. Objetivo geral- estudar a associação entre exposição a
chumbo e comportamento anti-social (CAS) em adolescentes brasileiros.
Objetivos específicos: a) analisar a associação entre exposição a chumbo
e CAS / cometimento de atos infracionais (CAI); b) estudar potenciais fontes
de exposição domiciliar a chumbo que mais estão associadas a altas
concentrações de chumbo no esmalte dentário (CCED) e; c) avaliar o
impacto de alterações metodológicas na técnica de microbiópsia ácida de
esmalte dentário superficial (MAEDS) sobre CCED e profundidade da
bíópsia. Métodos- Um estudo transversal foi conduzido com 173 jovens
(Bauru, SP). MAEDS foram realizadas nestes jovens por dois diferentes
protocolos metodológicos. Além disso, questionários sobre comportamento
dos adolescentes e exposição a possíveis fontes de contaminação por
chumbo foram aplicados a pais e adolescentes. Análises de regressão
logística, testes de Wilcoxon e testes t pareados foram aplicados aos dados.
Resultados- Odd ratios ajustados para covariáveis indicaram que alta
CCED está associada a risco aumentado de exceder o escore clínico para
queixas somáticas, problemas sociais, comportamento de quebrar regras e
problemas externalizantes (IC 95%). Alta CCED não foi associado com
escores elevados de CAI. Os fatores de risco mais associados com alta
CCED foram residir em área contaminada ou até 2 km da área contaminada
e trabalhar na fabricação de tintas, pigmentos, cerâmicas ou baterias. A
profundidade da biópsia, calculada pela fórmula da altura do cilindro, para
.
um dos protocolos, levou a resultados errôneos de profundidade da biópsia,
confirmados por testes de perfilometria. Conclusões- A exposição a altos
níveis de chumbo parece disparar o estabelecimento de CAS, o que alerta
para a necessidade de desenvolvimento e implantação de políticas públicas
de saúde que previnam o envenenamento da população por chumbo.
Adolescentes foram expostos ao chumbo por algumas fontes estudadas, no
Brasil. O esmalte dentário é um marcador fidedigno e a MAEDS é bastante
útil e confiável. No entanto, CCEDs não podem ser comparadas entre
resultados de pesquisas diferentes quando houver qualquer variação
metodológica entre os estudos, havendo a necessidade da padronização do
procedimento.
Descritores: Intoxicação por chumbo; Intoxicação do sistema nervoso por
chumbo; Transtorno da conduta; Transtornos do comportamento social,
Violência; Razão de chances; Fatores de risco; Esmalte dentário; Biópsia;
Biomarcador; Chumbo; Saúde ambiental.
.
ABSTRACT
Olympio KPK. Lead exposure and antisocial behavior in Brazilian
adolescents [Thesis]. São Paulo (BR): Faculdade de Saúde Pública da
Universidade de São Paulo; 2009.
Introduction- Lead poisoning is a long known public health problem. Thus,
lead exposure may cause damage to diverse organs, especially in the
Central Nervous System of children in developing process. Objectives- a) to
analyze the association between lead exposure and antisocial / delinquent
behavior; b) to study the potential sources of lead home exposure more
associated to high dental enamel lead levels (DELL) and c) to evaluate two
distinct enamel biopsy protocols in relation to biopsy depth and DELL.
Methods- A cross-sectional study was conducted with 173 adolescents
(Bauru, SP, Brazil). Surface dental enamel (SDE) etch-acid microbiopsies
were performed in upper central incisors of these youths by two different
methodological protocols. In addition, questionnaires about adolescents’
behavior and about possible sources of lead contamination were responded
by youths and their parents. Logistic regression, Wilcoxon and paired t tests
were applied to data. Results- Odd ratios adjusted for familial and social
covariates indicated that high DELL is associated with increased risk of
exceeding the clinical score for somatic complaints, social problems, rule-
breaking behavior (T≥70) and externalizing problems (T≥63) (CI 95%). High
DELL was not found to be associated with elevated SRD scores. The risk
factors associated to high DELL were residing in contaminated area or close
.
proximity and working in paints, pigments, ceramic or batteries
manufacturing. The biopsy depth, calculated by the cylinder formula, for
Protocol II induced misleading results, as confirmed by profilometry tests.
Conclusions- It seems that exposure to high lead levels can indeed trigger
antisocial behavior, which claims for public policies to prevent lead poisoning.
Adolescents were exposed to lead, by some studied sources, in Brazil. SDE,
measured by etch-acid microbiopsy, is a reliable biomarker, but DELL could
not be compared when there is some methodological variation among the
studies. A standardization of the procedure is necessary.
Descriptors: Lead poisoning; Lead poisoning, Nervous System; Conduct
disorder; Social behavior disorders; Violence; Odds ratio; Risk factors; Dental
enamel; Biopsy; Biomarker; Lead; Environmental health.
.
APRESENTAÇÃO
Esta tese de Doutorado é fruto de um longo e árduo trabalho
desempenhado durante quatro anos. Como cirurgiã-dentista, interessada
pelas questões de Saúde Pública, sempre senti necessidade de ter uma
experiência maior de interdisciplinaridade, já que o meu Mestrado em
Odontologia em Saúde Coletiva foi unicamente voltado para a Odontologia
sem um aprofundamento na interface com outras especialidades, interface
essa que a Saúde Pública apresenta como característica inerente da área.
Quando decidi que iria fazer meu Doutorado na Faculdade de Saúde Pública
da Universidade de São Paulo, resolvi que meu objetivo era ultrapassar a
Odontologia em Saúde Coletiva, alcançando a Saúde Coletiva em sua
essência. Mesmo assim, a princípio, nunca imaginei que este desejo seria
transformado em realidade, cujo resultado não foi um trabalho
interdisciplinar, mas um trabalho transdisciplinar, no qual as diversas áreas
envolvidas contribuiriam tanto e na mesma proporção para que os esforços
de todas estas subáreas unidas – Saúde Ambiental, Bioquímica, Química
Analítica, Epidemiologia, Estatística, Toxicologia, Psicologia, Medicina,
Odontologia, Sociologia, Matemática, se não me esqueço de mais nenhuma
– resultassem nos quatro artigos apresentados neste volume.
De forma a melhorar a compreensão do presente estudo, é importante
que seja entendida a morbidade psiquiátrica, cuja associação está sendo
estudada com relação à concentração de chumbo no esmalte dentário. O
.
comportamento anti-social é considerado como sendo a sintomatologia
central do transtorno da conduta, a mais freqüente categoria de transtornos
psiquiátricos em crianças e jovens. O quadro clínico do transtorno da
conduta é caracterizado por comportamento anti-social persistente com
violação de normas sociais ou direitos individuais. Crianças e adolescentes
com transtorno da conduta costumam agredir e maltratar pessoas e animais,
destruir propriedade alheia, envolver-se em roubos e assaltos e violar as
regras estabelecidas. Quando o quadro clínico é mais grave, pode ocorrer o
cometimento de assaltos com a utilização de armas, estupros e homicídios
(BORDIN 1996).
Inicialmente, o delineamento do projeto de pesquisa baseou-se em
um estudo de caso-controle, onde os casos foram criteriosamente definidos
como adolescentes julgados por cometimento de ato infracional e
sentenciados a regime de internato na antiga Fundação para o Bem Estar do
Menor (FEBEM), atual Fundação Casa. Três unidades foram selecionadas e
tiveram a anuência da instituição para que o trabalho fosse realizado:
unidade de Bauru-SP, Rio Dourado e Vitória Régia (Lins-SP). O trabalho de
campo foi iniciado na unidade de Bauru-SP. Todos os adolescentes desta
unidade foram examinados e entrevistados, assim como também foram
entrevistados seus responsáveis. No entanto, a grande maioria das
amostras de esmalte dentário coletadas na Fundação Casa apresentou
contaminação por chumbo e muitas amostras não puderam ser
consideradas na pesquisa. Na investigação da fonte de contaminação,
constatei que os tubos utilizados na coleta de material apresentavam
.
chumbo em concentrações aleatórias. Esta foi uma grande perda, pois o
trabalho na unidade havia sido extremamente desgastante e a
descontaminação prévia dos tubos já havia sido discutida antes do início do
trabalho, não sendo realizada e resultando no fato exposto. Além disso,
neste cenário, a presidência da FEBEM (nomeada assim, na época) foi
alterada e nossa permissão para entrada nas unidades foi suspensa. Mesmo
após todos nossos apelos para que nos fosse autorizada a continuação da
pesquisa, não pudemos concretizar o trabalho na instituição.
Nossos controles, segundo o projeto inicial, viriam de escolas da rede
pública de ensino, adolescentes com características semelhantes aos casos,
com exceção da condição de cometimento de ato infracional. A direção
regional de ensino de Bauru, na pessoa da Sra. Vera Nilce L. Jarussi Gomes
de Sá, negou a autorização para a realização da pesquisa nas escolas da
rede, alegando que a pesquisa geraria “polêmica entre os familiares dos
alunos e prejudicaria a rotina das secretarias das escolas com as dúvidas
que seriam levantadas pelos pais, os quais procurariam as secretarias para
tirar estas dúvidas”. A importância da pesquisa foi destacada, a manutenção
da ordem da rotina escolar foi assumida como compromisso, mas a dirigente
mostrou-se totalmente refratária a colaborar com o projeto, negando
bruscamente a autorização para o desenvolvimento da presente pesquisa.
Após todos estes fatos, excluindo a opção de mudança de projeto,
restou-me a opção de mudança de delineamento. O recrutamento de
adolescentes de bairros com altos índices de criminalidade surgiu, neste
contexto, como único cenário amostral possível. Assim, as visitas a projetos
.
sociais que atendiam jovens residentes destas áreas, com visitas
domiciliares nos locais onde as excursões pelo bairro eram possíveis,
agregando ainda jovens pela técnica epidemiológica da bola de neve foram
as estratégias utilizadas para que adolescentes em situação de risco de
apresentarem comportamento anti-social fossem encontrados para
comporem nossa amostra. No bairro onde as visitas domiciliares
representavam um risco à integridade dos pesquisadores, jovens da
comunidade foram contatados para a realização destas visitas e, assim, foi
composta a amostra apresentada nos artigos que compõem o capítulo de
resultados e discussão deste texto.
Esta apresentação se faz necessária para que os leitores
compreendam o que levou uma cirurgiã-dentista a se embrenhar por uma
pesquisa de caráter tão diferente das outras que vinha realizando até então,
apesar da Toxicologia estar presente nas minhas pesquisas, no que toca ao
estudo do flúor como causa da fluorose dentária. Além disso, a exposição de
todos os imprevistos ocorridos faz o leitor se lembrar dos caminhos
acidentados pelos quais muitas pesquisas avançam, cujos esforços e
méritos são coroados com a publicação dos resultados destas pesquisas em
revistas reconhecidas pela comunidade científica, ultrapassando a
impressão de volumes, os quais, muitas vezes, só servem para se juntar a
outros em prateleiras de bibliotecas, sendo consultados em uma freqüência
ínfima quando comparada à visibilidade dada a estes mesmos textos
transformados em artigos científicos.
.
Este volume é composto por quatro artigos submetidos ou que serão
submetidos a periódicos científicos. Um artigo de revisão de literatura
(Manuscrito I) substitui a introdução da tese. Esta revisão de literatura foi
redigida após a constatação de que há poucas publicações científicas que
se dedicaram a discutir o estabelecimento de comportamento anti-social
associado à exposição ao chumbo, além da necessidade do
desenvolvimento de políticas públicas que protejam a população brasileira
deste perigo. Esta revisão também é resultado de um trabalho realizado no
início do meu Doutorado para que eu me inteirasse do assunto e pudesse
estudar o conteúdo envolvido com o meu problema de pesquisa. Como era
nosso objetivo levantar a discussão entre os profissionais de Saúde Pública
e formuladores de políticas públicas de saúde, este artigo será publicado na
Pan American Journal of Public Health / Revista Panamericana de Salud
Pública, a qual é uma publicação da Organização Panamericana de Saúde
Pública (OPAS).
Os capítulos referentes aos resultados e discussão, estão sendo
representados por três artigos: Manuscrito II) “Surface dental enamel lead
levels and antisocial behavior in Brazilian adolescents” (preparado para a
Neurotoxicology and Teratology, FI=2,444); Manuscrito III) “Risk factors
associated with high lead levels measured in the surface dental enamel from
Brazilian youths” (submetido ao Bulletin of the World Health Organization,
FI=4,019); e Manuscrito IV) “Methodological alterations of surface dental
enamel microbiopsies for lead body burden measurement” (submetido à
Toxicological Sciences, FI=3,814). O primeiro destes artigos é fruto do nosso
.
problema de pesquisa principal – o estudo da associação entre exposição a
chumbo e comportamento anti-social. Como nós aplicamos um questionário
referente à identificação de possíveis fatores de confusão, aproveitamos
também para investigar possíveis fontes de exposição a chumbo que
estivessem presentes na rotina familiar daqueles adolescentes, o que
resultou no segundo artigo apresentado. No transcorrer do trabalho,
diferenças metodológicas e de resultados foram sendo detectados entre o
nosso trabalho e de outros grupos de pesquisa, o que gerou a curiosidade
científica de se estudar mais especificamente o esmalte dentário como
marcador biológico para chumbo, gerando o quarto artigo fruto da tese e
terceiro artigo do capítulo de resultados e discussão.
Espero com esta apresentação e após a realização desta pesquisa,
realizados com todo o rigor científico e carinho pessoal, poder ter contribuído
para a Ciência, além de tornar mais agradável o trabalho do leitor que se
aventurar a avaliar criticamente ou consultar displicentemente este volume.
.
ÍNDICE
1
INTRODUÇÃO
27
1.1 Neurotoxicity and aggressiveness triggered by low-level
lead in children – a review (Manuscrito I)
28
1.1.1 Abstract 29
1.1.2 Introduction 30
1.1.3 Lead toxicity: a short history 31
1.1.4 Major sources of lead exposure 33
1.1.5 Biochemical mechanisms of lead toxicity 37
1.1.6 Lead poisoning of the infant nervous system 39
1.1.7 Lead effects on IQ and social behavior 42
1.1.8 Primary prevention: benefits for public health 48
2
75
3
MÉTODOS
76
4
RESULTADOS E DISCUSSÃO
88
4.1 Surface dental enamel lead levels and antisocial behavior
in Brazilian adolescents (Manuscrito II)
89
4.2 Risk factors associated with high lead levels measured in
the surface dental enamel from Brazilian youths (Manuscrito
III)
120
4.3 Methodological alterations of surface dental enamel
microbiopsies for lead body burden measurement (Manuscrito
IV)
146
5
CONCLUSÕES
e RECOMENDAÇÕES
171
6
REFERÊNCIAS
173
ANEXOS
175
Anexo 1 – Carta de Informação e Consentimento Livre e Esclarecido 175
Anexo 2 – Inventário CBCL 178
Anexo 3 – Questionário SRD 179
Anexo 4 – Questionário Fatores de Confusão e Fontes de Exposição 181
INFORMAÇÕES
CURRICULARES
183
.
LISTA DE ABREVIATURAS
ADM - Assessment Data Manager
AFQT - Armed Forces Qualification Test
AIP - Acute Intermittent Porphyria
ALA - 5-aminolevulinic acid
ALAD - Delta-aminolevulinic acid dehydratase
CAS- Comportamento anti-social
CAI- Cometimento de atos infracionais
CBCL - Child Behavior Checklist
CCED - Concentração de Chumbo no Esmalte Dentário
CDC - Centers for Disease Control and Prevention
CEP - Cumulative Exposure Project
CETESB - Companhia de Tecnologia de Saneamento Ambiental
CI - Confidence Interval
CNPq - Conselho Nacional de Desenvolvimento Científico e
Tecnológico
CNS - Central Nervous System
CONAMA - Conselho Nacional do Meio Ambiente
DELL - Dental Enamel Lead Levels
DNA - Deoxyribonucleic acid
DSM - Diagnostic and Statistical Manual of Mental Disorders
EC-THGA - End-Capped Transverse-Heated Graphite Atomizers
.
EIA-RIMA - Estudo de Impacto Ambiental – Relatório de Impacto
Ambiental
EPA - Environmental Protection Agency
FAPESP - Fundação de Amparo à Pesquisa do Estado de São Paulo
FI - Fator de Impacto
GABA - Ácido gama-aminobutírico
GFAAS - Graphite Furnace Atomic Absorption Spectrometry
HCl - Cloridric acid
HNO
3 -
Nitric acid
5-HIAA - 5-hydroxyindoleacetic acid
IC - Intervalo de Confiança
ICP-OES - Inductively Coupled Plasma Optical Emission Spectrometry
IP - Instituto de Psicologia
IQ - Intelligence Quotient
KHN - Koop Hardness Number
KH
2
PO
4 -
Potassium dihydrogen phosphate
Km - Kilometer
LTCIP - Laboratório de Terapia Comportamental do Instituto de
Psicologia
MAEDS- Microbiópsia ácida de esmalte dentário superficial
Mg - Magnesium
Mg(NO
3
)
2 -
Magnesium nitrate
NHANES - National Health and Nutrition Examination Surveys
.
NLSY - National Longitudinal Survey of Youth
NMDA - N-Metil-D-Aspartato
OR - Odds ratio
P - Phosphorus
Pb - Lead
ppb - Parts per billion
ppm - Parts per million
Pb(NO
3
)
3 -
Lead nitrate
PNS - Peripheral Nervous System
Pd (NO
3
)
2 -
Palladium nitrate
SD - Standard Deviation
SDE - Surface Dental Enamel
SP - São Paulo
SRD - Self-Reported Delinquency
UN - United Nations
USA - United States of America
USP - Universidade de São Paulo
Zn - Zinc
WHO - World Health Organization
.
LISTA DE FIGURAS
Manuscrito I,
Figure 1 -
Effects of lead poisoning on human health………….
72
Manuscrito I,
Figure 2 -
Production of reactive oxygen species by the
aerobic oxidation of δ-aminolevulinic acid (ALA), a
heme precursor accumulated in lead poisoning,
ALA-driven oxidative damage to biomolecules, and
biological consequences observed in ALA-treated
rats and lead poisoned individuals……………………
73
Figura 1 - Local onde o adolescente respondia ao auto-relato
de cometimento de atos infracionais (Self-Reported
Delinquency)..............................................................
81
Figura 2 - Instrumental utilizado durante o exame clínico do
adolescente (A) e para posterior coleta de esmalte
dentário (B)................................................................
82
Figura 3 - Coleta da amostra de esmalte dentário.....................
83
Figura 4 – Vista do Bairro Ferradura Mirim, Bauru,
SP..............................................................................
84
Figura 5 - Centro Irmã Adelaide, localizado no Bairro
Ferradura Mirim, Bauru, SP......................................
85
Figura 6 - Vista do Núcleo Habitacional Fortunato Rocha
Lima, Bauru, SP........................................................
86
.
Figura 7 - Tipo de residência presente no Núcleo Fortunato
Rocha Lima...............................................................
87
Manuscrito
IV, Figure 1 -
Profilometry drawing (A. Protocol I: 4 mm in
diameter, 35 s, 10 uL HCl; B. Protocol II: 1.6 mm in
diameter, 20 s, 5 uL HCl)...........................................
170
.
LISTA DE TABELAS
Manuscrito I
Table 1 - Annual benefits of a 1 µg/dL reduction in the mean
blood lead concentration of US infant population,
according to Schwartz (1994)………………………...
74
Manuscrito II
Table 1 - Characteristics of the entire study sample and
according to sex subgroups…………………………...
114
Table 2 - Mean (µg/g, ±SD) (n) of dental enamel lead
concentrations (75
th
percentile) for clinical and
normal subjects, considering somatic, social,
conduct and externalizing problems and rule-
breaking behavior………………………………………
115
Table 3 - Number and percentage of normal and clinical
subjects, according to the CBCL profiles and the
SRD scores, in the low- and high-lead groups……...
117
Table 4 - Association between dental enamel lead
concentration and Child Behavior Checklist (CBCL)
profiles or Self-Reported Delinquency (SRD)
scores……………………………………………………
118
Manuscrito III
Table 1 - Descriptive variables (n) for all subjects and for
.
sample stratified by sex………………………………..
143
Table 2 - Mean (µg/g, ±SD) (n) of dental enamel lead
concentrations for exposed and non-exposed
subjects, considering risk factor A (to reside in
contaminated area or near of there); risk factor B (to
work in paints, pigments, ceramic or batteries
manufacturing), risk factor C (to do home use of
glazed ceramic, pirate toys, anticorrosive enamel
without covering paint in gates, and/or
commercializing used car batteries), and risk factor
D (smoking)……………………………………………..
144
Table 3 - Association between dental enamel lead
concentration and risk factor A (to reside in
contaminated area or near of there); risk factor B (to
work in paints, pigments, ceramic or batteries
manufacturing), risk factor C (to do home use of
glazed ceramic, pirate toys, batteries removed from
cars, and/or anticorrosive enamel without covering
paint in gates), and risk factor D (smoking)………….
145
Manuscrito IV
Table 1 - Means ± SD and medians of biopsy depth [µm] and
dental enamel lead levels (DELL) [ppm] found in
both maxillary central incisors by protocol I (4 mm in
.
diameter; 35 s, 10 µL HCl) in the microbiopsies
samples for first and second removed layers……….
167
Table 2 - Means ± SD and medians of dental enamel lead
levels (DELL) [ppm] and biopsy depth [µm] found by
protocol I (4 mm in diameter; 35 s, 10 µL HCl) and
by protocol II (1.6 mm in diameter, 20 s, 5 µL HCl)
in the microbiopsies samples for first and second
layer in each protocol…………………………………..
168
Table 3 - Individual and total means (µm, number of surface
scanning) of dental enamel wear, measured by
profilometry, after in vitro surface dental enamel
etch-acid microbiopsies in bovine enamel blocks…..
169
Introdução 27
1 INTRODUÇÃO
Artigo aceito para publicação na Pan American Journal of Public Health /
Revista Panamericana de Salud Pública em 23/09/2008 (Manuscrito I).
"(...) Portanto, não importa quão pequeno
seja o começo; o que é bem feito uma
vez, está feito para sempre."
Henry David Thoreau
Introdução – Manuscrito I – Pan American Journal of Public Health 28
Neurotoxicity and aggressiveness triggered by low-level lead in
children – a review
Kelly Polido Kaneshiro Olympio
1
, DDS, MSc
Claudia Gonçalves
2
, BSc
Wanda Maria Risso Günther
1
, DDS, PhD
Etelvino José Henriques Bechara
3,4
, DDS, PhD
1
Faculdade de Saúde Pública, Departamento de Saúde Ambiental,
Universidade de São Paulo, Brazil Av. Dr. Arnaldo, 715, Pinheiros – São
Paulo – SP 01246-904
2
Centro de Extensão Universitária, São Paulo, Brazil R. Maestro Cardim,
370 – Bela Vista, São Paulo – SP 01323-000
3
Instituto de Química, Departamento de Bioquímica, Universidade de São
Paulo, Brazil Av. Prof. Lineu Prestes, 748, bloco 10, sala 1074, Cidade
Universitária, São Paulo - SP 05508-900
4
Departamento de Ciências Exatas e da Terra; Universidade Federal de São
Paulo, Brazil Rua Prof. Artur Ridel, 275; Jardim Eldorado; Diadema - SP
09972-270
Introdução – Manuscrito I– Pan American Journal of Public Health 29
Abstract
Lead is a ubiquitous, silent and devastating metal toxin, used since
ancient times. Lead-induced neurotoxicity acquired by low-level long-term
exposure to the metal has special relevance for children. A plethora of recent
reports has demonstrated a direct link between low-level lead exposure and
deficits in the neurobehavioral-cognitive performance manifested from
childhood through adolescence. In many studies, aggressiveness and
delinquency have also been suggested as symptoms of lead poisoning.
Several environmental, occupational and domestic sources of contaminant
lead and consequent health risks are largely identified and understood, but
the occurrences of lead poisoning remain numerous. There is an urgent need
for public health policies to prevent lead poisoning so as to reduce individual
and societal damages and losses. In this paper we point out unsuspected
sources of contaminant lead, discuss the economic losses and urban
violence possibly associated with the metal and review the molecular bases
of lead-induced neurotoxicity, emphasizing its effects on children’s and
teenagers’ social behavior, delinquency and IQ.
Key words: lead poisoning, neurotoxicity syndromes, oxidative stress,
juvenile delinquency.
Introdução – Manuscrito I – Pan American Journal of Public Health 30
Introduction
Lead (Pb) is known to be toxic to human health since ancient times. In
200 BC, Dioscerides stated prophetically that “lead makes the mind give
way” (1). Indeed, lead is now recognized as a devastating neurotoxin. The
widespread contamination of the environment by lead, the metal’s propensity
to cause a wide spectrum of toxic effects, and the millions of people affected
worldwide, both in poor and developed nations, make this insidious and
ubiquitous neurotoxicant a public health problem of global magnitude and
concern (2).
The levels of lead considered tolerable and putatively non-toxic for
children have been repeatedly lowered over the last three decades (3,4,5).
Using venous blood lead levels as a lead poisoning marker, the upper
acceptable limit for children was 60 µg/dL in the early 1960s, a level capable
of engendering overt physical symptoms (6). In 1970, after the recognition
that even lower blood lead levels may not produce overt physical symptoms
but can cause brain damage (7), the lead threshold was reduced to 40 µg/dL.
Since then, the upper limit of “acceptable” blood lead levels has been
successively lowered. In 1975, it was reduced to 30 µg/dL, in 1985 to 25
µg/dL, and finally, in 1991, the Centers for Disease Control and Prevention
(CDCs) definition of childhood lead poisoning set 10 µg/dL as the screening
action guideline. According to Bellinger (8), although this value only intends
to serve for a risk guidance and management tool, it probably permeates
biological significance for the individual child. Indeed, 10 µg/dL blood may be
Introdução – Manuscrito I– Pan American Journal of Public Health
.
31
taken as a threshold; therefore, a level of <10 µg/dL can be viewed as safe
and a higher level as toxic.
In truth, no single number can be cited as a threshold without
considering contextual factors such as endpoint of interest, the age of the
individual at exposure and assessment, the duration of blood level elevation,
and characteristics of the child-rearing environment (8). Recently, studies by
Lanphear et al (9) and Canfield et al (10)
have shown intellectual impairment
in children with blood lead concentrations below 10 µg/dL, and Chiodo et al
(11) have demonstrated child neurobehavioral deficits linked to 3 µg/dL
concentrations.
We point out herein unexpected sources of contaminant lead and
review the molecular bases of the neurotoxicity induced in children by low-
level lead, emphasizing its effects on social behavior, criminality and IQ.
Finally we discuss the urgent need for public health policies designed to
prevent lead poisoning.
Lead toxicity: a short history
According to Needleman (5), childhood lead poisoning was first
recognized only one century ago. He formally divided the temporal
accumulation of scientific information on lead poisoning into four stages.
First, reports on lead-poisoned children in Brisbane (Australia) in 1892,
although having reached epidemic proportion, were received with widespread
disbelief. Many of the homes in Brisbane were raised on piles, with large
Introdução – Manuscrito I– Pan American Journal of Public Health
.
32
wooden-enclosed verandas that served as play areas for children. The rails
were painted with white lead, which chalked and powdered under the hot
Brisbane sun (12). The origin of the epidemic - lead-containing paint - was
established in 1904, and lead paint was banned for household use in
Brisbane in 1920 (5). Today, the Environmental Protection Agency (EPA,
USA) establishes that paints must not contain more than 0.06% of lead in
their formulation.
The first report of infantile lead poisoning in the United States was
presented by Blackfan (12), in 1914, according to Needleman (5). In this
second stage of the lead poisoning history, only two outcomes were
recognized: death or complete recovery without any sequelae. This
misconception was refuted in 1943 with the first follow-up of children who had
recovered from acute toxicity. Previously, Levinson and Harris (13)
recommended that children should perhaps be followed on a long-term basis
to ascertain possible neurobehavioral disturbances. In 1943, lead poisoning
sequelae were well documented by Byers and Lord, who studied 20 children
who had symptomatic lead poisoning in early childhood and were followed
until school age. They found that 19 out of the 20 children presented
aggressive, antisocial, and uncontrollable behavior (14). Thus, in this third
stage, it was generally accepted that lead toxicity caused long-term
neurological impairments, although these deficits were thought to occur only
in children who had displayed clinical signs of encephalopathy during the
acute episode. The fourth stage began in the 1970s, when studies of children
Introdução – Manuscrito I– Pan American Journal of Public Health
.
33
with no clinical signs of toxicity showed deficits in IQ scores, attention, and
language (15,16,17).
Major sources of lead exposure
Lead is naturally encountered in the Earth lithosphere at
concentrations of c.a. 13 mg/kg. From the early sixth millennium BC, several
ancient civilizations were already employing lead to manufacture tableware,
trays and other decorative objects. The Romans believed that lead - the
“oldest” metal - was a precious gift of the Gods’ father, Saturn (Khronos, for
the Greeks), as they used lead to construct aqueducts to draw water from the
hills to Rome and to prepare lead acetate, a sweetener of wine daily
consumed by the Roman aristocrats. The name saturnism for lead poisoning
was coined after Saturn.
Lead occurring naturally or anthropogenically is encountered in all
environmental compartments, including air, water, soil, biota and human
beings. Inhalation, intake and dermal contact are described as distinct
pathways of human exposure. Lead may be ingested directly from
contaminated water, air, and soil and indirectly by consuming animals, plants
and their derivates. Environmental or occupational exposure may be
aggravated by inadequate protective behaviors, habits as well as socio-
economic factors. Lead is encountered in food, batteries, solders, plastics,
household paints and gas, but also in pottery utensils, glass nursing-bottles,
toys, glazed pottery, granite floors, calcium supplements, herbal medicines,
Introdução – Manuscrito I– Pan American Journal of Public Health
.
34
wild game, facial make-up and cigars. Lead affects the brain, kidneys, liver,
blood and testicles, leading to disturbances of learning, attention, IQ,
memory, hearing, sociability, hypertension, anaemia, nephropathies, sterility
and encephalopathies, in a lead level-dependent poisoning degree (Figure
1). Human uses of lead increased during the Industrial Revolution and in the
early 20
th
century, when there was high demand for anti-knock leaded gas,
lead-containing paints, canned foods, and car batteries. Importantly, the
population of American children with blood lead levels over 10 µg/dL declined
by 80% since lead was banned from gas, solder in canned food, and house
paint (18). The World Health Organization recommends constant research on
the various “silent” sources of lead exposure (19). Vigilance is crucial and
must be shared through community awareness, and by the better control of
the use of products suspected to contain lead as well as stricter
surveillance/testing of imported goods (20).
According to Goyer (21), exposure to lead-contaminated food was
most likely to have occurred from cans containing lead solder in the joints.
Outlawing of lead solder in canned food is estimated to have reduced the
average dietary intake of lead in 2-year-old children from 30 µg/day in 1982
to approximately 2 µg/day in 1991 (21,22,23). While banned in the US, lead
solder continues to be used in other countries, resulting in elevated lead
levels in some imported canned foods (22,23). Additional but less common
than other sources of exposure to dietary lead are ethnic food, dietary
Introdução – Manuscrito I– Pan American Journal of Public Health
.
35
supplements, folk medicines and moonshine. Hair/eyelash/eyebrow dyes can
be sources of dermal lead poisoning (23,24).
Ingestion of lead does not occur solely through dietary sources.
Currently, lead-containing paint sold in the USA between 1884 and 1978 is
the major source of lead ingestion in young American children (22,23).
Although banned in household paint since 1971, 80% of US houses built
before 1950, or 23 million units, contain leaded paint (5). In 2002, it was
reported that 65% of 38 million housing units in the United States were
painted with products considered lead-based hazard (25). The replacement
of highly toxic lead compounds in white paints by very expensive titanium
oxide-based paints and, hopefully in the near future, for non-toxic and
inexpensive aluminum phosphate or polyphosphate-based pigments
(Biphor
®
, Bunge Co.) (26) represents novel technologies to prevent lead
poisoning from paints.
Drinking water can also be contaminated by lead, either at the source
due to deposition from environmental sources or in the water distribution
system. The U.S. National Primary Drinking Water Regulations for Lead and
Copper state that water is unsafe if 10% of a municipality’s test sample is
determined to have lead levels greater than 15 ppb (27,25). Several authors
have pointed out that the municipal infrastructure distribution systems contain
components that may leach lead, such as lead service lines that connect the
water main to the consumer’s residence, lead pipes that supply water to the
inside of the residence, copper supply pipes that have been joined using lead
Introdução – Manuscrito I– Pan American Journal of Public Health
.
36
solder and lead-containing brass pipes and fixtures that can contain up to 8%
lead (28,29). A recent survey of the Washington, DC area by the CDC (30),
the DC Department of Health and the US Public Health Service estimate that
18% of over 30% of the analyzed population residing in this area had blood
lead levels above 5 µg/dL, leading to cognitive deficits in children (9,10).
In Brazil, Teixeira et al (31) have found that 11% of pipes used in the
water distribution systems of 100 schools of São Paulo present lead levels
higher than the limit considered safe by the WHO. In 2% of the water
samples, the lead level was found to be five-fold higher than the
recommended value (<10 µg/L, according to the WHO), thereby threatening
children’s neuropsychiatric health. This information raises the possibility of
lead-containing pipes being used in the water distribution system of São
Paulo’s schools. According to the São Paulo State Health Department (18),
about 80% of the lead found in urban air samples before 1982 was derived
from leaded gas. Car battery manufacture is the main source of secondary
lead (32), but other sources cannot be discarded.
Several studies have shown that high blood lead levels of preschool
children are strongly correlated with high lead levels in house dust
(33,34,35). This association has been attributed to dust intake from the
frequent hand-to-mouth behavior of young children. Flaking lead-based paint,
road dust, garden soil and airborne lead-bearing particles are believed to be
the sources of lead in household dust (36).
Introdução – Manuscrito I– Pan American Journal of Public Health
.
37
Leaded gas has caused more exposure to the metal than any other
source worldwide (37). Thus, it is not surprising that there is consensus
among international bodies, such as the World Bank, WHO and the UN
Commission on Sustainable Development, that countries must give up
leaded gas for the sake of public health. In 1994, the UN commission called
on governments worldwide to switch from leaded to unleaded petrol.
Nevertheless, up to year 2000, only 42 countries, including China, New
Zealand, the US, some Western and Eastern European countries, and
several Latin American countries, had phased out or were phasing out lead
from gas. India and a dozen or more countries in Latin America and Western
Europe were committed to making the shift by 2005, while the remaining 150
or so countries in the world have still not had decided (38).
Biochemical mechanisms of lead toxicity
Lead is a heavy metal with no apparent biological function. In spite of
extensive documentation of the toxic effects of lead on human health, the
molecular mechanisms underlying its poisonous effects on the central
nervous system (CNS) have yet to be clarified (39,40).
Bioactive lead is a divalent cation that binds strongly to sulfhydryl
groups of cysteine residues of proteins and enzymes. Lead toxicity can be
largely attributed to conformational changes undergone by enzymes and
structural proteins upon binding the lead ion, but this versatile toxic agent has
other targets as well. For example, lead interferes in the endogenous opioid
Introdução – Manuscrito I– Pan American Journal of Public Health
.
38
system (41) and efficiently breaks the ribosyl phosphate group of tRNA (42).
Many toxic properties of lead are putatively due to the metal’s capacity to
mimic and compete with calcium and zinc ions in finger proteins dependent
on these metals. (43).
Recent studies have also focused on the heme biosynthetic pathway,
where many lead interference sites are encountered. Thus, lead poisoning
can be considered a chemical or acquired porphyria (44). The thiol enzymes
δ-aminolevulinic acid dehydratase (ALAD) and ferrochelatase of this pathway
are extremely sensitive to lead. Inhibition of these enzymes increases,
respectively, ALA and protoporphyrin IX concentrations in urine, blood and
other tissues. ALA has long been known to compete with γ-aminobutyric acid
(GABA), a neurotransmitter in the cortex, hypothalamus and other tissues of
the CNS and the peripheral nervous system (45).
An increase of ALA in the blood circulation and brain areas could
contribute to triggering behavior disorders in patients carrying genetic
porphyrias, including acute intermittent porphyria (AIP) and hereditary
tyrosinemia type 1, and also in lead poisoned individuals. This hypothesis is
based on the fact that ALA has been shown in vitro to exhibit pro-oxidant
properties towards biological molecules (proteins, membranes, DNA) and
supramolecular structures (mitochondria, synaptosomes), as well as, in vivo,
in brain, liver and red muscles of ALA- or lead-treated rats and in the blood of
lead exposed workers (46,47,48,49,50) (Figure 2). Of utmost importance was
the finding that ALA-driven oxidative injury to GABA receptors in synaptic
membranes, synaptosomes, and GABA-rich brain slices leads to a two-fold
Introdução – Manuscrito I– Pan American Journal of Public Health
.
39
increase of the dissociation constant of the receptor-GABA complex (51) and
a significant decrease of GABA receptor population (52).
Fundamental questions about the molecular bases of the ALA-induced
neurological lesions remain unanswered. It is worth noting that acute
porphyric attacks of inborn and acquired porphyria patients correlate with
elevation of blood and urinary ALA and that lead exposed subjects with high
levels of lead (> 60 µg/dL) and ALA (> 1 µM) in the blood present
neurological manifestations similar to those in AIP (53,54,55).
Lead poisoning of the infant nervous system
Studies in several countries have estimated that about 4% of their
children have high blood lead levels (19). Children living in inner-city areas of
the United States may reach even higher prevalence. According to data
collected between 1976 and 1980, 17% of children presented lead blood
levels above 15 µg/dL; 5.2% higher than 20 µg /dL and 1.4% higher than 25
µg/dL (56). Lead poisoning is not considered a significant environmental risk
for children in rural areas of developing countries. However, in a study with
children living in the rural Philippines, 21% (601 of 2861 children) had blood
lead levels higher than 10 µg/dL. Blood lead levels were associated
independently with age, hemoglobin concentration, water source, roofing
material, expenditures and history of breastfeeding. The authors evaluated
possible environmental exposures among a sub-sample of children with
elevated blood lead levels and found multiple potential sources, such as
Introdução – Manuscrito I– Pan American Journal of Public Health
.
40
fossil-fuel combustion, lead paint (in or around 38% of homes) and
household items (57).
Children are more sensitive to lead than adults for many reasons.
Their exposure to lead is favored by the habit of taking things to the mouth
(pica habit). A child’s intestine absorbs lead much faster than the adult’s and
the developing infant CNS is more vulnerable to toxic agents than the mature
CNS, especially in the case of undernourished children. Neural proliferation,
differentiation and plasticity are strongly impaired by lead.
In the United States, overall childhood blood lead levels have declined
as a result of federal regulatory measures to reduce population exposure to
environmental lead. Screening data from the late 1960s and early 1970s
found that 20% to 45% of children tested had blood lead levels ≥40 µg/dL.
Between 1976 and 1980, the weighted geometric mean of blood lead among
1 to 5-year-old children in the US was 15 µg/dL (58). Data from the Third
National Health and Nutrition Examination Survey (NHANES III), phase 1
(1988 – 1991), showed a decline in the geometric mean of lead level to 3.6
µg/dL (58). NHANES III (1991 – 1994) showed a further decline in this
biomarker to 2.7 µg/dL. The NHANES III, phase 2 data indicated that ~4.4%
of 1- to 5-year-old children (~890,000 children) had blood levels ≥ 10 µg/dL
(59). Bernard et al (28) analyzed the data from NHANES III (1988-1994) and
found that the overall prevalence of blood levels ≥5 µg/dL was 25.6%
although most (76%) of these children had lead levels <10 µg/dL.
Including Latin American experience data, in Argentina, studies carried
out in Cordoba and Buenos Aires showed that between 10 and 40% of
Introdução – Manuscrito I– Pan American Journal of Public Health
.
41
children aging less than 15 presented blood lead levels higher than 10 µg/dL
(60). In Uruguay (61), at beginning of 2001, blood lead levels higher than 25
µg/dL were detected in children in La Teja, Montevideo. In this place, several
metal smelting plants and other industrial segments were operating during
the last 50 years. Because that, Public Health Ministry created a special team
that carried out another study, which showed that 61% of the 2351 studied
children presented blood lead levels higher than 10 µg/dL.
In Brazil, studies on environmental lead exposure are rare, limiting a
comprehensive understanding about its impact on the Brazilian public health
(62). Silvany-Neto et al (63) found blood lead levels mean of 36.7±20.7 µg/dL
in children living close to a primary lead smelting plant in Santo Amaro,
Bahia. In 1996, the same authors (64), using the Zn-protoporphyrin method,
found lead levels as high as 65.5 µg/dL in children in the same area. These
levels have remained abnormally high since 1980 due to the contamination of
soil by lead. In 2003, those authors found blood lead levels of c.a. 17 µg/dL,
which were 5 µg/dL greater among children with pica habit, but independent
of age, visible presence of scum surrounding the home, employment status
of the father, family history of lead poisoning, and malnutrition (65).
In Cubatão (São Paulo), one of the most industrialized areas in Brazil,
Santos Filho et al. (66) found lead levels in the population blood averaging
17.8 µg/dL. Paoliello (67) assessed the blood lead level in children living in
the upper Ribeira do Iguape river valley and found a blood lead level median
value of 11.25 µg/dL. Freitas (2004) carried out an evaluation of lead
exposure in a contaminated area of Bauru which revealed that 311 out of 850
Introdução – Manuscrito I– Pan American Journal of Public Health
.
42
analyzed children presented blood lead levels above the limits established by
WHO (68).
In the mentioned investigation (Bauru, São Paulo), a battery recycling
plant contaminated its neighboring residential area with lead oxides during
the last 8 years. The environmental lead contamination has been assessed
by CETESB (State Authority for Environmental Control). The plant had its
activities suspended in 2002. Over half of the 311 studied children presented
blood lead levels between 15 and 19 µg/dL, 21% had levels between 20 and
39 µg/dL, and less than 1% (three children) showed blood lead levels of 40
µg/dL or higher. Comparing to other lead contaminated areas in Brazil, blood
lead levels found in Bauru study were rather low (median of 7.3 µg/dL) (69).
In Brazil, there are no public policies designed to establish official
procedures for sampling and analyzing lead in human tissues, nor to screen
lead in school children, even in the case of those who present psychomotor
and learning disabilities (18).
Lead effects on IQ and social behavior
Denno (70) traced the behavioral patterns of 987 African-American
youths from birth to age 22. She found that among the dozens of sociologic
and biologic correlates of delinquency, lead poisoning was among the
strongest for male subjects.
Introdução – Manuscrito I– Pan American Journal of Public Health
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43
Cognitive function, measured by psychometric IQ tests, has been the
major focus of most studies on lead exposure in childhood. There are
persuasive reasons to believe that cognitive dysfunction may not be the most
important effect of lead and that we may be entering a fifth stage of
understanding the biochemistry of lead toxicity, including the molecular
mechanisms by which lead triggers anti-social attitudes in children and adults
(5).
Lead-induced aggressiveness is not an entirely new notion. Parents
have frequently reported that after recovery from an episode of acute lead
poisoning, their children’s behavior changed dramatically to restless,
inattentive and aggressive conduct (5). In 1943, Byers and Lord reported
attention deficits and aggression in a sample of lead-poisoned children on
follow-up (14).
Needleman et al. (71) studied 301 primary-school students and found
that children with elevated bone lead levels scored higher on the attention
deficit, aggression, and delinquency clusters of the Child Behavior Checklist
after adjustment of covariates. Dietrich et al (72) found that prenatal lead
exposure was associated with parents’ reports of delinquency and
aggression, and postnatal lead exposure was associated with self-reports of
delinquent acts. A case-control study of 195 arrested and convicted
delinquent youths, conducted by Needleman et al. (73) on 194 adjudicated
youths (aged 12-18) and 146 non-delinquent controls, revealed an increased
risk of delinquency associated with bone lead concentrations measured by X-
ray fluorescence. The covariate-adjusted odds ratio was 4 (95% CL 1.4-
Introdução – Manuscrito I– Pan American Journal of Public Health
.
44
11.1). The population-attributable risk for delinquency due to lead exposure
ranged from 11% to 38% in this sample. In Brazil, a case-control study
survey of a connection between lead in the tooth enamel of adolescent
inmates and their anti-social behavior is presently underway in Brazil’s youth
re-educational system, Fundação Casa.
Recently, Chiodo et al. (74) showed a relation between blood lead
level and neurobehavioral outcome in African American 7-year-old children.
Among the studied variables, social problems, delinquent behavior and total
behavior problem were associated with blood lead levels (ß=0.10*, ß=0.09*
and ß=0.09*; *p<0.05, respectively).
A number of recent ecological investigations have correlated leaded
gasoline sales and lead in air particulates with crime rates, strongly indicating
an association between lead exposure and crime. Stretesky and Lynch (75),
when comparing homicide rates in 3111 counties in the United States and
adjusting 15 covariates, reported a four-fold increase in homicide rates in
those counties with the highest air lead levels compared to controls. Nevin
(76) correlated sales of leaded gasoline with violent crime rates and,
adjusting for unemployment and percentage of population in the high-crime
age group, found a statistically significant association between lead and
crime. It has been speculated that one of the reasons for the recent decline in
crime rates in the United States can be attributed to the adoption of public
policies to ban lead in paints, gas, and canned food, therefore reducing
exposure to lead. In 2007, Nevin (77) carried out single and combined nation
regressions, identifying “best-fit” lags for each crime analyzed, with the
Introdução – Manuscrito I– Pan American Journal of Public Health
.
45
highest significance (t-value) for blood lead and percent of crime rate
variation explained (R²). The results presented a strong association between
preschool blood lead and subsequent crime rate trends over several decades
in nine countries. Furthermore, regression analysis of average 1985-1994
murder rates across USA cities suggests that murder could be especially
associated with more severe cases of childhood lead poisoning.
Many studies provide evidence of an inverse relationship between
lead exposure and cognitive ability (78). There is, however, disagreement
about the IQ to blood lead slope (IQ points lost/one µg/dL increase in blood
lead) and the influence of confounding variables (79,80). There is strong
evidence that young children face the greatest risk of IQ losses due to lead
exposure, especially during the first three years of life, when basic cognitive
abilities develop (79). This information is very important because many
painted toys may contain lead, becoming a risk for children, especially
because children introduce toys into their mouths, sometimes biting them.
Cognitive losses due to lead exposure during the first three years of life
appear to be most evident in IQ tests carried out some years later, around
age 10 or older, when IQ scores are more stable and predictive of future
outcomes (78,79). There is no consensus, however, on whether lead
exposure is more strongly associated with verbal IQ, mathematical skills or
performance IQ. However, despite the similarity with a pattern of outcomes
typical of brain injury, detailed comparisons of children’s deficits indicate that
lead, like most other causes of brain injury, does not produce exactly the
same set of impairments in each patient (81).
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Although data on yearly changes in IQ are unavailable, temporal data
are available for specific types of social behavior associated with lower IQ
scores. Herrnstein and Murray (82), in their controversial book The Bell
Curve, cite data showing that individuals with lower IQ levels account for a
disproportionate share of violent crime and unwed births. Herrnstein and
Murray (82) associated IQ with social behavior based on data from the
National Longitudinal Survey of Labor Market Experience of Youth (NLSY), a
representative national sample of American youths. When the NLSY began
in 1979, the 12,686 participants were aged 14 to 22. In 1980, 94% of these
youths were given the Armed Forces Qualification Test (AFQT) in the IQ
metric (a mean of 100 and a standard deviation of 15), and these results are
referred to as IQ scores. To justify the use of IQ, the authors showed that this
test has a very high correlation with other IQ tests available for some NLSY
participants. In addition to the NSLY figures, the authors cite research from
Britain, Sweden, Denmark and New Zealand and conclude that the data as a
whole indicate that incarcerated offenders have an average IQ approximately
92 (eight points below the mean) (82).
In 1960, in the United States, the accepted threshold for blood lead in
children was 60 µg/dL. Follow-up studies carried out in American cities
showed 10 to 20% of the children of these cities presenting lead levels of up
to 40 µg/dL. This finding increased the conjecture that some learning
difficulties and behavior disorders (sub-clinical manifestations) may be
attributed to lead. Five studies on lead levels and behavior in children without
overt signs or symptoms of lead poisoning were carried out in the 70s. Three
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of them related an association between lead and IQ (15,17,16) and two did
not confirm this connection (83,84). These studies had limitations of design:
the number of patients in each study was small, and blood lead was used as
a biomarker. Blood lead can only be reliable as a biomarker of short term
exposures, as the metal has a half-life of 36 days in the blood (85).
Three meta-analysis studies confirmed that exposures to low levels of
lead may be associated with IQ deficiency (86,79,80). In response to these
data, in 1991, the Centers for Disease Control and Prevention – CDC-
reviewed the acceptable levels for blood lead and reduced the 60 µg/dL
blood threshold established in 1970 to the present accepted value of 10
µg/dL. More recent data point toward the manifestation of deficiencies in
cognition, attention and behavior in children presenting lead levels of
between 3 and 5 µg/dL of blood (9). In 2007, Chiodo et al (74) presented new
data showing that none of those studied neurobehavioral outcomes
evidenced a threshold below which blood lead levels do not be associated
with harmful outcomes. The authors suggest a reduction of the “acceptable”
level, considering the recent scientific evidences.
Dudek and Merecz (87) found that the most rapid deterioration of IQ
was observed at blood lead levels between 5-10 and 11-15 µg/dL, consistent
with the Schwartz’s (79) finding of an increased slope at lower blood lead
levels (76).
In recent decades, the social problems of violent crime and unwed
pregnancies have been associated with teenagers and young adults in poor
urban areas. Average blood lead for poor children in urban areas probably
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began to rise as early as the 1940s, due to the addition of tetraethyl lead to
gasoline combined with use of high lead-based pigments in house paints.
Therefore, temporal data on leaded gasoline consumption might serve as a
rough indicator for changes in blood lead in poor urban children from 1940 to
1987. If childhood lead exposure affects IQ, and IQ affects population rates
for crime and unwed pregnancy, then changes in crime and unwed
pregnancy rates from 1960 to the late 1990s could reflect changes in IQ
associated with temporal trends in leaded gasoline consumption from 1940
through the early 1980s (76).
Primary prevention: benefits for public health
A cost-benefit analysis carried out by the US Public Health Service
estimated the cost of abatement of old houses painted with lead-containing
paints over a 30-year period at $33.7 billion, in 1991. The estimated benefit
from avoided health care costs and increased income due to raised IQ was $
61.7 billion. This cost analysis may be conservative, as it does not include
avoided delinquency and cardiovascular disease, both demonstrated effects
of lead exposure, among other health effects (4).
According to Needleman (5), current analyses also demonstrate that
primary lead exposure prevention yields large economic benefits. Grosse et
al (88) calculated that each present-day preschool child’s IQ was increased
by 2.2-4.7 points above what it might have been if leaded gasoline and blood
lead had not been reduced. From this, they calculated the IQ-related
increase in income and estimated that the economic benefit for each year’s
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birth cohort was between $110 billion and $319 billion. Landrigan et al (89),
assuming no threshold for the lead-IQ association, estimated the loss of
future earnings for the one-year cohort of children aged 5 in 1997 at $43.4
billion.
The monetary cost associated with the ubiquitous exposure of fetuses
and children to lead industrialized societies has been also calculated by
Schwartz (90) estimating the benefits of a 1 µg/dL reduction in the population
mean blood lead concentration (Table 1). The analysis was based on the
monetary savings of reducing lead levels in children with blood lead
concentrations between 10 and 20 µg/dL. Schwartz (90) estimated medical
costs associated with treatment of children with undue lead exposure, the
increase in remedial education, and the costs associated with reduced birth-
weight and reduced gestational age, among other factors. The largest single
cost is earnings lost as a result of decreased intellectual capability. In a later
similar analysis using the comprehensive National Longitudinal Survey of
Youth database to give monetary value to the effect of decreased cognitive
ability on earning capacity, the estimated gain in earnings would be 7.5 billion
U.S. dollars per year for a decrease in blood lead levels of 1 µg/dL in the
U.S. population (91). It is obvious that the effect of lead on IQ is reflected in
an enormous cost to society in terms of lost potential and increased need for
medical care and special education.
According to Needleman (5), the evidence that lead toxicity extends
down to the lowest measurable levels, that pharmacological therapies are
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ineffective at preventing sequelae in those with low levels, and that reduction
of exposure yields huge economic as well as health benefits provides a
strong argument in favor of a systematic program of abatement of lead from
the single remaining major source in the United States: lead in older homes.
On the other hand, an association has been firmly established between air
lead concentrations and levels of lead in the body (92,93,94,95,96). To
determine if data on potential lead exposure drawn from the Environmental
Protection Agency's Cumulative Exposure Project (CEP) were correlated with
blood lead levels at the county level, Stretesky and Lynch (75) collected data
from the Ohio Department of Health on children younger than 6 years who
had blood lead levels above 10 µg/dL during 1998. The Centers for Disease
Control and Prevention (CDC) (Atlanta, GA) identified the data from Ohio as
more valid than data from most states because a large proportion of children
across all Ohio counties are tested for lead poisoning. The authors found that
across Ohio's counties, CEP-estimated air lead concentrations were
positively and significantly correlated with the percentage of children (of
those children screened) who had elevated blood lead levels (Pearson
correlation coefficient, 0.44; P<.001; n = 88). This relationship persisted
(Pearson correlation coefficient, 0.48; P<.001; n = 88) after adjustments from
the percentage of houses built before the 1950s (1990 census estimate),
reinforcing previous findings suggesting that lead exposure may result from a
variety of contamination sources, including lead-contaminated air.
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An important and interesting example of primary prevention is that of
the city of Hartford, Connecticut, USA. As part of a citywide effort to increase
lead poisoning awareness, the Hartford Health Department implemented a
multifaceted public health campaign involving several novel elements and
partnerships, including the use of municipal sanitation trucks to disseminate
lead-poisoning prevention messages throughout the city. Key results were as
follows: recall of campaign components ranged from 21.5 to 62.6%, with
newspaper advertisements and signs on buses and billboards recalled most
often and a video broadcast on public-access television recalled least often;
more than 45% of respondents reported that they took steps to prevent lead
poisoning because of at least one of the campaign components, with the
newspaper advertisements being the most effective component in terms of
prompting lead-poisoning prevention behavior; respondents’ awareness was
particularly low in terms of becoming more informed as to how medical
personnel and procedures can and cannot detect and prevent lead poisoning
in children. This campaign prompted caregivers to take steps to prevent lead
poisoning and may help public health professionals in other communities to
develop novel ideas by which to embark on similar initiatives (97).
In Brazil, according to the São Paulo State Health Department some
measures have been adopted for protecting the population, despite the lack
of an official program for environmental lead exposure prevention. Since
1978, tetraethyl lead has no longer been added to gasoline as an anti-
detonator. In addition, there are regulations for acceptable lead levels in food
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52
and water (98,99). Regarding acceptable levels in humans, only occupational
exposure is regulated.
Among the non-regulated sectors, some factories follow the
internationally accepted lead parameters. According to the Brazilian
Association of Paints Manufacturers, there is a trend that began in the 1990s
to substitute lead pigments in paints. At present, Brazilian domestic paints
are free of lead. However, lead is still used as an anti-corrosive, such as red
lead in iron gates, refrigerators, cars, stoves, bicycles, and many other
goods. In this case a covering paint should be applied over the red lead (18).
The aforementioned non-toxic and inexpensive Biphor
®
white pigment, based
on aluminum phosphates and polyphosphates, will greatly decrease paint
related lead poisoning (26).
Studies conducted at the Adolfo Lutz Institute (100) (Brazil) concluded
that lead may be found in pencils, pens, colored paints, erasers and other
school supplies. This study recommended that there should be regulatory
guidelines for the manufacture of such products.
The canned food manufacturers in Brazil have also substituted lead-
based solders. Regarding the contamination of fresh food by lead, Sakuma
(101) found in Brazil secure lead levels for human consumption. Glazed
ceramic containers can be a source of lead poisoning when lead leaches into
stored beverages, especially in the case of acidic fruit juices such as those
made from grapes and citrus fruit (102). Lead from glazed ceramics is
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53
promptly dissolved by the tartaric and citric acids present in the juices,
respectively, due to the chelation of the metal by these acids.
Since 1986, an Environmental Impact Report (EIA/RIMA) has been
officially required for the approval of potentially polluting industrial plants, as
an obligatory document to license these companies (103). The strategies
proposed in this report to minimize the pollution by the plant must be
analyzed and approved (99). Nevertheless, the companies built before 1986
are not obliged to follow this protocol, unless they have caused
environmental damage (104).
According to information from the Sanitary Vigilance Center, São
Paulo State Health Department (18), several small companies and domestic
sources of lead contamination do not issue warnings to prevent lead
exposure
Thus, considering that “the child is father of the man” and that a
healthy social tissue can be seriously harmed by lead, it is extremely
important to establish public policies against lead contamination and
guarantee an adult population that is socially well-balanced and productive.
In a recent paper, provocatively named “Childhood lead poisoning prevention
– too little, too late”, Lanphear (105) called attention to the importance of
preventing lead poisoning for the good of individuals and of society. In this
context, it is tempting to state that there is a high probability that many young
delinquents are actually victims of lead poisoning and not necessarily genetic
Introdução – Manuscrito I– Pan American Journal of Public Health
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54
or social criminals. We conclude with a plea for public health policies to
prevent lead poisoning in underdeveloped and developing countries, such as
those that have long been adopted in the U.S.A., Europe, and Japan
(www.cdc.gov, www.fda.gov, www.epa.gov).
Acknowledgements
This work was supported by grants from the Fundação de Amparo à
Pesquisa do Estado de São Paulo (FAPESP), the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), and the Projeto Milênio
Redoxoma. KPKO is recipient of a fellowship from the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior. The authors are indebted to
Prof. Abner Lall (Howard University, USA), and to Dr. Brian Bandy (University
of Saskatchewan, CA) for kindly reading this manuscript.
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55
References
1. Major RH. Some landmarks in the history of lead poisoning. Ann Med Hist
1931; 3:218-27.
2. Satcher DS. The surgeon general on the continuing tragedy of childhood
lead poisoning. Public Health Rep 2000; 115:579-80.
3. Lin-Fu JS. Health effects of lead, an evolving concept. In: Mahaffey KR,
editor. Dietary and environmental lead: human health effects. Amsterdam:
Elsevier; 1985. p. 58-9. (Topics in Environmental Health, v.7).
4. Centers for Disease Control and Prevention. Preventing lead poisoning in
young children: a statement. Atlanta: US Department of Health and
Human Services, Public Health Services; 1991.
5. Needleman H. Lead poisoning. Annu Rev Med 2004; 55:209-22.
6. Lidsky TI, Schneider JS. Lead neurotoxicity in children: basic mechanisms
and clinical correlates. Brain 2003; 126:1-19.
7. Lin-Fu JS. Undue absorption of lead among children: a new look at an old
problem. N Engl J Med 1972; 286:702-10.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
56
8. Bellinger DC. Lead. Pediatrics 2004; 113:1016-22.
9. Lanphear BP, Dietrich K, Auinger P, Cox C. Cognitive deficits associated
with blood lead concentrations <10 µg/dL in US children and adolescents.
Public Health Rep 2000; 115:521-9.
10. Canfield RC, Henderson CR, Cory-Slechta DA, Cox C, Jusko TA,
Lanphear BP. Intellectual impairment in children with blood lead
concentrations below 10 µg per deciliter. N Engl J Med 2003; 348:1517-
26.
11. Chiodo LM, Jacobson SW, Jacobson JL. Neurodevelopmental effects of
postnatal lead exposure at very low levels. Neurotoxicology 2004; 26:359-
71.
12. Blackfan KD. Lead poisoning in children with especial reference to lead
as a cause of convulsions. Am J Med Sci 1917; 53:877-87.
13. Levinson A, Harris LH. Lead encephalopathy in children. J Pediatr 1936;
8:315-29.
14. Byers RK, Lord EE. Late effects of lead poisoning on mental
development. Am J Dis Child 1943; 66:471-83.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
57
15. De La Burne B, NcLin S, Choate S. Does asymptomatic lead exposure in
children have latent sequelae? J Pediatr 1972; 81:1088-91.
16. Perino J, Ernhart CB. The relation of subclinical lead level to cognitive
and sensorimotor impairment in black preschoolers. J Learn Disabil 1974;
7:26-30.
17. Landrigan PJ, Baloh RW, Barthel WF. Neuropsychological dysfunction in
children with low level lead absorption. Lancet 1975; 1:708-12.
18. Freitas CU. Estratégias de abordagem para a exposição ambiental ao
chumbo no Estado de São Paulo. Available from:
http://www.cve.saude.sp.gov.br/htm/doma/chumbo.htm [Accessed 2006
Dec 1].
19. World Health Organization. Human exposure to lead. In: Human
Exposure Assessment Series 1992.
20. Mangas S, Fitzgerald DJ. Exposures to lead require ongoing vigilance.
Bull World Health Organ 2003; 81:847.
21. Goyer RA. Results of lead research: prenatal exposure and neurological
consequences. Environ Health Perspect 1996; 104:1050-4.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
58
22. Mielke HW. Lead in inner cities. Am Sci 1999; 87:62-73.
23. United States. Food and Drug Administration. FDA consumer. Dangers of
lead still linger. Available from:
http://www.cfsan.fda.gov/~dms/fdalead.html [Accessed 2006 Oct 23].
24. Lynch RA, Boatright DT, Moss SK. Lead-contaminated imported tamarind
candy and children’s blood lead levels. Public Health Rep 2000; 115:537-
43.
25. Jacobs DE, Clickner RP, Zhou JY, Viet SM, Marker DA, Rogers JW, et al.
The prevalence of lead-based paint hazards in U.S. housing. Environ
Health Perspect 2002; 110:A599-A606.
26. Galembeck F, De Brito J. Aluminum phosphate or polyphosphate
particles for use as pigments in paints and method of making same. U.S.
Pat Appl Publ 2006; Application US 2005-215312 20050830, 14 pp.
27. United States. Environmental Protection Agency. National primary
drinking water regulations: consumer confidence; proposed rule, in CFR
1998; 40:141-142. Available from:
http://www.regulations.gov/fdmspublic/component/main [Accessed 2007
Jan 24].
Introdução – Manuscrito I– Pan American Journal of Public Health
.
59
28. Bernard SM, Samet JM, Grambsch A, Ebi KL, Romieu I. The potential
impacts of climate variability and change on air pollution related health
effects in the United States. Environ Health Perspect 2001; 109:199-209.
29. Maas RP, Patch SC, Parker AF. An assessment of lead exposure
potential from residential cutoff valves. J Environ Health 2002; 65:9-14.
30. Centers for Disease Control and Prevention. Blood lead levels in
residents of homes with elevated lead in tap water – District of Columbia.
MMWR Morb Mortal Wkly Rep 2004; 53:268-70.
31. Teixeira P. Encanamentos inadequados podem contaminar a água com
chumbo. Agência USP de notícias 2002. Available from:
http://www.usp.br/agen/ [Accessed 2006 Dec 6].
32. Paoliello MMB, Capitani EM. Chumbo. In: Azevedo FA, Chasin AM.
Metais: gerenciamento da toxicidade. São Paulo: Atheneu Inter Tox;
2003. p. 359-60.
33. Charney E, Kessler B, Fartel M, Jackson D. Childhood lead poisoning: a
controlled trial of the effect of dust-control measures on blood lead levels.
N Engl J Med 1983; 309:1089-93.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
60
34. Tornton I; Davies DJA; Watt JM; Quinn MJ. Lead exposure in young
children from dust and soil in the United Kingdom. Environ Health
Perspect 1990; 89:55-60.
35. Rhoads GG, Ettinger AS, Weisel CP, Buckley TJ, Goldman KD, Lioy PJ.
The effect of dust lead control on blood lead in toddlers: a randomized
trial. Pediatrics 1999; 103:551-5.
36. Yiin LM, Rhoads GG, Lioy PJ. Seasonal influences on childhood lead
exposure. Environ Health Perspect 2000; 108:177-82.
37. Landrigan PJ. The worldwide problem of lead in petrol. Bull World Health
Organ 2002; 80:768.
38. Gavaghan H. Lead, unsafe at any level. Bull World Health Organ 2003;
80:82.
39. Toscano CD, Guilarte TR. Lead neurotoxicity: from exposure to molecular
effects. Brain Res Rev 2005; 49:529-54.
40. Moreira FR, Moreira JC. Os efeitos do chumbo sobre o organismo
humano e seu significado para a saúde. Rev Panam Salud Publica 2004;
15: 119-29.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
61
41. Bailey C, Kitchen I. Ontogenesis of proenkephalin products in rat striatum
and the inhibitory effects of low-level lead exposure. Dev Brain Res 1985;
22:75-9.
42. Brown RS, Hingerty BE, Dewan JC. Pb (II)-catalyzed cleavage of the
sugar-phosphate backbone of yeast tRNA (Phe) – implications for lead
toxicity and self-splicing RNA. Nature 1983; 303:543-6.
43. Markovac J, Goldstein GW. Picomolar concentrations of lead stimulate
brain protein kinase C. Nature 1988; 334:71-3.
44. Chisolm Jr JJ. Lead poisoning. Sci Am 1971; 224:15-23.
45. Monteiro HP, Bechara EJH, Abdalla DSP. Free radicals involvement in
neurological porphyrias and lead poisoning. Mol Cell Biochem 1991;
103:73-83.
46. Bechara EJH. Oxidative stress in acute intermittent porphyria and lead
poisoning may be trigged by 5-aminolevulinic acid. Braz J Med Biol Res
1996; 29:841-51.
47. Costa CAC, Trivelato GC, Pinto AMP, Bechara EJH. Correlation between
plasma 5-aminolevulinic acid concentrations and indicators of oxidative
stress in lead–exposed workers. Clin Chem 1997; 43:1196-202.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
62
48. Gurer H, Ercal N. Can antioxidants be beneficial in the treatment of lead
poisoning? Free Radic Biol Med 2000; 29:927-45.
49. Aykin-Burns N, Laegeler A, Kellogg G, Ercal N. Oxidative effects of lead
in young and adult Fisher 344 rats. Arch Environ Contam Toxicol 2003;
44:417-20.
50. Rocha MEM, Dutra F, Bandy B, Baldini RL, Gomes SL, Faljoni-Alário A,
et al. Oxidative damage to ferritin by 5-aminolevulinic acid. Arch Biochem
Biophys 2003; 409:349-56.
51. Demasi M, Costa CA, Pascual C, Lesuy S, Bechara EJH. Oxidative
tissue response promoted by 5-aminolevulinic acid promptly induces the
increase of plasma antioxidant capacity. Free Rad Res 1996; 26:235-46.
52. Adhikari A, Penatti CA, Resende RR, Ulrich H, Britto LR, Bechara EJ. 5-
Aminolevulinate and 4,5-dioxovalerate ions decrease GABA(A) receptor
density in neuronal cells, synaptosomes and rat brain. Brain Res 2006;
1093:95-104.
53. Anderson KE. Doenças das porfirinas ou metais. In: Wyngaarden JB,
Lloyd H, Bennett JC, editors. Cecil tratado de medicina interna. Rio de
Janeiro: Guanabara Koogan; 1993. p. 1146-51.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
63
54. Bechara EJH, Medeiros MHG, Monteiro HP, Hermes Lima M, Pereira B,
Demasi M, et al. A free radical hypothesis of lead poisoning and inborn
porphyries associated with 5-aminolevulinic acid overload. Quim Nova
1993; 16:385-92.
55. Bechara EJH, Medeiros MHG, Catalani LH, Soares CHL, Monteiro HP,
Abdalla DSP, et al. Enolizable carbonyl and imino metabolites may act as
endogenous sources of reactive oxygen species. Cienc Cult 1995;
47:346-57.
56. Centers for Disease Control and Prevention. Current trends childhood
lead poisoning: reports to the Congress by the Agency for toxic
substances and disease registry. MMWR Morb Mortal Wkly Rep 1988;
37:481-5.
57. Riddell TJ, Solon O, Quimbo SA, Tan CM, Butrick E, Peabody JW.
Elevated blood-lead levels among children living in the rural Philippines.
Bull World Health Organ 2007; 85:674-80.
58. Pirkle JL, Brody DJ, Gunter EW, Kramer RA, Paschal DC, Flegal KM, et
al. The decline in blood lead levels in the United States: the National
Health and Nutritional Examination Surveys (NHANES). JAMA, J Am Med
Assoc 1994; 272:284-91.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
64
59. Centers for Disease Control and Prevention. Update: blood lead levels –
United States, 1991 – 1994. MMWR Morb Mortal Wkly Rep 1997; 46:141-
6.
60. Garcia SI, Mercer R. Salud infantil y plomo en Argentina. Salud Publ
Mex 2003; 45: s252-5.
61. Manay N, Alonzo C, Dol I. Contaminación por plomo en el bairro La
Teja; Montevideo, Uruguay. Salud Publ Mex 2003; 45: s268-s75.
62. Franco-Netto G, Alonzo MD, Cancio J, Jost M, Souza-Oliveira S. Human
health risk reduction due to lead exposure in Brazil. Salud Publ Mex 2003;
45: s255-s7.
63. Silvany-Neto AM, Carvalho FM, Chaves MEC, Brandão AM, Tavares TM.
Repeated surveillance of lead poisoning among children. Sci Total
Environ 1989; 78: 179-86.
64. Silvany-Neto AM. Evolução da intoxicação por chumbo em crianças de
Santo Amaro, Bahia 1980, 1985 e 1992. Bol of Sanit Panam 1996;
120:11-2.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
65
65. Carvalho FM, Silvany Neto AM, Tavares TM, Costa ACA, Chaves CER,
Nascimento LD, et al. Chumbo no sangue de crianças e passivo
ambiental de uma fundição de chumbo no Brasil. Rev Panam Salud
Publica 2003; 13:19-23.
66. Santos Filho E, Souza e Silva R, Barretto HHC, Inomata ONK, Lemes
VRR, Sakuma AM, et al. Concentrações sanguíneas de metais pesados
e praguicidas organoclorados em crianças de 1 a 10 anos. Rev Saúde
Pública 1993; 27:59-67.
67. Paoliello MMB. Human lead exposure in mining areas, Vale do Ribeira,
Brazil. [Tese de Doutorado]. Campinas:Universidade Estadual de
Campinas; 2002
68. Freitas CU. Vigilância de população exposta a chumbo no município de
Bauru – São Paulo: investigação de fatores de risco de exposição e
avaliação da dinâmica institucional [Tese de Doutorado]. São Paulo:
Faculdade de Saúde Pública, Universidade de São Paulo; 2004.
69. Freitas CU, Capitani EM, Gouveia N, Simonetti MH, Paula e Silva MR,
Kira CS et al. Lead exposure in an urban community: investigation of risk
factors and assessment of the impact of lead abatement measures.
Environ Res 2007; 103:338-44.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
66
70. Denno DW. Biology and violence. New York: Cambridge University
Press; 1990.
71. Needleman HL, Riess JA, Tobin MJ, Biesecker GE, Greenhouse JB.
Bone lead levels and delinquent behavior. JAMA, J Am Med Assoc 1996;
275:363-9.
72. Dietrich KN, Ris MD, Succop PA, Berger OG, Bornschein RL. Early
exposure to lead and juvenile delinquency. Neurotoxicol Teratol 2001;
23:511-8.
73. Needleman HL, McFarlandC, Ness RB, Fienberg SE, Tobin MJ. Bone
lead levels in adjudicated delinquents. A case control study. Neurotoxicol
Teratol 2002; 24:711-7.
74. Chiodo LM, Covington C, Sokol RJ, Hannigan JH, Jannise J, Ager J et al.
Blood lead levels and specific attention effects in young children.
Neurotoxicol Teratol 2007; 29:538-46.
75. Stretesky PB, Lynch MJ. The relationship between lead exposure and
homicide. Arch Pediatr Adolesc Med 2001; 155:579-82.
76. Nevin R. How lead exposure relates to temporal changes in IQ, violent
crime and unwed pregnancy. Environ Res 2000; 83:1-22.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
67
77. Nevin R. Understanding international crime trends: the legacy of
preschool lead exposure. Environ Res 2007; 104: 315-36.
78. National Research Council. Committee on Measuring Lead in Critical
Populations. Measuring lead exposure in infants, children and other
sensitive populations. Washington DC: National Academy Press; 1993.
79. Schwartz J. Low-level lead exposure and children’s IQ: a meta-analysis
and search for a threshold. Environ Res 1994; 65:42-55.
80. Pocock SJ, Smith M, Baghurst P. Environmental lead and children’s
intelligence: a systematic review of the epidemiological evidence. BMJ [Br
Med J] 1994; 309:11889-97.
81. Lidsky TI, Schneider JS. Adverse effects of childhood lead poisoning: the
clinical neuropsychological perspective. Environ Res 2006; 100:284-93.
82. Herrnstein R, Murray C. The Bell curve: intelligence and class structure in
American life. New York: Free Press; 1994.
83. Kotok D. Development of children with elevated blood levels: a controlled
study. J Pediatr 1972; 80:57-61.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
68
84. Lansdown RG, Shepherd J, Clayton BE. Blood lead levels, behavior and
intelligence. A population study. Lancet 1974; 1:538-41.
85. World Health Organization. Environmental health criteria 165 – Inorganic
lead. Geneva: WHO, 1995. 300 p. Publish under the joint sponsorship of
the United Nations environment programme, the International Labour
Organization, and the World Health Organization.
86. Needleman HL, Gatsonis C. Low level lead exposure and the IQ of
children. JAMA, J Am Med Assoc 1990; 263:673-8.
87. Dudek B, Merecz D. Impairment of psychological functions in children
environmentally exposed to lead. Int J Occup Med Environ Health 1997;
10:37-46.
88. Grosse SD, Matte TD, Schwartz J, Jackson RJ. Economic gains resulting
from the reduction in children’s exposure to lead in the United States.
Environ Health Perspect 2002; 110:563-70.
89. Landrigan PJ, Schechter CB, Lipton JM, Fahs MC, Schwartz J.
Environmental pollutants and disease in American children: estimates of
morbidity, mortality, and costs for lead poisoning, asthma, cancer and
developmental disabilities. Environ Health Perspect 2002; 110:721-8.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
69
90. Schwartz J. Societal benefits of reducing lead exposure. Environ Res
1994; 66: 105-24.
91. Salveker DS. Updated estimates of earnings benefits from reduced
exposure of children to environmental lead. Environ Res 1995; 70:1-6.
92. Brunkenreff B. The relationship between air lead and blood lead in
children. Sci Total Environ 1984; 38:79-123.
93. Nriagu JO, Kim MJ. Emissions of lead and zinc from candles with metal-
core wicks. Sci Total Environ 2000; 250:37-41.
94. Hayes EB, McElvaine MD, Hyman GO, Fernandez AM, Lyne S, Matte
TD. Long-term trends in blood lead levels among children in Chicago:
relationship to air lead levels. Pediatrics 1994; 93:195-200.
95. Lai JS, Wu TN, Liou SH, Shen CY, Gun CF, Ko KN, et al. A study of the
relationship between ambient lead and blood lead among lead battery
workers. Int Arch Occup Environ Health 1997; 69:295-300.
96. Thomas VM, Socolow RH, Fanelli JJ, Spiro TG. Effects of reducing lead
in gasoline: an analysis of the international experience. Environ Sci
Technol 1999; 33:3942-84.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
70
97. McLaughlin T; Humphries Jr O, Nguyen T, Maljanian R, McCormack K.
“Getting the lead out” in Hartford, Connecticut: a multifaceted lead-
poisoning awareness campaign. Environ Health Perspect 2004; 112:1-5.
98. Portaria nº 16, de 13 de março de 1990. Autoriza a inclusão na tabela II,
como preceitua o artigo 26 do decreto 55871, de 26 de março de 1965
dos limites máximos de tolerância de chumbo em alimentos. Diário Oficial
da União 1990; 15 mar. Seção 1, p. 5436.
99. Resolução CONAMA nº 357. Dispõe sobre a classificação dos corpos de
água e diretrizes ambientais para o seu enquadramento, bem como
estabelece as condições e padrões de lançamento de efluentes, e dá
outras providências. Diário Oficial da União 2005; 18 mar.
100. Garrido NS, Pregnolatto NP, Murata LTF, Silva MR, Nunes MCD,
Engler VM, et al. Determinação de chumbo e cádmio em artigos
escolares. Rev Inst Adolfo Lutz 1990; 50:291-6.
101. Sakuma AM, Scorsafava MA, Zenebon O, Tiglea P, Fukumoto CJ.
Hortaliças comercializadas em São Paulo: aspectos da contaminação de
chumbo, cádmio e zinco. Rev Inst Adolfo Lutz 1989; 49:81-4.
102. Browder AA. Lead poisoning from glazes. Ann Intern Med 1972; 76:665.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
71
103. Resolução CONAMA nº 237. Dispõe sobre a revisão e
complementação dos procedimentos e critérios utilizados para o
licenciamento ambiental. Diário Oficial da União 1997; 22 dez.
104. Machado PAL. Direito ambiental brasileiro. São Paulo: Revista dos
Tribunais; 1982.
105. Lanphear BP. Childhood lead poisoning prevention – too little, too late.
JAMA, J Am Med Assoc 2005; 293:2274-6.
106. Bechara EJH, Dutra F, Cardoso VES, Sartori A, Olympio KPK, Penatti
CAA, et al. The dual face of alfa endogenous aminoketones: pro-
oxidizing metabolic weapons Comp Biochem Physiol, Part C: Toxicol
Pharmacol 2006; 146:88-110.
Introdução – Manuscrito I– Pan American Journal of Public Health
.
72
Children
Learning disabilities
Attention & IQ deficits
Anti-social behavior
Headache & seizure
Hearing & growth
Mental retardation
Abdominal & joint pain
Hemoglobin
Anemia
Nephropathy
Encephalopathy
Death
Figure 1. Effects of lead poisoning on human health. Adapted from Gurer
and Ercal, 2000 (48).
= reduction
10
5
0
10
0
µg Pb/dL blood
Adults
Hypertension
Neuropathy
Memory
Irritability
Headache
Hearing acuity
Nephropathy
Sterility/Impotence
Hemoglobin
Longevity
Anemia
Encephalopathy
1
50
Introdução – Manuscrito I– Pan American Journal of Public Health
.
73
Figure 2. Production of reactive oxygen species by the aerobic oxidation of δ-
aminolevulinic acid (ALA), a heme precursor accumulated in lead poisoning,
ALA-driven oxidative damage to biomolecules, and biological consequences
observed in ALA-treated rats and lead poisoned individuals (106).
HO
HO
O
O
O
O
O
ALA
DOVA
NH
2
O
2
O
2
●
-
+ H
2
O
2
NH
4
+
HO
●
DNA guanine oxidation
to 8-HOdG in vitro
and rat liver
HO radical
in vivo
detection in ALA-treated
rats
IRP-1 activation
(iron regulatory protein)
GABAergic receptor
damage in synaptic
membranes, neuronal cells
and brain
Iron mobilization
to liver and brain of
succinylacetone-treated
rats
Iron release
from ferritin
Elevated levels of
SOD and Gpx in
AIP and plumbism
patients
In vitro single-strand
breaks in plasmid DNA
Disruption of mitochondrial
membrane potential,
matrix Ca
2+
release,
and increased state-4
respiration
Lipid peroxidation
In cardiolipin-rich
liposomes (mitochondria
model) catalyzed by ferritin
Increased glycolytic
metabolism in chronically
ALA-treated rats
DOVA-DNA
adduct formation
Introdução – Manuscrito I– Pan American Journal of Public Health
.
74
Table 1. Annual benefits of a 1 µg/dL reduction in the mean blood lead
concentration of US infant population, according to Schwartz (1994) (90).
Annual Benefits US$, million
Medical costs 189
Compensatory education 481
Infant mortality 1,140
Neonatal care 67
Earnings 5,060
Total 6,937
Objetivos
.
75
2 OBJETIVOS
Objetivo geral:
Estudar a exposição ao chumbo associada a comportamento anti-
social em adolescentes brasileiros e desenvolver metodologias para
determinação de biomarcadores que retratem a exposição ao
chumbo.
Objetivos específicos:
Avaliar a associação entre altas concentrações de chumbo presentes
no esmalte dentário superficial e estabelecimento de comportamento
anti-social / cometimento de atos infracionais em adolescentes
(Manuscrito II);
Investigar potenciais fatores de risco domiciliares à contaminação por
chumbo mais associados a altas concentrações de chumbo no
esmalte dentário (Manuscrito III);
Verificar o impacto de alterações metodológicas da técnica de
microbiópsia ácida de esmalte dentário superficial nos valores obtidos
para a profundidade da biópsia, calculada pela fórmula da altura do
cilindro, e sobre conseqüentes interpretações das concentrações de
chumbo no esmalte dentário (Manuscrito IV).
Métodos
.
76
3 MÉTODOS
.
Este estudo foi aprovado pelo Comitê de Ética em Pesquisa da
Faculdade de Saúde Pública da Universidade de São Paulo (Proc. No.
244/05). O modelos das cartas de informação e termos de consentimento
livre e esclarecido assinados pelos adolescentes e responsáveis estão
apresentados no ANEXO 1.
Os procedimentos adotados para atender aos objetivos da pesquisa
estão resumidamente descritos abaixo. Todos os procedimentos são
detalhados nos manuscritos que compõem o capítulo RESULTADOS e
DISCUSSÃO. Assim, para facilitar a leitura do texto, o manuscrito e o
número da página onde a respectiva metodologia pormenorizada pode ser
encontrada estão indicados entre parênteses.
Um estudo transversal foi conduzido com 173 jovens residentes na
cidade de Bauru, SP, Brasil. MICROBIÓPSIAS ÁCIDAS DE ESMALTE
DENTÁRIO SUPERFICIAL foram realizadas nos incisivos centrais
superiores destes jovens por dois diferentes protocolos metodológicos. O
Protocolo I (n=114) consistiu em uma biópsia circular de 4 mm de diâmetro,
35 s de exposição do dente ao ácido [HCl 1,6 M em 70% de glicerol], 10 µL
HCl (Manuscritos II (p. 95), III (p. 125) e IV (p. 150 )) e o Protocolo II (n=59)
consistiu de uma biópsia de 1,6 mm de diâmetro, 20 s de exposição ao
mesmo ácido, 5 µL HCl (Manuscrito IV, p. 150 ). Todos os adolescentes
participantes da pesquisa receberam profilaxia dentária, raspagem e
Métodos
.
77
alisamento dentário, quando necessário, e aplicação tópica de flúor gel,
quando indicado clinicamente.
Além disso, QUESTIONÁRIOS SOBRE COMPORTAMENTO DOS
ADOLESCENTES (Child Behavior Checklist - CBCL e Self-Reported
Delinquency - SRD) (Manuscrito II, p. 93) e EXPOSIÇÃO A POSSÍVEIS
FONTES CONTAMINADORAS DE CHUMBO (Manuscrito III, p. 125) foram
aplicados aos pais e aos adolescentes.
As ANÁLISES QUÍMICAS foram realizadas no Laboratório de
Química Analítica do Instituto de Química da USP/SP. As amostras foram
analisadas para o fósforo (P) por espectroscopia de emissão ótica com
plasma indutivamente acoplado e, para o chumbo, por espectrometria de
absorção atômica com forno de grafite (Manuscritos II, p. 96; III, p. 126 e IV,
p. 152 ).
Para atender aos objetivos tratados no Manuscrito IV, TESTES DE
PERFILOMETRIA (p. 154) foram realizados em blocos de esmalte dentário
bovino, após realização de biópsias de esmalte utilizando-se, para isso,
perfilômetro da Clínica de Odontologia Preventiva, Periodontia e Cariologia
da Universidade de Zurique, Suíça.
QUESTIONÁRIOS
Para a avaliação do comportamento social, foram utilizados dois
questionários: “Child Behavior Checklist” (CBCL) (ACHENBACH, 2001)
(ANEXO 2), validado no Brasil e adaptado transculturalmente por BORDIN et
Métodos
.
78
al., 1995, e “Self-Reported Delinquency” (SRD) (LOEBER et al., 1989)
(ANEXO 3).
O SRD é um auto-relato de comportamento composto por 36 questões
no qual, para cada questão, o jovem atribui uma escala de 0 a 4, onde os
escores significam: 0= nunca; 1= uma vez; 2= 2-5 vezes; 3= 6-10 vezes e 4=
mais de 10 vezes, referindo-se ao número de vezes que o adolescente
praticou cada ação questionada nos últimos 6 meses. O escore final é obtido
pela soma total de todos os 36 escores.
O CBCL é um questionário empiricamente baseado, ou seja: a) os
dados levantados são baseados nas experiências de pessoas que vêem o
adolescente em um determinado contexto, no caso desta pesquisa, o
contexto familiar; b) “os dados obtidos para uma grande amostra de clientes
foram analisados estatisticamente para identificar os padrões de problemas
que realmente acontecem nas pontuações dadas por diferentes avaliadores”
(Laboratório de Terapia Comportamental do Instituto de Psicologia da
Universidade de São Paulo (LTCIP-IP-USP, 2006)); e c) “os padrões
derivados das análises estatísticas foram usados para construir as escalas
síndromes para marcar os conjuntos que co-ocorrem” (LTCIP-IP-USP,
2006).
Os dados obtidos a partir de itens de competência social são
convertidos em escores de 0 a 4, segundo as instruções do manual do
CBCL (ACHENBACH, 2001). Estes escores são chamados de “escores
crus” que, registrados em escalas, fornecem o perfil social da criança ou do
adolescente. O perfil social aplica-se à faixa etária de 6 a 18 anos de idade e
Métodos
.
79
conta com três escalas individuais: atividades, sociabilidade e escolaridade.
A soma dos escores crus obtidos nas escalas sociais individuais indica a
Competência Social Total do indivíduo.
Os 113 itens relativos a problemas de comportamento constituem
descrições de comportamentos que podem estar presentes ou ausentes na
vida da criança ou adolescente. O informante pode classificar tais
comportamentos de acordo com três variáveis: item falso ou comportamento
ausente (escore=0); item parcialmente verdadeiro ou comportamento às
vezes presente (escore=1) ou comportamento freqüentemente presente
(escore=2).
Os escores de 0 a 2 para problemas de comportamento são chamados
de “escores crus” que, registrados em escalas, fornecem o perfil
comportamental da criança ou adolescente. O perfil comportamental aplica-
se à faixa de 6 a 18 anos de idade e é constituído de oito escalas. As oito
escalas correspondem às seguintes síndromes: I. Ansiedade / Depressão, II.
Isolamento / Depressão, III. Queixas Somáticas, IV. Problemas sociais, V.
Problemas de pensamento, VI. Problemas de atenção, VII. Comportamento
de quebrar regras, VIII. Comportamento agressivo. As escalas I, II e III são
chamadas de problemas internalizantes, quando consideradas em conjunto
e as escalas VII e VIII são nomeadas problemas externalizantes, quando
agrupadas. A soma dos escores crus obtidos nas escalas comportamentais
individuais corresponde ao total de problemas de comportamento (BORDIN
et al., 1995).
Métodos
.
80
Os fatores de confusão foram avaliados por meio de perguntas sobre
endereço; tempo de residência no endereço atual; locais anteriores onde o
jovem residiu; número de pessoas que moram na casa (crianças e adultos);
se os pais moram juntos; escolaridade do pai e da mãe; se fumante ou não
(pais e filhos); local de trabalho dos pais; se alguém da casa trabalhou em
fábricas de baterias, pigmentos, tintas e cerâmica; utilização doméstica de
cerâmica vitrificada, brinquedos pirateados e de baixa qualidade, baterias de
carros e zarcão (ANEXO 4). Este questionário foi baseado na tese de
Doutorado de FREITAS, 2004.
Todos os entrevistadores foram selecionados e treinados pela
pesquisadora responsável (KPKO), anteriormente ao início das entrevistas,
de maneira a padronizar os procedimentos adotados. Entre os
entrevistadores havia estudantes de Relações Públicas (UNESP-Bauru) e
uma cirurgiã-dentista, Mestre em Odontologia em Saúde Coletiva (USP-
Bauru).
A Figura 1 mostra a urna utilizada para colocação das respostas do
SRD, o que foi realizado com o intuito de diminuir possíveis
constrangimentos dos adolescentes em relatar cometimento de atos
infracionais.
Métodos
.
81
Figura 1- Local onde o adolescente respondia ao auto-relato de cometimento
de atos infracionais (Self-Reported Delinquency).
A urna foi utilizada para que o adolescente não se sentisse constrangido em
entregar suas respostas diretamente para a pesquisadora.
MATERIAL UTILIZADO PARA OS PROCEDIMENTOS CLÍNICOS
Métodos
.
82
Figura 2 - Instrumental utilizado durante o exame clínico do adolescente (A)
e para posterior coleta de esmalte dentário (B).
A
A
B
A
B
Métodos
.
83
Figura 3 - Coleta da amostra de esmalte dentário.
A fita foi colorida de preto para melhor visualização da imagem.
Originalmente, a fita adesiva é transparente. Após a realização da profilaxia, o
dente a ser biopsiado é isolado com roletes de algodão, a fita perfurada é
fortemente aderida à superfície dentária e a biópsia é realizada (Fotografias
gentilmente cedidas pela Profa. Dra. Maria Fernanda Borro Bijella).
Métodos
.
84
BAIRROS ONDE OS ADOLESCENTES RESIDEM E LOCAIS DE
COLETAS DE AMOSTRAS
Figura 4 – Vista do Bairro Ferradura Mirim, Bauru, SP.
Métodos
.
85
Figura 5 - Centro Irmã Adelaide, localizado no Bairro Ferradura Mirim.
Neste local, projetos sociais são desenvolvidos como o Programa Primeiro
Emprego voltado aos jovens de 15 a 17 anos de idade. A maioria deles
integrou a presente pesquisa.
Métodos
.
86
Figura 6 – Vista do Núcleo Habitacional Fortunato Rocha Lima, Bauru, SP.
Este núcleo habitacional é oriundo de um projeto de desfavelamento da
cidade de Bauru – SP. Neste local, funcionam o Projeto Girassol e a Creche
São José, ambos atendendo a comunidade carente que lá reside.
Métodos
.
87
Figura 7 – Tipo de residência presente no Núcleo Fortunato Rocha Lima.
ANÁLISE ESTATÍSTICA
Análises de regressão logística foram realizadas para verificação das
síndromes psiquiátricas (p. 98) e fontes de exposição (p. 128) mais
associadas a altas CCED. Para verificação da diferença da CCED e
profundidade de biópsias obtidas por protocolos metodológicos diferentes (I
e II), testes de Wilcoxon e testes t pareados foram aplicados aos dados (p.
155).
Resultados e Discussão
.
88
4 RESULTADOS e DISCUSSÃO
Artigos:
Manuscrito II. “Surface dental enamel lead levels and antisocial behavior in
Brazilian adolescents” (última versão redigida para submissão
à Neurotoxicology and Teratology, FI=2,444);
Manuscrito III. “Risk factors associated with high lead levels measured in the
surface dental enamel from Brazilian youths” (versão
submetida ao Bulletin of the World Health Organization,
FI=4,019);
Manuscrito IV. “Methodological alterations of surface dental enamel
microbiopsies for lead body burden measurement” (versão
submetida à Toxicological Sciences, FI=3,814).
Manuscrito II – Neurotoxicology and Teratology
.
89
Surface dental enamel lead levels and antisocial behavior in Brazilian
adolescents
Kelly Polido Kaneshiro Olympio
a
Pedro Vitoriano de Oliveira
b
Juliana Naozuka
b
Maria Regina Alves Cardoso
c
Antonio Francisco Marques
d
Wanda Maria Risso Gunther
a
Etelvino José Henriques Bechara
e
*
a
Faculdade de Saúde Pública, Departamento de Saúde Ambiental, Universidade de
São Paulo, Av. Dr. Arnaldo, 715, Cerqueira César, 01246-904, São Paulo, Brazil.
b
Instituto de Química, Departamento de Química Analítica, Universidade de São
Paulo, Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo, Brazil.
c
Faculdade de Saúde Pública, Departamento de Epidemiologia, Universidade de
São Paulo, Av. Dr. Arnaldo, 715, Cerqueira César, 01246-904, São Paulo, Brazil.
d
Faculdade de Ciências, Departamento de Educação, Universidade Estadual
Paulista, Av. Eng. Luiz Edmundo Carrijo Coube, 14-01, 17033-360, Bauru – SP,
Brazil.
e
Instituto de Química, Departamento de Bioquímica, Universidade de São Paulo,
Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo, and Departamento de Ciências
Exatas e da Terra, Universidade Federal de São Paulo, Rua Prof. Artur Riedel, 275,
09972-270, Diadema, Brazil.
Manuscrito II – Neurotoxicology and Teratology
.
90
Abstract
Lead poisoning is reportedly linked to a high risk for learning disabilities, aggression
and criminal offenses. Thus, to study the association between lead exposure and
antisocial/delinquent behavior, a cross-sectional study was conducted with 173
Brazilian youths aged 14-18 and their parents (n=93), living in impoverished
neighborhoods of Bauru-SP, with high criminality indices. Self-Reported
Delinquency (SRD) and Child Behavior Checklist (CBCL) questionnaires were used
to evaluate delinquent/antisocial behavior. Body lead burdens were evaluated in
surface enamel acid-etch microbiopsies. The dental enamel lead levels (DELL) were
quantified by graphite furnace atomic absorption spectrometry (GFAAS) and the
phosphorus content was measured using inductively coupled plasma optical
emission spectrometry (ICP-OES). Logistic regression was used to identify
associations between DELL and each scale defined by CBCL and SRD scores. Odd
ratios adjusted for familial and social covariates, considering a group of children
exposed to high lead levels of lead (≥75 percentile), indicated that high DELL is
associated with increased risk of exceeding the clinical score for somatic
complaints, social problems, rule-breaking behavior (T≥70) and externalizing
problems (T≥63) (CI 95%). High DELL was not found to be associated with elevated
SRD scores. In conclusion, it seems that exposure to high lead levels can indeed
trigger antisocial behavior, which calls for public policies to prevent lead poisoning.
Keywords: lead poisoning; dental enamel; biopsy; antisocial behavior; juvenile
delinquency; aggressiveness, d-aminolevulinic acid.
Manuscrito II – Neurotoxicology and Teratology
.
91
1. Introduction
Lead is a long known devastating neurotoxicant [Toscano and
Guilarte, 2005]. The specific biological mechanisms underlying lead´s effect
on social behavior have not been completely established. Many toxic
properties of lead are putatively due to the metal’s capacity to mimic and
compete with calcium and zinc ions in finger proteins dependent on these
metals [Markovac and Goldstein, 1988]. Studies have also focused on the
heme biosynthetic pathway, where many lead interference sites are
encountered. Thus, lead poisoning can be considered a chemical or acquired
porphyria [Chisolm, 1971].
The thiol enzymes delta-aminolevulinic acid dehydratase (ALAD) and
ferrochelatase of this pathway are extremely sensitive to lead. ALA has long
been known to compete with γ--aminobutyric acid (GABA), a neurotransmitter
in the cortex, hypothalamus and other tissues of the Central Nervous System
(CNS) and the peripheral nervous system (PNS) [Monteiro et al, 1991]. An
increase of ALA in the blood circulation and brain areas could trigger
behavior disorders in patients carrying genetic porphyries [Bechara, 1996;
Costa et al, 1997; Gurer and Ercal, 2000; Aykin-Burns et al, 2003; Rocha et
al, 2003]. Of utmost importance was the finding that ALA-driven oxidative
injury to GABA receptors in synaptic membranes, synaptosomes, and GABA-
rich brain slices leads to a two-fold increase of the dissociation constant of
the receptor-GABA complex [Demasi et al, 1996] and a significant decrease
of the GABA receptor population [Adhikari et al., 2006].
Manuscrito II – Neurotoxicology and Teratology
.
92
Three meta-analysis studies confirmed that low lead levels exposures
were associated with reduced IQ [Needleman and Gatsonis, 1990; Schwartz,
1994; Pocock et al. 1994]. More recent data indicated cognition, attention
and behavior disturbances in children presenting lead levels in order of 3 - 5
µg/dL [Lanphear et al. 2000; Chiodo et al, 2004; Chiodo et al, 2007]. In the
recent years, ecological [Denno, 1990; Nevin 2000; Stretesky; Lynch, 2001;
Nevin, 2007] and observational [Needleman et al, 1996; Dietrich et al, 2001;
Needleman et al, 2002; Whright et al, 2008] studies relating lead exposure to
antisocial, aggressive, delinquent and criminal behavior have been carried
out. These studies showed an association between these disorders and lead
intoxication.
Thus, considering evidence linking lead exposure to a higher risk for
aggression and antisocial problems [Olympio et al, 2009], the aim of this
study was to determine lead concentrations in poverty-stricken Brazilian
adolescents using a biological marker of chronic exposure (surface dental
enamel) to evaluate the association between lead exposure and antisocial/
delinquent behavior in Brazilian youths.
2. Methods
2.1. Subjects
Volunteers were youths residing in a settlement of shacks (Ferradura
Mirim); in a housing complex, originated from an urban renovation project to
remove shantytown squatters from the city (Fortunato Rocha Lima); and in
Manuscrito II – Neurotoxicology and Teratology
.
93
the Fundação Casa (a unit serving court-ordered juvenile delinquents). All
these sites are located in the city of Bauru, Southeast Brazil, and were
selected for this study due to the high criminality indices. In Ferradura Mirim,
a census was recently carried out by the School of Sciences of the University
of the State of São Paulo and, considering these data, only the houses with
adolescents aged 14-18 years were visited. In Fortunato Rocha Lima, where
no prior census data existed, two social projects (Girassol Project and Youth
Agent) that youths attended were visited and the adolescents who
participated of these projects were enrolled. These youths were encouraged
to invite other adolescents resident in the same area to compose the sample
(snowball technique sampling). In both areas, meetings were held with the
parents and youths to explain the purpose of the research. Afterwards,
informed consent forms were presented and the adolescents whose parents
signed them were scheduled for an interview.
The Ethical Committee of the School of Public Health (University of
São Paulo) reviewed and approved this research (Proc. No. 244/05). All
youths participants received a dental cleaning and neutral fluoride gel
application, if indicated. A clinical exam was carried out and the conditions of
oral health were informed to the adolescent. When a curative treatment was
necessary it was explained to the participant and a written orientation was
provided with a list of local public institutions offering treatment. This
procedure was adopted for all subjects participating in this study.
2.2. Measures of antisocial / delinquent behavior
Manuscrito II – Neurotoxicology and Teratology
.
94
The Self-Reported of Delinquency (SRD) [Loeber et al, 1989] was
administered to the adolescents. SRD is a 36-item inventory of delinquent
acts committed over the past 6 months, scaled from 0 to 4 depending on the
frequency of acts committed. To prevent disingenuous answers to questions
that may be possibly embarrassing or incriminating, the interviewer (KPKO)
gave a copy of the SRD to the adolescent and stayed distant from him or her.
All the questions were then read aloud by the interviewer and, if the
adolescent had any questions while filling out the inventory, the interviewer
was available. When the inventory was completely filled out, the youth
deposited it into a slot in a large sealed box. The volunteers were cleared up
that the inventories were codified and it was not necessary to write their
names on the paper.
The CBCL/6-18 is a revision of the CBCL/4-18 [Achenbach, 1991]. It is
an inventory containing 113-item, with a three point scale (scored “never,
some, often”), used widely in diagnosis and assessment of psychopathology
[Achenbach; Rescorla, 2001]. CBCL/6-18 investigates competence items,
illness, disabilities and behavior, emotional and social problems. In the
present study, parents or guardians answered this inventory. As the literacy
and comprehension level of the parents were low, the interviewer read the
questions and the respondent followed the reading with a copy of the
inventory. The answers were given in a clear audible voice and the
interviewer recorded them. This process was adopted to avoid
disconcertedness or confusion and it is recommended by Achenbach and
Rescorla (2001).
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2.3. Covariates
To allow for social and familial context variables in the analysis, we
included in this study questions about maternal educational level, occupation
of the head of the household (Hollingshead, 1958), number of children and
number of people present in the house, age and sex of the adolescent, and
parents living together or not.
2.4. Dental enamel biopsy
To measure the dental enamel lead levels (DELL), surface enamel
acid-etch microbiopsies were performed. To prevent possible prior
contamination, all vials and polypropylene flasks used to prepare and store
solutions were cleaned with detergent solution, rinsed with 10 % (v/v) HNO
3
overnight, rinsed with deionized water, dried and stored in a closed
polypropylene container. High purity water provided by a Milli-Q water
purification system (Millipore, Bedford, MA, USA) was used throughout.
Analytical-grade Tritisol solutions of 1000 mg L
-1
of Pb (Merck, Darmstadt,
Germany) was used to prepare the reference analytical solutions. All
reagents used were analytical-grade. The biopsy procedure was performed
at the dental clinic. Adolescents were positioned on the dental chair and all
the procedures were performed by a dentist (KPKO). The teeth were cleaned
with a rotary brush and pumice slurry, washed and dried. The maxillary right
incisor was isolated with cotton rolls prior to the biopsy and an adhesive tape
(Magic Tape, 810 Scotch -3M) containing a circular perforation of 4.0 mm in
diameter was firmly pressed on to the labial surface of one of the central
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maxillary incisors (11), delimiting the biopsy site. The sampling site was
etched once according to the following procedure: 10 µL 1.6 mol/L HCl in
70% (v/v) glycerol were applied to the area for 35 s [Cleymaet et al, 1991].
The biopsy solution was then transferred to centrifuge tube (Axygen
Scientific, Inc., Union City, USA), containing 200 µL high purity water. The
surface was then rinsed twice for 10 s with 10 µL high purity water, which
was quantitatively transferred to the centrifuge tube, making a final volume
of 230 µL. The tape was then removed and the tooth was washed with water
for 30 s and dried with an air jet to receive a topical fluoride application.
Biopsies were also performed on various surfaces at the dental clinic to
evaluate lead contamination in the environment where the procedures were
carried out.
2.5. Chemical Analysis
2.5.1 Pb determination
A SIMAA-6000 graphite furnace atomic absorption spectrometer with a
longitudinal Zeeman-effect background correction system, Echelle optical
arrangement, solid state detector, end-capped transversal heating graphite
tubes (EC-THGA) with integrated pyrolytically coated platforms (Perkin-
Elmer, Norwalk, CT) and hollow cathode lamp was used for the Pb
determination. Solutions were delivered into the graphite tube by means of
an AS-72 autosampler. The instrumental conditions for the spectrometer
were 15 mA of current lamp, 0.7 nm of bandpass and 283.3 nm of
wavelength. The heating program consisted of 5 steps (temperature/
o
C,
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ramp/s, hold/s): 1 (130, 10, 10); 2 (200, 5, 20); 3 (800, 5, 20); 4 (2100, 0, 5);
and 5 (2400, 1, 2). Aliquots of 10 µL of samples or analytical solutions were
introduced into the graphite furnace with 10 µL of chemical modifier (5 µg Pd
+ 3 µL Mg). This chemical modifier was prepared using suprapur solutions of
10 g/L Pd in 15 % (v/v) HNO
3
and 10 g/L Mg, from the salts Pd(NO
3
)
2
and
Mg(NO
3
)
2
(Merck, Darmstadt, Germany), respectively.
The calibration curve (2 – 40 µg/L) was constructed using analytical-
grade Tritisol solutions of 1000 mg L
-1
of Pb (Pb(NO
3
)
3
), conveniently diluted
in 1.6 mol/L HCl in 70% glycerol (v/v).
The samples analyses were performed without previous pretreatment.
Dilution procedure with deionized water (2-5 times) were done for samples
with high concentrations of Pb (>40 µg/L). For each sample, the analytical
signals were obtained in triplicate.
The accuracy of analytical procedure was checked by analysis of
standard reference material of animal bone (H-5, IAEA from Austria). The
comparison between Pb concentrations obtained experimentally (3.08 ± 0.16
mg/kg) and the certified value (3.10 ± 0.18 mg/kg) showed good agreement,
considering Student’s t test at significance level of 95 %.
2.5.2 P determination
A Modula ICP optical emission spectrometer (Spectro Analytical
Instruments, Kleve, Germany) equipped with radial-viewed plasma torch was
used for phosphorus determination. The setting of the instrumental conditions
for the analyses is 1400 W of power, cross-flow nebulizer, double pass
(Scott-type) spray chamber, 12 L/min of outer gas flow, 1.0 L/min of
Manuscrito II – Neurotoxicology and Teratology
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98
intermediate and nebulizer gas flow, 1.5 mL/min of sample uptake rate and
213.618 nm of atomic P analytical wavelength.
The calibration curve was obtained using analytical-grade Tritisol
solutions of 1000 mg/l of P (KH
2
PO
4
) from Spex (Spex Sample Preparation,
Metuchen, USA) after appropriate dilution (20 times) in water. The analytical
range was 0.5-10 mg/L. For each sample, the analytical signals were
obtained in triplicate.
2.6. Statistical Analysis
The Assessment Data Manager (ADM) software (ASEBA, Burllington,
VT) was used to type and analyze data obtained from inventories.
Syndromes scales (withdrawn/depressed (counterpart of the 1991 Withdrawn
Scale), somatic complaint, anxious/depressed, social problems, thought
problems, attention problems, rule-breaking behavior (counterpart of 1991
Delinquent Behavior Scale), aggressive behavior), internalizing, externalizing
problems, and DSM (Diagnostic and Statistical Manual) - oriented scales
(affective problems, anxiety problems, somatic problems, attention
deficit/hyperactivity problems, oppositional defiant problems and conduct
problems) scored from the CBCL and the SRD scores were considered as
dependent variables. The independent variables were DELL, maternal
educational levels, head of the household’s occupational status, adolescent’s
sex and age, presence of two paternal figures at home (if living together or
not). CBCL clusters were dichotomized in clinical (score T exceeding the
clinical score) or normal profile.
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To deal with the large standard deviation in DELL, and considering
that there is not an established prevalence of DELL in the population, we
decided to consider the 75
th
percentile (217.35 ppm) as a cut-off point to
more appropriately study the extreme cases. Thus, DELL was analyzed as a
dichotomic variable (DELL≥217.37 ppm = high exposed-lead group and
DELL<217.35ppm = low exposed lead group).
The SRD scores were also dichotomically analyzed [≥ 75
th
percentile
(SRD≥15) or < 75
th
percentile (SRD<15)], because our sampling was not
previously selected to find people presenting higher rates of criminal
infractions, except for the selection of the areas recognized as having higher
criminality indices. Bivariate analyses were performed to identify associations
between the independent variables and the outcomes. To take into account
the influence of potential confounding factors, multiple logistic regression
models were used.
The software Stata, version 9.1, was used for the analysis.
3. Results
During the entire period, 313 adolescents were invited to participate in
this study. Of these eligible subjects, 234 (75%) parents signed the informed
consent. Nevertheless, 94 parents did not attend the interviews and 6
adolescents did not attend the exams, even after a minimum of three
attempts by home visit or telephone. Four volunteers could not be submitted
to lead exams because they presented dental caries or orthodontic
appliances on both the maxillary central incisors. Fifty one adolescents were
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not examined for lead because the Fundação Casa canceled the
authorization for the research with its adolescents due to a change in the
institution administration. Thus, 173 (55%) subjects were examined for lead
and filled out the Self-Reported of Delinquency (SRD). Out of 140 (45%)
parents who answered the Child Behavior Checklist (CBCL), 93 children
were examined for lead. Table 1 shows the data characterizing the sample.
Table 2 shows the mean ± SD of DELL for all volunteers and for sex
subgroups. Subjects presenting clinical scores had significantly higher DELL
than those with normal scores. There were no significant statistically
differences between sex subgroups (p> 0.05).
Tests χ
2
were applied and the Table 3 shows the number and
percentage of normal and clinical subjects distributed in the low- and high-
lead groups. There were significant differences for somatic complaints,
somatic problems, social problems and externalizing between low and high-
lead groups (p< 0.05).
Because the biopsy depth is critical to evaluate the DELL, all models
were adjusted for biopsy depth. Table 4 presents the odds ratios adjusted
only for biopsy depth and adjusted for biopsy depth, familial and social
variables. The only influential variable was “parents living together” in the
models with the following dependent variables: conduct problems, thought
problems, attention problems, SRD, aggressive behavior, rule-breaking
behavior, and externalizing problems. The other covariates were not
statistically significant. The bold variables in the table showed the stronger
associations, adjusted only for biopsy depth and adjusted for depth biopsy,
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familial and social variables. Adolescents’ SRD was not significantly related
to DELL.
4. Discussion
The results of the present study support the previous findings
[Needleman et al, 1996; Dietrich et al, 2001; Needleman et al, 2002]
indicating an association between exposure to high lead levels and
antisocial/delinquent behavior. In all the models, where the associations were
found significant, DELL was the strongest risk factor with small effects of the
covariates (Table 4). Despite that Byers and Lord’s early case studies
identified severe behavior problems as prominent outcomes of lead
poisoning, epidemiological studies have only recently begun to focus in detail
on psychopathological outcomes (Bellinger, 2008).
“From a public health standpoint, a major concern is a possible “silent
pandemic” [Grandjean and Landrigan, 2006] of neurodevelopmental
disorders resulting from children’s continuing exposure to low lead levels”
[Bellinger, 2008]. The form in which neurodevelopmental toxicity is expressed
depends on factors such as: age at exposure, coexposures to other
neurotoxicants, nutritional status, genotype and the characteristics of the
home environment [Hubbs-Tait et al, 2005; Weiss and Bellinger, 2006].
Unfortunately, we could not continue the study with arrested and
adjudicated adolescents because the Fundação Casa institution canceled the
authorization previously conferred to perform the study there. If we could
have continued the study there, a case-control design would have been
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performed. Thus, we choose to study adolescents living in adverse
socioeconomic conditions because we hypothesized that maybe the so many
social problems present in their lives, such as low-income; bad conditions of
dwelling-place; biological parents living separated; conflicting, aggressive
and violent familial environment [Loeber, 1990; Farrington, 1995] could be
more significantly associated to behavior problems than exposure to lead.
However, even in these severe conditions, the lead exposure was found to
be associated with somatic complaints, social problems, rule-breaking
behavior and externalizing problems. In this respect, it is important to
mention that externalizing is a grouping consisting of the two syndromes that
comprise problems that mainly involve conflicts with other people (aggressive
behavior and rule-breaking behavior) [Achenbach and Rescorla, 2001 and
2007]. In addition, the rule-breaking behavior scale, in this CBCL version
used, is the counterpart version of the 1991 Delinquent behavior scale.
Conduct problems is a DSM-oriented scale that group problems such as
vandalism, rule-breaking, fighting and stealing, to name a few.
Recently, Wright et al. (2008) published a prospective study, in
which they followed subjects measuring blood lead levels from pregnancy
until the adulthood. In their conclusions, prenatal and postnatal blood lead
concentrations were associated with higher rates of total arrests and/or
arrests for offenses involving violence, being the first study that reported an
association between developmental exposure to lead and adult criminal
behavior. Following this same cohort, Dietrich et al. (2001) had previously
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showed an association between lead exposure and antisocial behavior,
independent on other social and biomedical covariates.
An experimental study supports the controversial [McCall et al, 2004]
association between lead exposure and aggressiveness. This study used a
validated animal model to test the hypothesis that there is a causal
relationship between lead exposure and aggression in the absence of
confounding variables. The results showed that lead exposure enhances
predatory aggression in the cats and provided experimental support for a
causal relationship between lead exposure and aggressive behavior in
humans (Li et al., 2003). Previously, another experimental study indicated
this causal relationship. Golden hamsters exposed to lead became faster and
more likely to attack and bite their intruders [Delville, 1998]. These results are
very important because epidemiological studies have reported associations
between environmental lead exposure and aggressive behavior in children,
but could not establish a causal relationship.
In spite of extensive documentation of the toxic effects of lead on
human health, the molecular mechanisms underlying its poisonous effects on
the central nervous system (CNS) have yet to be clarified [Toscano and
Guilarte, 2005]. The specific biological mechanisms underlying lead’s effect
on aggression and impulsivity are not completely known. “Lead acts on a
large number of CNS sites, some which are involved in impulse control. Lead
interferes with synaptogenesis [Averill and Needleman, 1980], reduces the
inhibition of brain phosphokinasie C [Markovac and Goldstein, 1988], and
decreases norepinephrine-induced inhibition [Taylor et al., 1978] and lowers
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brain levels of serotonin or 5-HIAA [Lasley et al., 1984; Widmer et al., 1991].
Lead exposure is associated with increased levels of D-aminulevulinic acid,
which may antagonize GABA inhibition [Meredith, 1978]. Lead also enhances
both D1 and D2 dopamine sensitivity, and alters NMDA receptor sensitivity
[Cory-Slechtas et al., 1992]” (Needleman et al., 2002).
Recently, the neuroanatomical basis for deficits in cognition, executive
functions, social behaviors and motor abilities was studied through the
relationship between childhood lead exposure and adult brain volume using
magnetic resonance imaging. Higher mean childhood lead concentrations
were associated with significant decrements in brain volume. The most
affected regions were frontal gray matter, specifically the anterior cingulated
cortex, responsible for executive functions, mood regulation, and decision-
making. [Cecil et al., 2008].
In this study, no association was found between self-reported
delinquency, measured by SRD, and DELL. In a case-control study,
adjudicated delinquents had significantly higher mean concentrations of lead
in their bones than controls and the adjusted odds ratio was 4.0 (95% CI: 1.4-
11.1) [Needleman et al, 2002]. However, in a previous cohort study,
Needleman et al (1996) found an association between SRD and bone lead
levels, but this finding was slightly altered by entering covariates into the
model (p=0.07). Some reasons may explain why high DELL had presented
itself as a protection factor for high SRD in the present study. Firstly, we used
the snow ball technique, but, even so, the participants resided in districts with
high indices of criminality; the participation in the study was voluntary, which
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may explain the small number of offenders in the sample; increasing the
need for a larger sample size. Secondly, although great care was taken to
avoid possible embarrassment in responding to questions about offences
committed, some youths may have omitted facts, which would diminish the
SRD score.
A limitation of this study was the small number of participants whose
parents responded to the CBCL. It is important to remember that statistical
significance and the width of the confidence intervals are strongly dependent
on the sample size [Szklo and Nieto, 2007]. Thus, a smaller number of
clinical conditions of a given syndrome could explain why an association
such as, for example, conduct problems vs DELL was not found to be
statistically significant. However, inferring that there is no association when
the association is not significant or when the confidence interval overlaps the
null hypothesis value fails to consider the important fact that the likelihood of
the values within the confidence interval is the maximum for the point
estimate [Szklo and Nieto, 2007].
To our knowledge, this is the first study showing an association
between antisocial/delinquent behavior and exposure to high lead levels in
Brazilian adolescents. In Brazil, only recently a law setting the maximum limit
of lead in the manufacturing of varnishes, furniture, toys and other materials
used by children in educational settings was approved [Brazil, 2008].
Considering the current high criminality indices existing in Brazil and the
results of our study, we substantiate the need for establishing a strong
primary prevention policy for preventing lead poisoning in the country. The
Manuscrito II – Neurotoxicology and Teratology
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scientific evidence presented is more than sufficient to justify this urgent
social action.
Acknowledgements
The authors acknowledge the valuable collaboration of all directors
and teachers from the Projeto Girassol, the Projeto Agente Jovem and the
Centro Irmã Adelaide, the volunteers and their families involved in this
investigation. We are indebted to Profa. Dra. Cássia Maria Buchalla
(Faculdade de Saúde Pública, Universidade de São Paulo) for her essential
collaboration during the design of this study and to Drs. Herbert L.
Needleman (University of Pittsburgh, School of Medicine) and Dr. Kim N.
Dietrich (University of Cincinnati, College of Medicine) for encouragement
and kindly providing us information about questionnaires. This research was
supported by Fundação de Amparo à Pesquisa do Estado de São Paulo
(FAPESP, Grants 01/09641-1 and 06/56530-4), the Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq), and the Projeto Milênio
Redoxoma. KPKO is recipient of a fellowship from the Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior. MRAC is an investigator for
the CNPq (scholarship 303049/2007-3).
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References
T.M. Achenbach, L.A. Rescorla, Multicultural understanding of child and
adolescent psychopathology. Implications for mental health assessment, The
Guilford Press, New York, NY, 2007.
T.M. Achenbach, L. Rescorla, Manual for the ASEBA school-age forms &
profiles, ASEBA, Burlington, VT, 2001.
T.M. Achenbach, Manual for the Child Behavior Checklist/4-18 and 1991
profile. Burlington, VT: University of Vermont, Department of Psychiatry.
A. Adhikari, C.A. Penatti, R.R. Resende, H. Ulrich, L.R. Britto, E.J. Bechara
,
5-Aminolevulinate and 4,5-dioxovalerate ions decrease GABA(A) receptor
density in neuronal cells, synaptosomes and rat brain, Brain Res. 1093
(2006) 95-104.
N. Aykin-Burns, A. Laegeler, G. Kellogg, N. Ercal, Oxidative effects of lead in
young and adult Fisher 344 rats, Arch. Environ. Contam. Toxicol. 44 (2003)
417-420.
R.K. Byers, E.E. Lord, Late effects of lead poisoning on mental development,
Am. J. Dis. Child 66 (1943) 471-483.
Manuscrito II – Neurotoxicology and Teratology
.
108
E.J.H. Bechara, Oxidative stress in acute intermittent porphyria and lead
poisoning may be trigged by 5-aminolevulinic acid, Braz. J. Med. Biol. Res.
29 (1996) 841-851.
D. Bellinger, Very low lead exposures and children’s neurodevelopment,
Curr. Opin. Pediatr. 20 (2008) 172-177.
Brasil. Lei nº 11.762 de 1 de agosto de 2008. Fixa o limite máximo de
chumbo permitido na fabricação de tintas imobiliárias de uso infantil e
escolar, vernizes e materiais similares e dá outras providências. Available
from: http://www.planalto.gov.br/ccivil_03/_Ato2007-
2010/2008/Lei/L11762.htm [Accessed 2008 Oct 7].
K.M. Cecil, C.J. Brubaker, C.M. Adler, K.N. Dietrich, M. Altaye, J.C. Egelhoff,
S. Wessel, I. Elangovan, R. Hornung, K. Jarvis, B.P. Lanphear, Decreased
brain volume in adults with childhood lead exposure, PLoS Med. 5 (2008)
741-750.
L.M. Chiodo, S.W. Jacobson, J.L. Jacobson, Neurodevelopmental effects of
postnatal lead exposure at very low levels, Neurotoxicology 26 (2004) 359-
371.
Manuscrito II – Neurotoxicology and Teratology
.
109
L.M. Chiodo, C. Covington, R.J. Sokol, J.H. Hannigan, J. Jannise, J. Ager,M.
Greennwald, V. Delaney-Black , Blood lead levels and specific attention
effects in young children, Neurotoxicol. Teratol. 29 (2007) 538-546.
R. Cleymaet, E. Quartier, D. Slop, D.H. Retief, J. Smeyers-Verbeke, D.
Coomans, Model for assessment of lead content in human surface enamel,
J. Toxicol. Environ. Health 32 (1991) 111-127.
C.A.C. Costa, G.C. Trivelato, A.M.P. Pinto, E.J.H. Bechara, Correlation
between plasma 5-aminolevulinic acid concentrations and indicators of
oxidative stress in lead–exposed workers, Clin. Chem. 43 (1997) 1196-1202
.
J.J. Chisolm Jr, Lead poisoning, Sci. Am. 224 (1971) 15-23.
Y. Delville, exposure to lead during development alters aggressive behavior
in golden hamsters, Neurotoxicol. Teratol. 21 (1999) 445-449.
M. Demasi, C.A. Costa, C. Pascual, S. Lesuy, E.J.H. Bechara, Oxidative
tissue response promoted by 5-aminolevulinic acid promptly induces the
increase of plasma antioxidant capacity, Free Rad. Res. 26 (1996) 235-246.
D.W. Denno. Biology and violence, Cambridge Univ. Press, New York, NY,
1990.
Manuscrito II – Neurotoxicology and Teratology
.
110
K.N. Dietrich, M.D.Ris, P.A. Succop, O.G. Berger, R.L. Bornschein, Early
exposure to lead and juvenile delinquency, Neurotoxicol. Teratol. 23 (2001)
511-518.
D.P. Farrington, The Twelfth Jack Tizard Memorial Lecture. The development
of offending and antisocial behavior from childhood: key findings from the
Cambridge Study in Delinquent Development, J. Child Psychol. Psychiatry.
36 (1995) 929-64.
H. Gurer, N. Ercal, Can antioxidants be beneficial in the treatment of lead
poisoning?, Free Radic. Biol. Med. 29 (2000) 927-945.
R. Herrnstein, C. Murray, The Bell curve: intelligence and class structure in
American life, Free Press, New York, NY, 1994.
A.B. Hollingshead. The index of social position, in: A.B. Hollingshead, F.C.
Redlich, Social class and mental illness: a community study, John Willey &
Sons, USA, 1958.
B.P. Lanphear, K. Dietrich, P. Auinger, C. Cox, Cognitive deficits associated
with blood lead concentrations <10 µg/dL in US children and adolescents,
Public Health Rep. 115 (2000) 521-529.
Manuscrito II – Neurotoxicology and Teratology
.
111
W. Li, S. Han, T.R. Gregg, F.W. Kemp, A.L. Davidow, D.B. Louria, A. Siegel,
J.D. Bogden, Lead exposure potentiates predatory attack behavior in the
cat, Environ. Res. 92 (2003)197-206.
R. Loeber, M. Stouthhammer-Loeber, W. VonKammen, D. Farrington,
Development of a new measure of self-reported crime and delinquency,
Kluwer Academic Publishers, Dordrecht, 1989, pp. 203-225.
P.L. MacCall, K.C. Land, Trends in environmental lead exposure and
troubled youth, 196-1995: an age-period-cohort-characteristic analysis, Soc.
Sci. Res. 33 (2004) 339-359.
J. Markovac, G.W. Goldstein. Picomolar concentrations of lead stimulate
brain protein kinase C. Nature 334 (1988)71-73.
H.P. Monteiro, E.J.H. Bechara, D.S.P. Abdalla, Free radicals involvement in
neurological porphyrias and lead poisoning, Mol. Cell. Biochem. 103 (1991)
73-83.
H.L. Needleman, C. Gatsonis, Low level lead exposure and the IQ of
children. J. Am. Med. Assoc. 263 (1990) 673-678.
Manuscrito II – Neurotoxicology and Teratology
.
112
H.L. Needleman, J.A. Riess, M.J. Tobin, G.E. Gretchen, J.B. Greenhouse,
Bone lead levels and delinquent behavior, J. Am. Med. Assoc. 275 (1996)
363-369.
H.L. Needleman, C. McFarland, R.B. Ness, S.E. Fienberg, M.J. Tobin, Bone
lead levels in adjucated delinquents. A case control study, Neurotoxicol.
Teratol. 24 (2002) 711-717.
R. Nevin, How lead exposure relates to temporal changes in IQ, violent
crime, and unwed pregnancy, Environ. Res. 83 (2000) 1-22.
R. Nevin, Understanding international crime trends: the legacy of preschool
lead exposure, Environ. Res. 104 (2007) 315-336.
K.P.K. Olympio, C. Gonçalves, W.M.R. Günther, E.J.H. Bechara,
Neurotoxicity and aggressiveness triggered by low-level lead in children – a
review, Rev. Panam. Salud Publica (2009) [in press].
S.J. Pocock, M. Smith, P. Baghurst, Environmental lead and children’s
intelligence: a systematic review of the epidemiological evidence, Br. Med. J.
309 11994) 11889-11897.
Manuscrito II – Neurotoxicology and Teratology
.
113
M.E.M. Rocha, F. Dutra, B. Bandy, R.L. Baldini, S.L. Gomes, A. Faljoni-Alári,
C.W. Líria, M.T.M. Miranda, E.J.H. Bechara, Oxidative damage to ferritin by
5-aminolevulinic acid, Arch. Biochem. Biophys. 409 (2003) 349-356.
J. Schwartz, Low-level lead exposure and children’s IQ: a meta-analysis and
search for a threshold, Environ. Res. 65 (1994) 42-55.
P. Stretesky, M.J. Lynch, The relashionship between lead exposure and
homicide, Arch. Pediatr. Adolesc. Med. 155 (2001) 579-582.
M. Szklo, J. Nieto. Communicating results of epidemiologic studies, in: M.
Szklo, J. Nieto. Epidemiology beyond the basics 2nd ed, Jones and Barlett
Publishers, Sudbury, MA, 2007.
C.D. Toscano, T.R. Guilarte, Lead neurotoxicity: from exposure to molecular
effects, Brain Res. Rev. 49 (2005) 529-554.
J.P. Wright, K.N. Dietrich, M.D. Ris, R.W. Hornung, S.D. Wessel, B.P.
Lanphear, M. Ho, M.N. Rae, Association of prenatal and childhood blood
lead concentrations with criminal arrests in early adulthood, PLoS Med. 5
(2008) 732-740.
Manuscrito II – Neurotoxicology and Teratology
.
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Table 1. Characteristics of the entire study sample according to sex
subgroups
Variable
All
(n)
Boys (n)
Girls (n)
p-
value
Mean ± SD of age 15.6±1.3
(179)
15.5±1.3
(103)
15.6 ± 1.2
(75)
0.51
Mean ± SD of
schooling (years)
8.1±1.8
(111)
8.0±1.9
(81)
8.5±1.7
(30)
0.16
Mean± SD and
Median of maternal
schooling
1.77± 1.46(159)
Median= Up to
4 years
1.98±145 (90)
Median = Up to
4 years
1.51±1.43 (69)
Median= Up to
4 years
0.04
Occupation of the
head of the family*
(Median)
Unskilled work
(160)
Unskilled work
(90)
Unskilled work
(70)
0.60
Mean ± SD of number
of children living at
home
1.4±1.2
(157)
1.3±1.3
(90)
1.5±1.2
(67)
0.21
Mean ± SD of number
of people living at
home
5.3±1.6
(158)
5.3±1.7
(89)
5.3±1.6
(69)
0.70
Percentage of parents
living together
57.5%
(160)
57.8%
(90)
57.1%
(70)
0.08
Self-Reported
Delinquency Score
12.4±16.0 (173)
Median = 7
15.9±18.8 (97)
Median = 10
8.0±9.9 (76)
Median = 5
0.001
p-values express the statistical differences between boys and girls.
*
Hollingshead classification
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Table 2. Mean (µg/g, ±SD) (n) of dental enamel lead concentrations (75
th
percentile) for clinical and normal subjects, considering somatic, social,
conduct and externalizing problems and rule-breaking behavior.
CBCL
cluster
Clinical
Normal
All
Boys
Girls
All
Boys
Girls
Somatic
problems
197.1±157.0
(22)
Median=
188.8
206.0±159.0
(18)
Median=
226.8
156.6±163.5
(4)
Median=
102.6
177.5±347.0
b
(71)
Median=
89.0
138.1±136.3
(59)
Median=
81.0
126.8±133.7
(12)
Median=
101.1
Social
problems
231.1±482.6
(32)
Median=
114.7
240.5±503.4
(29)
Median=
148.5
140.3±220.3
(3)
Median =
22.3
156.4±164.04
(61)
Median=
89.5
159.9±174.0
(48)
Median=
88.7
143.3±125.3
(13)
Median=
122.0
Rule-
breaking
behavior
293.56±632.25
(18)
Median=
120.0
288.0±651.3
(17)
Median=
76.9
387.4
(1)
Median=
387.4
155.37±159.38
(75)
Median=
89.5
162.6±166.8
(15)
Median=
94.8
126.4±125.8
(15)
Median=
80.4
Externalizing 232.6±445.6
(38)
Median=
156.4
236.0±467.2
(34)
Median=
156.4
203.8±216.3
(4)
Median=
207.2
147.2 ±163.3
(55)
Median=
82.0
154.1±176.3
(43)
Median=
82.0
122.4±106.7
(12)
Median=
101.2
Mann-Whitney test (between clinical and normal groups): Somatic
problems
p=0.01; Social problems p=0.09; Rule-breaking behavior p=0.08;
Externalizing p=0.47
Mann-Whitney test (between clinical boys and girls): Normal somatic
problems p=0.78; Clinical Somatic problems p= 0.60; Normal Social
problems p= 0.99; Clinical Social problems p= 0.49; Normal rule-breaking
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behavior p= 0.40; Clinical rule-breaking behavior p=0.14; Normal
externalizing p=0.79; Clinical externalizing p=0.77.
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Table 3. Number and percentage of normal and clinical subjects, according
to the CBCL profiles and the SRD scores, in the low- and high-lead groups.
CBCL cluster
Low
-
Lead Group
High
-
Lead Group
p
Normal
Clinical
Normal
Clinical
Withdrawn 33 (35.5%) 33 (35.5%) 12 (12.9%) 15 (16.1%) 0.63
Somatic complaints
45 (48.4%)
21 (22.6%)
11 (11.8%)
16 (17.2%)
0.01
Somatic problems
55 (59.2%)
11 (11.8%)
16 (17.2%)
11 (11.8%)
0.01
Anxious/depressed 28 (30.1%) 38 (40.9%) 11 (11.8%) 16 (17.2%) 0.88
Social competences 46 (49.5%) 20 (21.5%) 15 (16.1%) 12 (12.9%) 0.19
Social problems
44 (47.3%)
22 (23.7%)
12 (12.9%)
15 (16.1%)
0.04
Thought problems 43 (46.2%) 23 (24.7%) 17 (18.3%) 10 (10.8%) 0.84
Attention problems 52 (55.9%) 14 (15.1%) 19 (20.4%) 8 (8.6%) 0.38
Self-Reported Delinquency 100 (57.8%) 34 (19.7%) 30 (17.3%) 9 (5.2%) 0.77
Aggressive behavior 51 (54.8%) 15 (16.1%) 18 (19.4%) 9 (9.7%) 0.29
Rule-breaking behavior 56 (60.2%) 10 (10.8%) 19 (20.4%) 8 (8.6%) 0.11
Conduct problems 54 (58.1%) 12 (12.9%) 19 (20.4%) 8 (8.6%) 0.22
Internalizing 14 (15.1%) 52 (55.9%) 3 (3.2%) 24 (25.8%) 0.25
Externalizing
44 (47.3%)
22 (23.7%)
11 (11.8%)
16 (17.2%)
0.02
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Table 4. Association between dental enamel lead concentration and Child
Behavior Checklist (CBCL) profiles or Self-Reported Delinquency (SRD)
scores.
CBCL Cluster
or
SRD Score
Adjusted for biopsy
depth
Adjusted for biopsy
depth, familial and
social variables
Withdrawn 1.38 (0.54 – 3.49) 1.18 (0.44 – 3.20)
a
Somatic complaints
3.09 (1.20
–
7.98)
2.93 (1.09
-
7
.91)
b
Anxious/depressed 1.15 (0.45 – 2.96) 1.13 (0.41 – 3.09)
c
Social problems
2.61 (1.00
–
6.84)
3.04 (1.07
-
8.64)
d
Thought problems 1.06 (0.39 – 2.83) 0.99 (0.34 – 2.88)
e
Attention problems 1.42 (0.49 – 4.08) 1.47 (0.47 – 4.62)
f
SRD 0.93 (0.40 – 2.20) 0.84 (0.32 – 2.22)
g
Aggressive behavior 1.51 (0.54 – 4.20) 1.31 (0.42 – 4.09)
h
Rule-breaking behavior
2.77 (0.90
–
8.51)
3.72 (0.99
–
14.04)
i
Conduct problems 2.38 (0.79 - 7.17) 3.01 (0.83 - 10.89)
j
Internalizing 2.40 (0.61 – 9.46) 2.17 (0.54 – 8.83)
l
Externalizing
3.06 (1.17
–
8.04)
2.87 (1.05
–
7.85)
m
Odds ratios (OR) and their 95% confidence interval (CI)
a
adjusted for biopsy depth, sex, age, number of children at
home and head of the family’s occupation;
b
adjusted for biopsy depth, head of the family’s occupation and parents
living together;
c
adjusted for biopsy depth, age, parents living together and number of children at home;
d
adjusted
for biopsy depth, sex, head of the family’s occupation, parents living together and number of children at home;
e
adjusted for biopsy depth and parents living together;
f
adjusted for biopsy depth, parents living together, head of the
family’s occupation and number of children at home;
g
adjusted for biopsy depth, parents living together and
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maternal schooling;
h
adjusted for biopsy depth, parents living together, head of the family’s occupation and number
of children at home;
i
adjusted for biopsy depth, parents living together, head of the family’s occupation and number
of children at home
;
j
adjusted for biopsy depth, parents living together, head of the family’s occupation and number
of children at home;
l
adjusted for biopsy depth, parents living together and maternal schooling;
m
adjusted for
biopsy depth and parents living together.
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Risk factors associated with high lead levels measured in the
superficial dental enamel from Brazilian youths
Kelly Polido Kaneshiro Olympio
a
Juliana Naozuka
b
Pedro Vitoriano de Oliveira
b
Maria Regina Alves Cardoso
c
Etelvino José Henriques Bechara
d
Wanda Maria Risso Günther
a
*
a
Faculdade de Saúde Pública, Departamento de Saúde Ambiental, Universidade de
São Paulo, Av. Dr. Arnaldo, 715, Cerqueira César, 01246-904, São Paulo, Brazil.
b
Instituto de Química, Departamento de Química Analítica, Universidade de São
Paulo, Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo, Brazil.
c
Faculdade de Saúde Pública, Departamento de Epidemiologia, Universidade de
São Paulo, Av. Dr. Arnaldo, 715, Cerqueira César, 01246-904, São Paulo, Brazil.
d
Instituto de Química, Departamento de Bioquímica, Universidade de São Paulo,
Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo, and Departamento de Ciências
Exatas e da Terra, Universidade Federal de São Paulo, Rua Prof. Artur Riedel, 275,
09972-270 Diadema, Brazil.
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Abstract
Objective To investigate the domestic risk factors associated with high lead levels
in the surface dental enamel (SDE).
Methods A cross-sectional study was conducted with 179 Brazilian youths aged 14-
18 and their parents living in impoverished neighborhoods (Bauru-SP). The parents
responded a questionnaire on exposure to possible exposure sources of lead. Body
lead burdens were evaluated in SDE acid-etch microbiopsies. The dental enamel
lead levels (DELL) were quantified by graphite furnace atomic absorption
spectrometry (GFAAS) and the phosphorus content was measured using inductively
coupled plasma optical emission spectrometry (ICP-OES). Logistic regression was
used to identify associations between DELL and each risk factor evaluated (A,
residing in a contaminated area or close proximity; B, working with paints, pigments,
ceramic or battery manufacturing; C, to do home-based use of lead-glazed ceramic,
low-quality pirated toys, anticorrosive enamel in gates and/or selling of used car
batteries, and D= smoking. Social and familial covariates were entered in the
models.
Findings The risk factors associated with high DELL were A (OR=4.49;
95%CI=1.69-11.97); B (OR=3.43; 95%CI= 1.31-9.00). C and D were not found to be
associated with high DELL (OR=1.31; 95%CI= 0.56-3.03) and (OR=1.66;
95%CI=0.52-5.28), respectively.
Conclusion This work demonstrated that Brazilian youth continue to be exposed to
lead by the studied sources. The companies that utilize lead in their manufacturing
process must prevent their employees from carrying contamination home on their
bodies, clothes or shoes. The surveillance on these companies must be reinforced
so that people living near of them are not contaminated and undergo the lead
poisoning.
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Introduction
Lead poisoning is a long known public health problem. There is not a
lead dose considered safe for human health
1
. Thus, lead exposure may
cause damage in diverse organs, especially in the Central Nervous System
(CNS) of children in developing process.
Many countries still add tetraethyl lead to gasoline. In Brazil, although
tetraethyl lead is no longer added to gasoline as an anti-detonator since
1978, and there are also regulations for acceptable lead levels in food and
water
2,3
, regarding acceptable levels in humans, until a recent past, only
occupational exposure was regulated. Recently, a law was approved fixing
the maximum limit of lead in the manufacturing of toys and other material for
children in educational settings, as well as varnishes and furniture
4
.
Among the non-regulated sectors, some factories follow the
internationally accepted lead parameters. According to the Brazilian
Association of Paint Manufacturers, there has been a trend beginning in the
1990s to substitute lead pigments in paints. At present, Brazilian domestic
paints are lead free. However, lead is still used as an anti-corrosive, such as
red lead in iron gates, refrigerators, cars, stoves, bicycles, and many other
goods. In this case a protective paint coating should be applied over the red
lead
5
.
The canned food manufacturers in Brazil have also substituted lead-
based solders. Glazed ceramic containers can be a source of lead poisoning
when lead leaches into stored beverages, especially in the case of acidic fruit
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juices such as those made from grapes and citrus fruits
6
. Lead from glazed
ceramics is promptly dissolved by the tartaric and citric acids present in the
juices, respectively, due to the chelation of the metal by these acids.
Since 1986, an Environmental Impact Report (EIA/RIMA) has been
officially required for the approval of potentially polluting industrial plants, as
an obligatory document to license these companies
7
. The strategies
proposed in this report to minimize the pollution by the plant must be
analyzed and approved
8
. Nevertheless, the companies built before 1986 are
not obliged to follow this protocol, unless they have caused environmental
damage
8
. Unfortunately, several small companies and domestic sources of
lead contamination do not issue warnings to prevent lead exposure
5
.
Thus, this study aimed to measure the lead levels present in surface
dental enamel from Brazilian adolescents and to compare them between
youths exposed and non-exposed to potential risk factors, such as: a)
residing in contaminated area or close proximity; b) having someone from
their family who worked/is working in paint, pigment, ceramic or battery
manufacturing; c) home-based use of glazed ceramic kitchenware, low-
quality/pirated toys, anticorrosive enamel (red lead) in iron gates without
protective paint coating, and/or selling used car batteries; and d) smoking.
Methods
Subjects
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Volunteers were youths aged 14-18, residents in the municipality of
Bauru, Southeast Brazil. Some of these adolescents attended a social project
called “First Job”, offered by the Irmã Adelaide Center and resided in a
shantytown (Ferradura Mirim), in a neighborhood 2 km from a battery
recycling plant (Tangarás). These two areas were considered “contaminated
or close proximity”. A housing state, originating from an urban renewal
project aimed at removing shantytowns from the city (Fortunato Rocha Lima)
was considered a non-contaminated area. This housing state is located
around 11 km from the battery recycling plant.
In Ferradura Mirim, a census was recently carried out by the School of
Sciences of the University of the State of São Paulo and, considering the
results of this census, only the houses where adolescents aged 14-18 years
resided were visited. In Fortunato Rocha Lima, previous data did not exist,
two social projects (“Girassol Project” and “Youth Agent”) that youths
attended were visited, and the adolescents that participated in these projects
were enrolled. These youths were motivated to invite other adolescent
residents in the same area to join and compose the sample (snowball
technique sampling). In both areas, meetings were held with the parents and
youths to explain the purpose of the research. Afterwards, informed consent
forms were distributed and those who signed them were scheduled for an
interview.
The Ethical Committee of the School of Public Health, University of
São Paulo, reviewed and approved this research (Proc. No. 244/05).. All
youth participants received a dental cleaning and neutral fluoride gel
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application, if indicated. A clinical exam was carried out, and when a curative
treatment was necessary, it was explained to the study participants and a
written orientation was delivered with a list of local public institutions offering
treatment. This process was adopted for all subjects participating of this
study.
Measures of risk factors
Parents of the adolescents completed a questionnaire focusing on:
location of actual and prior residences (risk factor A); whether someone from
home had worked or works in ceramic, paint, pigment or battery
manufacturing (risk factor B); whether their home contains/contained glazed
ceramic for hot/acidic food/beverage, low-quality/pirated-toys, anticorrosive
enamel on gates without other enamel covering it, and or someone who sells
used car batteries (risk factor C); and if the adolescent was a smoker (risk
factor D).
Covariates
To allow for social and familial context variables in the analysis, we
included in this study questions about maternal educational level, occupation
of the head of the family (Hollingshead classification
9
), number of children
and number of people present in the house, age and sex of the adolescent.
Dental enamel biopsy
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To measure the dental enamel lead levels (DELL), surface dental
enamel acid-etch microbiopsies were performed. Material used during the
procedure was cleaned in 10% nitric acid (v/v) to avoid possible prior
contamination. All reagents were tested for contamination with lead. The
biopsy procedure was performed at the dental clinic. Adolescents were
positioned on the dental chair and all the procedures were performed by a
dentist (KPKO, first author). The teeth were cleaned with a rotary brush and
pumice slurry, then washed and dried. The maxillary right incisor was
isolated with cotton rolls prior to the biopsy and an adhesive tape (Magic
Tape, 810 Scotch -3M) containing a circular perforation of 4.0 mm in
diameter was adhered on to the labial surface of the tooth, delimiting the
biopsy site. The sampling site was etched once according to the following
procedure: 10 µL 1.6 mol/L HCl in 70% glycerol (v/v) were applied to the area
for 35 s
10
. The biopsy solution was then transferred to a 0.2 mL centrifuge
tube (Axygen Scientific, Inc., Union City, USA) containing 200 µL ultrapurified
water. The surface was then rinsed twice for 10 s with 10 µL ultrapurified
water which was then transferred to the centrifuge tube, making a final
volume of 230 µL. The tape was then removed and the tooth was washed
with water for 30 s and dried with an air jet to receive a neutral topical fluoride
application. Biopsies were also performed on various sites at the dental clinic
bench or at a centrifuge tube rack to evaluate lead contamination in the
environment where the procedures were carried out.
Chemical Analysis
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Lead (Pb) determination
A SIMAA-6000 graphite furnace atomic absorption spectrometer with a
longitudinal Zeeman-effect background correction system, Echelle optical
arrangement, solid state detector, end-capped transversal heating graphite
tubes (EC-THGA) with integrated pyrolytically coated platforms (Perkin-
Elmer, Norwalk, CT) and hollow cathode lamp was used for the Pb
determination. Solutions were delivered into the graphite tube by means of
an AS-72 autosampler. The instrumental conditions for the spectrometer
were 15 mA of current lamp, 0.7 nm of bandpass and 283.3 nm of
wavelength. The heating program consisted of 5 steps (temperature/
o
C,
ramp/s, hold/s): 1 (130, 10, 10); 2 (200, 5, 20); 3 (800, 5, 20); 4 (2100, 0, 5);
and 5 (2400, 1, 2). Aliquots of 10 µL of samples or analytical solutions were
introduced into the graphite furnace with 10 µL of chemical modifier (5 µg Pd
+ 3 µL Mg). This chemical modifier was prepared using suprapur solutions of
10 g/L Pd in 15 % (v/v) HNO
3
and 10 g/L Mg, from the salts Pd(NO
3
)
2
and
Mg(NO
3
)
2
(Merck, Darmstadt, Germany), respectively.
The calibration curve (2 – 40 µg/L) was constructed using analytical-
grade Tritisol solutions of 1000 mg L
-1
of Pb (Pb(NO
3
)
3
), conveniently diluted
in 1.6 mol/L HCl in 70% glycerol (v/v).
The samples analyses were performed without previous pretreatment.
Dilution procedure with deionized water (2-5 times) were done for samples
with high concentrations of Pb (>40 µg/L). For each sample, the analytical
signals were obtained in triplicate.
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The accuracy of analytical procedure was checked by analysis of
standard reference material of animal bone (H-5, IAEA from Austria). The
comparison between Pb concentrations obtained experimentally (3.08 ± 0.16
mg/kg) and the certified value (3.10 ± 0.18 mg/kg) showed good agreement,
considering Student’s t test at significance level of 95 %.
Phosphorus (P) determination
A Modula ICP optical emission spectrometer (Spectro Analytical
Instruments, Kleve, Germany) equipped with radial-viewed plasma torch was
used for phosphorus determination. The setting of the instrumental conditions
for the analyses is 1400 W of power, cross-flow nebulizer, double pass
(Scott-type) spray chamber, 12 L/min of outer gas flow, 1.0 L/min of
intermediate and nebulizer gas flow, 1.5 mL/min of sample uptake rate and
213.618 nm of atomic P analytical wavelength.
The calibration curve was obtained using analytical-grade Tritisol
solutions of 1000 mg/l of P (KH
2
PO
4
) from Spex (Spex Sample Preparation,
Metuchen, USA) after appropriate dilution (20 times) in water. The analytical
range was 0.5-10 mg/L. For each sample, the analytical signals were
obtained in triplicate.
2.6. Statistical Analysis
The data from questionnaires were typed using the software Excel
2003 (Microsoft Corporation, Redmond, WA, USA). Each risk factor
evaluated was dicotomically analyzed. For risk factor A, to reside in
Ferradura Mirim, Tangarás or close to companies that possibly contaminate
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the environment with lead were considered as 1 and residing in Fortunato
Rocha Lima or locals where there are not industries was considered as 0.
For risk factor B, when someone at home had worked/ was working in
factories that possibly use lead in their manufacturing process, it was
considered 1 and, when nobody had worked in this type of work, it was
considered 0. For risk factor C, 0 was adopted for none use of glazed
ceramic kitchenware, pirated toys, anticorrosive enamel in gates without
covering paint, and selling of used car batteries and 1 when one or more of
those items was/were utilized. For risk factor D, smokers were considered 1
and non-smokers were designated 0. Afterwards, dental enamel lead levels
(DELL) were considered as a dependent variable and each risk factor as
independent variables.
To deal with the large standard deviation in DELL, and considering
that there is not an established DELL prevalence in the population, we
decided to consider the 75
th
percentile (217.35 ppm) as a cut-off point to
study more appropriately the extreme cases. Thus, DELL was analyzed as a
dichotomic variable (DELL≥217.37= high exposed-lead group and
DELL<217.35= low exposed-lead group). All the models were controlled by
the biopsy depth.
Bivariate analyses were performed to identify associations between
the independent variables and the outcome. To take into account the
influence of potential confounding factors, multiple logistic regression models
were used.
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Mann-Whitney tests were used to analyze the difference between sex
subgroups; and between exposed and non-exposed groups (p<0.05).
The software Intercooled Stata version 9.1 was used for the analysis.
Results
During the entire period, 262 adolescents were invited to participate in
this study. Of these eligible subjects, 183 (70%) parents signed informed
consent forms. Nevertheless, 6 adolescents did not attend the exams, even
after a minimum of three attempts at home visits or telephone contact. Four
volunteers could not be given lead exams because they presented dental
caries or orthodontic appliances on both the maxillary central incisors. Thus,
179 (68%) subjects were examined for lead and 160 parents (61%) filled out
the questionnaires. The principal motives of the youth who did not want to
participate in the study were: the adolescent had left home; the adolescent
could not attend the exam because he/she worked the whole day or did not
have interest in participating.
Table 1 shows the covariates structure and data characterizing the
sample. As can be seen, the only variable significantly different between
males and females was the mean of the maternal educational level (p=0.04).
DELL only was statistically different between exposed and non-
exposed subjects, when risk factor A was analyzed (p=0.0006). In relation to
risk factor B, although the medians found for adolescents whose parents
worked in ceramic, pigment, battery or paint manufacturing were found to be
higher than DELL median from adolescents whose parents did not work in
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this type of work, the difference was not statistically significant (p=0.0908).
Nor there was significant differences of DELL means between exposed and
non-exposed for risk factor C (exposure to glazed ceramic kitchenware, red
lead in gates without protective paint coating, batteries removed from cars
and pirated-toys, nor risk factor D (smoking) (p=0.9022; p=0.4528,
respectively) (Table 2).
Table 3 shows the association between DELL and the risk factors
analyzed. High DELL was significantly associated with residing in
contaminated areas or in close proximity and (risk factor A) and someone at
home working in ceramic, pigment, battery or paint manufacturing (risk factor
B). The adjusted odds ratios for social and familial variables (95% CI) were
4.49 (1.69-11.97) and 3.43 (1.31-9.00) for A and B risk factors, respectively.
The odds ratios for DELL vs. risk factor C or risk factor D were not
significantly associated with high DELL.
Discussion
This study demonstrated a strong association between high DELL and
residing next to or in close proximity (around 2 kilometers) to companies
using lead or even living with someone who worked in such places. These
findings are not original
11,12,13,14,15,16
, but their importance lies in that they
substantiate that lead exposure is preventable, and that the consequences of
lead poisoning are very serious and, furthermore, people continue to be
contaminated in the studied area.
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Probably, unsuspected sources of lead poisoning are present in the
daily routine of millions of people worldwide. Recently, a study concluded that
one-fifth of both US-manufactured and Indian-manufactured Ayurvedic
medicines purchased via the Internet contained detectable lead, mercury, or
arsenic
17
. However the principal danger resides in those products destined
for children. Toys may contain lead in their paints and small children have the
habit of placing objects in their mouth. This can be grave because a child’s
intestine absorbs lead much faster than an adult’s and the developing infant
CNS is more vulnerable to toxic agents than the mature CNS, especially in
the case of undernourished children. Neural proliferation, differentiation and
plasticity are strongly impaired by lead.
The presence of smokers in the familial environment was also
identified as a risk factor for higher blood lead levels in children living close to
a smelting plant in Sweden
18
. In the present study, we did not find an
association between high DELL and the adolescent’s smoking habit. Dental
enamel contamination by lead contained in the cigarettes would show a post-
eruptive uptake of lead to dental issue and there is no data that definitely
demonstrate that it occurs. From our results, smokers did not present higher
DELL than non-smokers; in spite of there are only a few smokers in our
sample. The pre-eruptive lead uptake seems to be predominant.
The use of low-quality/pirated-toys, lead-glazed ceramic kitchenware,
red lead for iron gates, and/or selling used car batteries was not found to be
associated with high DELL. Admittedly, our sample was small and we did not
measure the lead content of the toys and glazed ceramic used. This was
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partly because the questionnaire asked about early use of the objects in the
period when the adolescents were children, which we had not means of
measuring. In addition, in Brazil, the “invasion” of imported and low-quality
toys is recent. Another interesting fact is that the majority of the people
interviewed stated having not lived in houses with iron gates because they
lived in shacks, which typically would not have iron gates associated with
higher quality housing.
On this point, the socioeconomic status must be stressed. The form in
which neurodevelopmental toxicity is expressed depends on factors such as
age at exposure, coexposure to other neurotoxicants, nutritional status,
genotype and the characteristics of the home environment
19,20
. Low-
socioeconomic status implies residing in risk areas or in areas surrounding
contaminated factories, which is aggravated by unpaved streets. These
places lack recreation options, and children frequently play in the streets in
direct contact with the lead contaminated soil and dust which, then
accumulates in their homes. Playing with soil and dust containing lead were
indeed found to be risk factors to lead poisoning
21,22,23,24
. In the studied area,
parents related that their children barely had toys, and, the few toys that they
had had been bought from street vendors, who sell pirated low-quality toys.
The association found in this study between living with someone
working with lead and high DELL has been demonstrated in previous
studies
11,13,25,26,27,28,29
. In the Freitas’ study, although the company provided
separate bathrooms and lockers, 20% of the studied workers said they use to
bring home the clothes they had worked in. Some adolescents studied by us
Manuscrito III – Bulletin of the World Health Organization
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134
were residents from the same area or their relatives worked in the same
company studied by Freitas et al
11
.
Lead is long known as a ubiquitous, insidious and devastating
neurotoxicant. Lead poisoning is reportedly linked to a high risk of learning
disabilities, aggressiveness and criminal offenses
30,31,32,33,34,35
.
One of the many positive consequences of preventing lead exposure
has substantial economic benefits. Grosse et al
36
calculated that US
preschool children would experience a 2.2-4.7 point IQ increase above what
it might have been if leaded gasoline and blood lead had not been reduced.
From this, they calculated the IQ-related increase in income and estimated
that the economic benefit for each year’s cohort of 3.8 million of children
aged 2 ranges from $110 billion and $319 billion. Landrigan et al
37
, assuming
no threshold for the lead-IQ association, estimated the loss of future earnings
for the one-year cohort of children aged 5 in 1997 at $43.4 billion.
In summary, DELL reflected risk factors more associated with lead
exposure among the studied sources. Because lead exposure is preventable,
we conclude by calling for public health policies that protect the population of
these poisoning risks and avoid individual and national economic losses.
Government supported education campaigns should inform the public of the
dangers of lead exposure. Such public initiatives for primary prevention
already exist in developed countries (www.cdc.gov, www.fda.gov,
www.epa.gov); nevertheless, very little has been done in Brazil, where the
vast majority of the population is not aware of the dangers of lead poisoning
Manuscrito III – Bulletin of the World Health Organization
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135
and it is not known what portion of the population is in danger of lead
exposure.
Funding
The authors acknowledge the collaboration of all directors and
teachers from Projeto Girassol, Projeto Agente Jovem, Centro Irmã Adelaide
and Creche São José, the volunteers and their families involved in this
investigation. We thank Dr. Clarice Umbelino de Freitas (Centro de Vigilância
Epidemiológica, Secretaria de Saúde do Estado de São Paulo, Brazil) for
valuable contribution during the design of this study and Dr. Marília Afonso
Rabelo Buzalaf (Faculdade de Odontologia de Bauru, Universidade de São
Paulo, Brazil) for critical review of the manuscript and helpful suggestions.
This research was supported by Fundação de Amparo à Pesquisa do Estado
de São Paulo (FAPESP, Grants 01/09641-1 and 06/56530-4), the Conselho
Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and the
Projeto Milênio Redoxoma. KPKO is recipient of a fellowship from the
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior.
Competing interests: None declared
References
Manuscrito III – Bulletin of the World Health Organization
.
136
1. Chiodo LM, Covington C, Sokol RJ, Hannigan JH, Jannise J, Ager J et
al. Blood lead levels and specific attention effects in young children.
Neurotoxicol Teratol 2007; 29:538-46.
2. Brasil. Portaria nº 16, de 13 de março de 1990. Autoriza a inclusão na
tabela II, como preceitua o artigo 26 do decreto 55871, de 26 de
março de 1965 dos limites máximos de tolerância de chumbo em
alimentos. Diário Oficial da União 1990; 15 mar. Seção 1, p. 5436.
3. Resolução CONAMA nº 357. Dispõe sobre a classificação dos corpos
de água e diretrizes ambientais para o seu enquadramento, bem
como estabelece as condições e padrões de lançamento de efluentes,
e dá outras providências. Diário Oficial da União 2005; 18 mar.
4. Brasil. Lei nº 11.762 de 1 de agosto de 2008. Fixa o limite máximo de
chumbo permitido na fabricação de tintas imobiliárias de uso infantil e
escolar, vernizes e materiais similares e dá outras providências.
Available from: http://www.planalto.gov.br/ccivil_03/_Ato2007-
2010/2008/Lei/L11762.htm [Accessed 2008 Oct 7].
5. Freitas CU. Estratégias de abordagem para a exposição ambiental ao
chumbo no Estado de São Paulo. Available from:
http://www.cve.saude.sp.gov.br/htm/doma/chumbo.htm [Accessed
2006 Dec 1].
Manuscrito III – Bulletin of the World Health Organization
.
137
6. Browder AA. Lead poisoning from glazes. Ann Intern Med 1972;
76:665.
7. Resolução CONAMA nº 237. Dispõe sobre a revisão e
complementação dos procedimentos e critérios utilizados para o
licenciamento ambiental. Diário Oficial da União 1997; 22 dez.
8. Machado PAL. Direito ambiental brasileiro. São Paulo: Revista dos
Tribunais; 1982.
9. A.B. Hollingshead. The index of social position, in: A.B. Hollingshead,
F.C. Redlich, Social class and mental illness: a community study, John
Willey & Sons, USA, 1958.
10. Brudevold, F. Reda, A. Aasenden, R. Bakhos, Y. Determination of
trace elements in surface enamel of human teeth by a new biopsy
procedure. Arch Oral Biol 1975; 20: 667 –73.
11. Freitas CU, Capitani EM, Gouveia N, Simonetti MH, Paula e Silva MR,
Kira CS, et al. Lead exposure in an urban community: investigation of
risk factors and assessment of the impact of lead abatement
measures. Environ Res 2007; 103:338-44.
Manuscrito III – Bulletin of the World Health Organization
.
138
12. Carvalho FM, Silvany Neto AM, Tavares TM, Costa ACA, Chaves
CER, Nascimento LD, et al. Chumbo no sangue de crianças e passivo
ambiental de uma fundição de chumbo no Brasil. Rev Panam Salud
Publica 2003; 13:19-23.
13. Paoliello MMB, De Capitani EM, Cunha FG, Matsuo T, Carvalho MF,
Sakuma A., et al. Exposure of children to lead and cadmium from
mining area of Brasil. Environ Res 2002; A 88: 120–8.
14. Suplido ML, ONG CN. Lead exposure among small-scale battery
reciclers, automobile radiators mecanics, and their children in Manila,
the Philippines. Environ Res 2000; A82: 231-8.
15. Baghurst PA, Tong S, Sawyer MG, Burns J, Mc Michael AJ.
Sociodemographic and behavioral determinants of blood lead
concentrations in children aged 11-13 years. The Port Pirie cohort
study. MJA 1999; 170:63-67.
16. Silvany Neto AM, Carvalho FM, Tavares TM, Guimarães GC, Amorim
CJB, Peres MFT et al. Lead poisoning among children of Santo
Amaro, Bahia, Brazil in 1980, 1985, and 1992. Bulletin of PAHO 1996;
30: 51-63.
Manuscrito III – Bulletin of the World Health Organization
.
139
17. Saper RB, Phillips RS, Sehgal A, Khouri N, Davis RB, Paquin J et al.
Lead, mercury, and arsenic in US- and Indian-Manufactured ayurvedic
medicines sold via the internet. J Am Med Assoc 2008; 300:915-23
doi:10.1001/jama.300.8.915
18. Berglund M, Lind B, Sorensen S, Vahter M. Impact of and dust lead on
children’s blood lead in contaminated areas of Sweden. Arch of
Environ Health 2000; 55:93-7.
19. Weiss B, Bellinger DC, Social ecology of children’s vulnerability to
environmental pollutants. Environ Health Perspect 2006; 114: 1479-
85.
20. Hubbs-Taitt L, Nation JR, Krebs NF, Bellinger DC. Neurotoxicants,
micronurients, and social environments: individual and combined
effects on children’s development. Psychol Sci Public Interest 2005;
6:57-121.
21. Rhoads GG, Ettinger AS, Weisel CP, Buckley TJ, Goldman KD, Lioy
PJ. The effect of dust lead control on blood lead in toddlers: a
randomized trial. Pediatrics 1999; 103:551-5.
Manuscrito III – Bulletin of the World Health Organization
.
140
22. Tornton I, Davies DJA, Watt JM, Quinn MJ. Lead exposure in young
children from dust and soil in the United Kingdom. Environ Health
Perspect 1990; 89:55-60.
23. Charney E, Kessler B, Fartel M, Jackson D. Childhood lead poisoning:
a controlled trial of the effect of dust-control measures on blood lead
levels. N Engl J Med 1983; 309:1089-93.
24. Ordonez BR, Romero LR, Mora R. Investigacion epidemiológica sobre
niveles de plomo em La populacion infantil y em El médio ambiente
domiciliário de Ciudad Juarez, Chihuahua, em relacion com uma
fundicion de El Paso, Texas. Bol Oficina Sanit Panam 1976; 80:303-
17.
25. Leroyer A, Nisse C, Hemon D, Gruchociak A, Salomez JL, Haguenoer
JM. Environmental lead exposure in a population of children in
Northern France: factors affecting lead burden. Am J Ind Med 2000;
38: 281–9.
26. Trepka MJ, Heinrich J, Krause C, Schulz C, Lippold U, Meyer E, et al.
The internal burden of lead among children in smelter town—a small
area analysis. Environ Res 1997; 72: 118–30.
Manuscrito III – Bulletin of the World Health Organization
.
141
27. Cook M., Chappell WR, Hoffman RE, Manglone EJ. Assessment of
blood lead levels in children living in a historic mining and smelting
community. Am J Epidemiol 1993; 137: 446–55.
28. Silvany-Neto AM, Carvalho FM, Chaves MEC, Brandão AM, Tavares
TM. Repeated surveillance of lead poisoning among children. Sci
Total Environ 1989; 78: 179-86.
29. Tavares TM, Brandão AM, Carvalho FM. Lead in hair of children
exposed to gross environmental pollution. Int J Environ Anal Chem
1989; 36: 221–30.
30. Olympio KPK, Gonçalves C, Günther WMR, Bechara EJH,
Neurotoxicity and aggressiveness triggered by low-level lead in
children – a review. Rev Panam Salud Publica 2009; [in press].
31. Wright JP, Dietrich KN, Ris D, Hornung RW, Wessel SD, Lanphear
BP, et al. Association of prenatal and childhood blood lead
concentrations with criminal arrests in early adulthood. PLoS Med
2008; 5: 732-40.
32. Nevin R. Understanding international crime trends: the legacy of
preschool lead exposure. Environ Res 2007; 104: 315-36.
Manuscrito III – Bulletin of the World Health Organization
.
142
33. Needleman HL, McFarlandC, Ness RB, Fienberg SE, Tobin MJ. Bone
lead levels in adjudicated delinquents. A case control study.
Neurotoxicol Teratol 2002; 24:711-7.
34. Dietrich KN, Ris MD, Succop PA, Berger OG, Bornschein RL. Early
exposure to lead and juvenile delinquency. Neurotoxicol Teratol 2001;
23:511-8.
35. Needleman HL, Riess JA, Tobin MJ, Biesecker GE, Greenhouse JB.
Bone lead levels and delinquent behavior. J Am Med Assoc 1996;
275:363-9.
36. Grosse SD, Matte TD, Schwartz J, Jackson RJ. Economic gains
resulting from the reduction in children’s exposure to lead in the United
States. Environ Health Perspect 2002; 110:563-70.
37. Landrigan PJ, Schechter CB, Lipton JM, Fahs MC, Schwartz J.
Environmental pollutants and disease in American children: estimates
of morbidity, mortality, and costs for lead poisoning, asthma, cancer
and developmental disabilities. Environ Health Perspect 2002;
110:721-8.
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Table 1. Descriptive variables (n) for all subjects and for sample stratified by sex.
Variable
All (n)
Boys (n)
Girls (n)
P
value
Mean ± SD of age
(years)
15.6±1.3
(179)
15.5±1.3
(103)
15.6 ± 1.2
(75)
0.51
Mean ± SD (years)
of adolescent’s
educational level
8.1±1.8
(111)
8.0±1.9
(81)
8.5±1.7
(30)
0.16
Maternal educational
level (mean ± SD and
median, years)
1.77±1.46(159)
Median= Up to
4 years
1.98±1.45 (90)
Median = Up to
4 years
1.51±1.43 (69)
Median= Up to
4 years
0.04
Occupation of the
head of the
household* (Median)
Unskilled work
(160)
Unskilled work
(90)
Unskilled work
(70)
0.60
Mean ± SD of
number of children
living at home
1.4±1.2
(157)
1.3±1.3
(90)
1.5±1.2
(67)
0.21
Mean ± SD of
number of people
living at home
5.3±1.6
(158)
5.3±1.7
(89)
5.3±1.6
(69)
0.70
*Hollingshead classification
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Table 2. Mean (µg/g, ±SD) (n) of dental enamel lead concentrations for
exposed and non-exposed subjects, considering risk factor A (to reside in
contaminated area or near of there); risk factor B (to work in paints,
pigments, ceramic or batteries manufacturing), risk factor C (to do home use
of glazed ceramic, pirate toys, anticorrosive enamel without covering paint in
gates, and/or commercializing used car batteries), and risk factor D
(smoking).
Risk
factor
Exposed
Non
-
exposed
All
Boys
Girls
P
All
Boys
Girls
P
A
222.8±363.0
(65)
Median=
139.5
530.7±784.4
(11)
Median=
248.4
262.1±287.7
(4)
Median=
188.6
0.59 129.9±189.0
(95)
Median=
62.5
147.1±215.2
(44)
Median=
52.3
115.1±163.8
(51)
Median=
62.5
0.40
B
274.3±500.4
(31)
Median=
148.0
356.4±640.5
(18)
Median=
204.8
160.6±143.7
(13)
Median=
129
0.47 142.0±181.3
(128)
Median=
79.3
149.7±187.0
(72)
Median=
78.2
132.2±175.1
(56)
Median=
79.4
0.32
C
178.2±314.0
(112)
Median=
88.7
215.1±392.6
(62)
Median=
88.7
132.4±166.4
(50)
Median=
90.2
0.13 143.1±155.6
(48)
Median=
76.8
137.8±143.5
(28)
Median=
61.5
150.5±174.7
(20)
Median=
99.0
0.94
D
158.2±134.2
(16)
Median=
155.8
155.3±130.2
(14)
Median=
155.8
178.2±221.1
(2)
Median=
178.2
0.75 168.7±287.9
(144)
Median=
84.0
197.6±362.0
(76)
Median=
81.5
136.4±168.0
(68)
Median=
90.2
0.25
Mann-Whitney test (between exposed and non-exposed groups): A -
p=0.0006; B - p=0.0908; C - p=0.9022; D - p= 0.4528
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Table 3. Association between dental enamel lead levels and risk factor A (to
reside in contaminated area or near of there); risk factor B (to work in paints,
pigments, ceramic or batteries manufacturing), risk factor C (to do home use
of glazed ceramic, pirate toys, batteries removed from cars, and/or
anticorrosive enamel without covering paint in gates), and risk factor D
(smoking).
Risk factor
Adjusted by biopsy
depth
Adjusted by biopsy
depth, familial and
social variables
A
4.25 (1.63
-
11.13)
4.49 (1.69
-
11.97)
a
B
2.30 (0.96
-
5.49)
3.43 (1.31
-
9.00)
b
C
1.24 (0.55-2.82) 1.31 (0.56-3.03)
c
D
2.06 (0.67-6.28) 1.66 (0.52-5.28)
d
a
adjusted for biopsy depth, number of children at home and head of the
family’s occupation;
b
adjusted for number of people at home, head of the
family’s occupation, adolescent’s sex and adolescent’s age;
c
adjusted for
number of people at home, head of the family’s occupation and adolescent’s
sex;
d
adjusted for biopsy depth, mother’s schooling and sex.
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Methodological alterations of surface dental enamel microbiopsies for
lead body burden measurement
Kelly Polido Kaneshiro Olympio,* Juliana Naozuka,
§
Ana Carolina
Magalhães,
†
Manuel Valentim de Pera Garcia,
‡
Pedro Vitoriano de Oliveira,
§
Marília Afonso Rabelo Buzalaf,
†
Etelvino José Henriques Bechara,
Wanda
Maria Risso Günther
*,1
*
Faculdade de Saúde Pública, Departamento de Saúde Ambiental, Universidade
de São Paulo, Av. Dr. Arnaldo, 715, Cerqueira César, 01246-904, São Paulo-SP,
Brazil;
§
Instituto de Química, Departamento de Química Analítica, Universidade de
São Paulo, Av. Prof. Lineu Prestes, 748, 05508-900, São Paulo-SP, Brazil;
†
Faculdade de Odontologia de Bauru, Departamento de Ciências Biológicas,
Universidade de São Paulo, Al. Octávio Pinheiro Brisolla, 9-75, Jd. Aeroporto,
17012-901, Bauru-SP, Brazil;
‡
Instituto de Matemática e Estatística, Departamento
de Matemática Aplicada, Universidade de São Paulo, Rua do Matão, 110, sala B-
111, 05508-900, São Paulo-SP, Brazil;
Instituto de Química, Departamento de
Bioquímica, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, 05508-900,
São Paulo-SP, and Departamento de Ciências Exatas e da Terra, Universidade
Federal de São Paulo, Rua Prof. Artur Riedel, 275, 09972-270 Diadema, Brazil.
.
Short title: Surface dental enamel microbiopsies
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ABSTRACT
Albeit lead poisoning is preventable, it continues to be a public health
problem in several countries. Measuring lead on the surface dental enamel (SDE) is
rapid, safe and painless. However, the methodological protocol for SDE etch-acid
microbiopsies must be calibrated to render studies comparable among diverse
populations. This study aimed to compare two distinct enamel biopsy protocols. For
this, two consecutive enamel layers were removed by each protocol, in the same
subject group (n=138). To confirm the in vivo results, profilometry tests were
performed in bovine enamel blocks. Protocol I consisted of a biopsied site of 4 mm
in diameter after application of 10 µL HCl for 35 s. Protocol II was based on a
biopsied site of 1.6 mm in diameter after application of 5 µL HCl for 20 s. Next, the
dental enamel lead levels (DELL) and biopsy depth were compared: a) first and
second layers under the same protocol; b) right and left incisors by the same
protocol; c) protocols I and II for two removed layers. The adopted significance level
was 5%. The biopsy depth, calculated by the cylinder formula, for Protocol II led to
misleading results, as confirmed by profilometry tests. This study demonstrated that
SDE, analyzed by etch-acid microbiopsy, is a reliable biomarker, but DELL could not
be compared when there was any methodological variant among studies. In
addition, the cylinder formula does not seem to be adequate for calculating the
depth of the removed layer in all biopsy protocols.
Key words: biopsy; dental enamel; environmental health; lead; toxicology; public
health
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INTRODUCTION
Lead is the most abundant environmental contaminant in industrialized
areas worldwide (Cleymaet, 1991). In addition, “silent” human poisoning
occurs from unsuspected sources, such as paints covering low-quality and
pirated toys, lead-glazed ceramic kitchenware, anticorrosive red lead used on
iron gates, to name a few (Olympio et al, 2009a).
For surveillance of the lead levels in the population, blood is the most
commonly used biomarker (Rabinowitz, 1995). Although blood is a reliable
marker for recent exposures, it is not a perfect biomarker. Recently, an
increased interest in lead content of human dental tissues has been clearly
demonstrated by the Archaeological Sciences (Budd et al, 1998; Budd et al,
2000; Budd et al, 2004). Unlike bone, dental enamel is not remodeled in later
life and the human dental tissues allow for the historic reconstruction of the
early exposure (Budd et al, 1998).
Particularly, dental enamel is the most appropriated material to
sampling when the aim is to describe the natural history of lead exposure.
Dental enamel is highly mineralized and its dense structure makes it resistant
to diagenesis in the burial environment. Concomitantly, surface dental
enamel acid-etch microbiopsies have been used to measure the body burden
lead levels in vivo (Almeida et al, 2007; Almeida et al, 2008; Cleymaet,
1991a, 1991b, 1991c, 1991d; Gomes et al, 2004; Olympio et al, 2009b).
Surface dental enamel acid-etch microbiopsies have been carried out
by different protocols in which a number of variables are modified. Dental
enamel, even though it is not widely utilized, is a promising biomarker
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because the surface dental enamel microbiopsies are minimally invasive,
easy to operate and inexpensive. However, a trustworthy protocol must be
established for comparisons among diverse studies and populations.
During a previous study carried out by our research group (Olympio et
al, 2009b), we found notable differences between dental enamel lead levels
(DELL) and biopsy depth described in the diverse studies that used surface
dental enamel etch-acid biopsy (Almeida et al, 2007; Almeida et al, 2008;
Cleymaet, 1991a, 1991b, 1991c, 1991d; Gomes et al, 2004). This finding
pointed out for us a research problem – evaluating the accuracy of the
formula used to calculate the biopsy depth because DELL is directly
dependent on this variable. Lead levels in dental enamel decreases from the
outermost superficial layer to the inner layer of dental enamel (Brudevold et
al, 1975; Cleymaet et al, 1991). Biopsy depth is commonly calculated by the
cylinder formula in surface dental enamel etch-acid biopsies.
Thus, the general objective of this study was to verify whether DELL
found in studies that used surface dental enamel etch-acid biopsy with
methodological variants in their protocol can be compared. In addition, the
specific aims of the present study were: a) to compare DELL and biopsy
depths between homologous teeth biopsied by the same protocol; b) to
compare DELL and biopsy depths between homologous teeth biopsied by
different protocols; and c) to compare DELL and biopsy depths between two
consecutive layers biopsied by protocols I and II.
MATERIAL AND METHODS
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Subjects. Volunteers were 138 youths aged 14-18, residents of the
municipality of Bauru, Southeast São Paulo State, Brazil. Part of this group
was composed of adolescents attending in a social project “First Job”, offered
by the Irmã Adelaide Center. Some adolescents of this group resided in
Tangarás, a neighborhood where a battery recycling plant existed (closed in
2002) and most of the youth lived in Ferradura Mirim, a settlement of shacks,
distant around 2 kilometers from the plant. These two areas were considered
as “contaminated or close proximity”, respectively. A housing complex,
originated from an urbanistic project for resettling shantytown squatters from
the city (Fortunato Rocha Lima) was included as a non-contaminated area.
This housing complex is located far (11 kilometers) from the battery recycling
plant. Of the 138 subjects included in this study, 59 adolescents composed
the sample to compare the Protocols I and II and 79 youths composed the
sample to compare the Protocol I between homologous teeth. This study was
approved by the Institutional Review Board of the Public Health School,
University of São Paulo (Proc. No. 244/05).
In vivo human surface dental enamel biopsy. To measure the
dental enamel lead levels (DELL), surface enamel acid-etch microbiopsies
were performed. The material used during the procedure was cleaned in 10%
(v/v) nitric acid to avoid possible prior contamination. The biopsy procedure
was performed at the dental clinic. Adolescents were positioned on the dental
chair and all the procedures were performed by a dentist (KPKO). The teeth
were cleaned with a rotary brush and pumice slurry, washed and dried. For
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the protocol I group (n=59), the right maxillary central incisor (11) was
isolated with cotton rolls prior to the biopsy and adhesive tape (Magic Tape,
810 Scotch -3M) containing a circular perforation of 4.0 mm in diameter was
adhered onto the labial surface of the tooth, delimiting a window for the
biopsy site. This window was etched twice according to the following
procedure: 10 µL 1.6 mol/L HCl in 70% (v/v) glycerol were applied to the area
for 35 s [Brudevold, 1975]. The biopsy solution was then quantitatively
transferred to centrifuge tube (Axygen Scientific, Inc., Union City, USA),
containing 200 µL Millipore Alpha Q water. Afterwards, the surface was
rinsed twice for 10 s each with 10 µL Millipore Alpha Q water which was then
transferred to the respective centrifuge tube, making a final volume of 230
µL. This procedure was repeated in the same window to remove the second
sampling (inner layer). For the protocol II group (n=59), the contra lateral
tooth (21) was biopsied. For this, the adhesive tape (Magic Tape, 810 Scotch
-3M) containing a circular perforation of 1.6 mm in diameter was adhered
onto the labial surface of the left maxillary central incisor, delimiting a window
for the biopsy site. That window was also etched twice according to the
following procedure: 5 µL 1.6 mol/L HCl in 70% (v/v) glycerol were applied to
the area for 20 s [Gomes et al, 2004]. The solution of biopsy was then
transferred to centrifuge tube (Axygen Scientific, Inc., Union City, USA),
containing 200 µL Millipore Alpha Q water. Next, the surface was rinsed once
for 10 s with 5 µL Millipore Alpha Q water which was then transferred to the
respective centrifuge tube, making a final volume of 210 µL. For both
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protocols (I and II), after the described procedures, the tape was then
removed and the teeth were washed with water for 30 s and dried with air
jets to receive neutral topical fluoride applications. Biopsies were also
performed in various sites at the dental clinic bench or at the centrifuge tube
rack to evaluate lead contamination in the environment where the procedures
were carried out.
To compare DELLs and biopsies depths between homologous teeth
by the same biopsy protocol, 79 adolescents had both maxillary central
incisors biopsied by protocol I.
Lead and phosphorus determinations. To avoid contamination, all
vials and polypropylene flasks used to prepare and store solutions were
cleaned with detergent solution, rinsed with 10 % (v/v) HNO
3
overnight,
rinsed with deionized water, dried and stored in a closed polypropylene
container. High purity water provided by a Milli-Q water purification system
(Millipore, Bedford, MA, USA) was used throughout. All reagents used were
analytical-grade.
Lead determination was done by graphite furnace atomic absorption
spectrometry (GF AAS). For this, a graphite furnace atomic absorption
spectrometer, model SIMAA-6000, with a longitudinal Zeeman-effect
background correction system, Echelle optical arrangement, solid state
detector, end-capped transversal heating graphite tubes (EC-THGA) with
integrated pyrolytically coated platforms (Perkin-Elmer, Norwalk, CT) and
hollow cathode lamp was used. Solutions were delivered into the graphite
Manuscrito IV – Toxicological Sciences
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153
tube by means of an AS-72 autosampler. The instrumental conditions for the
spectrometer were 15 mA of current lamp, 0.7 nm of bandpass and 283.3 nm
of wavelength. The heating program consisted of 5 steps (temperature/
o
C,
ramp/s, hold/s): 1 (130, 10, 10); 2 (200, 5, 20); 3 (800, 5, 20); 4 (2100, 0, 5);
and 5 (2400, 1, 2). Aliquots of 10 µL of samples or analytical solutions were
introduced into the graphite furnace with 10 µL of chemical modifier (5 µg Pd
+ 3 µL Mg). This chemical modifier was prepared using Suprapur solutions of
10 g/L Pd in 15% (v/v) HNO
3
and 10 g/L Mg, prepared from the inorganic
salts Pd(NO
3
)
2
and Mg(NO
3
)
2
(Merck, Darmstadt, Germany). The calibration
curve (2 – 40 µg/L) was prepared in the ausampler vials using analytical-
grade Tritisol solutions of 1000 mg L
-1
of Pb (Pb(NO
3
)
3
) diluted in 0.06 mol/L
HCl in 7% glycerol (v/v). The samples analyses were performed without
previous pretreatment. Dilution procedure with high pure water (2-5 times)
was done for samples with high concentrations of Pb (>40 µg/L). For each
sample, the analytical signals were obtained in triplicate. The accuracy of the
GF AAS analytical procedure was checked by analysis of standard reference
material of animal bone (H-5, IAEA from Austria).
A Modula ICP optical emission spectrometer (Spectro Analytical
Instruments, Kleve, Germany) equipped with radial-viewed plasma torch was
used for phosphorus determination. The setting of the instrumental conditions
for the analyses is 1400 W of power, cross-flow nebulizer, double pass
(Scott-type) spray chamber, 12 L/min of outer gas flow, 1.0 L/min of
intermediate and nebulizer gas flow, 1.5 mL/min of sample uptake rate and
213.618 nm of atomic P analytical wavelength. The calibration curve was
Manuscrito IV – Toxicological Sciences
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154
obtained using analytical-grade Tritisol solutions of 1000 mg/l of P (KH
2
PO
4
)
from Spex (Spex Sample Preparation, Metuchen, USA) after appropriate
dilution (20 times) in water. The analytical range was 0.5-10 mg/L. For each
sample, the analytical signals were obtained in triplicate.
Biopsy depth calculation. The calculus, previously described by
Cleymaet et al (1991), is based in the information that 17.4% of the enamel
weight is phosphorus and the mean density of the dental enamel is 2.95
g/cm
3
. Biopsy depths are estimated according to the following equation:
Biopsy depth = mass enamel (µg) / 2.95 x biopsy area (mm
2
)
Measurement of biopsy depth after the in vitro bovine dental
enamel biopsy. Sixteen sound crowns of bovine incisors were embedded in
acrylic resin cylinders (Paladur, Heraeus Kulzer, Wehrheim, Germany), and
the labial surfaces were ground flat and polished with water-cooled
carborundum paper (500, 800, 1200, 2400 and 4000 grit, waterproof silicon
carbide paper; Struers, Erkrat, Germany), approximately 200 µm of the outer
enamel being removed. Surface hardness of enamel specimens was chosen
as the criterion for stratified allocation of the specimens to the protocol I and
protocol II groups.
Prior to the experiment, baseline scans were obtained from the
specimens with a contact profilometer (Mahr Perthometer, Göttingen,
Germany). After preparation, the specimens were stored in water until used
in the experiment to avoid dehydration.
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155
In 8 samples, the adhesive tape (Magic Tape, 810 Scotch -3M)
containing a circular perforation of 4.0 mm in diameter was adhered on to the
labial surface of the tooth, delimiting a window for the biopsy site (Protocol I),
while another 8 samples had an adhesive tape containing a circular
perforation of 1.6 mm in diameter, delimiting a window for the biopsy site
(Protocol II). The biopsies were performed as described earlier. The biopsies
were repeated twice in each specimen.
After each etch-acid biopsy, enamel loss was determined by contact
profilometry. (Mahr Perthometer, Göttingen, Germany). The reference areas,
which remained protected by tape during the biopsy, were marked with a
scalpel blade on the outer surface to allow for exact reposition of the tape.
Prior to the experiment, five and three equidistant baseline surface scans of
each specimen from Protocol I and II were performed, respectively. To
determine the enamel loss, the tape was removed and five profiles were
recorded at exactly the same sites as the baseline measurement. For this,
the enamel specimens were provided with identification marks, which allowed
the stylus to be re-positioned accurately at each measurement. The profile
scans were performed in the center of each specimen at intervals of 250 µm.
Pre- and post-treatment scans were superimposed on graphs and the
average depth of the area under curve in the eroded area was calculated
with specially designed software (Marh, Göttingen, Germany). The results of
the five scans were averaged for each specimen (Magalhães et al., 2008).
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156
Statistical Analysis. For statistical analysis, Graph Pad Instat
statistical software was used. DELL and biopsy depth data did not pass
normality test (Kolmogorov-Smirnov test) and were then analyzed by
Wilcoxon matched-pairs signed ranks tests. To compare the loss of enamel
between first and second layers from each protocol paired t tests were
applied and t test was used to compare the protocols I and II. A significance
level of 5% was adopted.
RESULTS
The comparison between lead concentrations obtained experimentally
(3.08 ± 0.16 mg/kg) and the certified value (3.10 ± 0.18 mg/kg) showed good
agreement, considering Student’s t test at significance level of 95 %.
In the 79 subjects examined, whose both maxillary central incisors
were biopsied by protocol I, the biopsy depths ranged between 0.48 and 2.04
µm for the first removed layer, and between 0.65 and 1.55 µm for the second
removed layer (n=70). In the 59 subjects whose right maxillary central
incisors were biopsied by protocol I, the biopsy depths ranged between 0.26
and 0.99 µm for the first removed layer, and between 0.20 and 0.99 µm for
the second layer (n=58). In the same subjects, whose left maxillary central
incisors were biopsied by the protocol II, the biopsy depths ranged between
0.08 and 4.99 for the first layer, and 0.13 to 5.64 µm for the second removed
layer (n=57). The different numbers sampling between first and second
layers are due to losses during the collecting procedures. When we
compared the results of biopsy depth and DELLs between homologous teeth,
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biopsied by the same protocol (protocol I), there were not significant
differences between right and left maxillary central incisors (p> 0.05). To
calculate the DELL means, we excluded the DELL values that were below
the detection limit (0.5 µg/L) of the equipment (GFAAS) (Table 1).
Table 2 shows the means ± SD and medians of DELLs and biopsy
depths found by protocols I and II in the biopsy samples. Both protocols were
performed in the same adolescent, for the first and second layers in each
protocol. As can be observed, there were significant differences in biopsy
depths in the first and second layers between both protocols (p<0.05). The
biopsy depths were found to be different according to the protocol followed
for both layers. Interestingly, the biopsy depths found by the protocol II
(dental enamel exposed to acid for a lesser time) were significantly higher
than those found by the protocol I, which was contrary to what was to be
expected. Thus, we decided to perform an in vitro study to confirm these
data. The in vitro study results will be shown later on.
Albeit the mean biopsy depths were different, mean DELL were not
significantly different when the results from protocols I and II were compared
(p>0.05). There was a significant difference between DELL in the first and
second layers for both protocols (I and II) (Table 2).
Table 3 presents the individual and total means of enamel wear
caused by the in vitro biopsies carried out in bovine enamel blocks. The
enamel wear seen in protocol I was significantly higher than that observed for
protocol II, for both layers (t=5.737, p<0.0001 and t=6.239, p<0.0001, for the
first and second layers), respectively. In addition, the enamel wear seen in
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the second layer was significantly higher compared to that observed in the
first layer, for each protocol (t=27.463, p<0.0001 and t=20.980, p<0.0001 for
the protocols I and II) respectively.
Figure 1 shows the surface scanning of eroded areas after enamel
biopsies performed according to protocols I (A) or II (B). It can be observed
that the worn area obtained for A has a shape very similar to a cylinder, while
that seen for B has a very different shape.
DISCUSSION
The results of the present study showed that SDE is a reliable
biomarker of lead exposure because both biopsy depths and DELLs were not
different between homologous teeth from the same individual, examined by a
single methodology. However, when different methodological protocols were
applied in the same individual, biopsy depths were found to be significantly
different between homologous teeth. Biopsy depth depends on both pH
solution, which was the same for both protocols, as well as exposure time of
the SDE to acid etch. When the SDE is exposed to acid for shorter periods, it
is expected that the removed layer is smaller. However, when we calculated
the biopsy depths (removed by protocols I and II) by the cylinder formula, the
layer removed by the acid exposure for 20 s was deeper and DELL was
individually higher (although the mean values were not found to be
significantly different) than when SDE was exposed to acid for a longer time.
This result is not biologically plausible because deeper biopsies contain less
lead than more superficial biopsies, since there is a very steep lead gradient
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in the surface enamel (Brudevold, 1977). Thus, these results suggested that
the protocol II led to an erroneous result for permanent teeth, overestimating
DELL, since biopsy depth is critical to calculate it. The profilometry test was
then conducted in bovine enamel crowns to help explain these discrepant
results. It was observed that when SDE was exposed to acid for a shorter
time period, a more superficial layer was removed than when SDE was
exposed to acid for a longer period. Then, in fact, DELL was higher because
the protocol II biopsy was more superficial, which confirmed the expected
results (Brudevold, 1975; Gomes et al., 2004). The profilometry drawings
(Figure 1) suggest that the shorter time period of exposure of SDE to acid
etch in protocol II was not sufficient to promote homogenous erosion as
occurred in protocol I. As shown in Figure 1B, it can be inferred that it is
impossible to calculate the biopsy depth by the cylinder formula in this case,
because there is no defined height to be considered in the calculations, since
the basis of the geometric figure obtained is quite irregular.
It is known that protocol II is suggested to be followed when deciduous
teeth are biopsied (Gomes et al., 2004). Deciduous enamel can be
considered to be less mineralized than permanent teeth (Wilson and Beynon,
1989), since it has been shown that the superficial microhardness of
deciduous teeth (358 KHN) is significantly lower than that found for
permanent (368 KHN) (Magalhães et al., 2008). As the crown of deciduous
teeth is very small, it was not possible for us to obtain a section with
extension sufficient to conduct the profilometry tests using both protocols.
Consequently, we decided to use bovine enamel crowns for this purpose. It
Manuscrito IV – Toxicological Sciences
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160
must be acknowledged that a definitive conclusion regarding the suitability of
the protocol II for deciduous teeth can only be drawn if a study of the worn
area conducted in deciduous teeth is done. Considering the area limitation
mentioned above, one possible alternative would be the use of atomic force
microscopy as a response variable. In this case, it would be possible to
evaluate the topography profile of the worn area after the acid biopsy using
deciduous enamel and this experiment will be conducted by our research
group.
There are many variations among published microbiopsy
methodologies. Cleymaet et al (1991) applied a small synthetic fiber pellet to
dry the biopsied surface which was added to a tube together with the
collected SED sample. This detail is the only difference from protocol I of the
present study. Almeida et al (2004) modified the Brudevold’s technique to be
used in deciduous teeth, as the protocol II in the present study. These
authors did not mention how the modifications made to the original technique
were evaluated. In relation to the original technique (similar to protocol I), the
modified technique (similar to protocol II), the acid etch exposure time of SDE
was decreased, as it was the biopsy site diameter, and the volume of the
acid applied. Since many variables were modified simultaneously, it could be
expected that the geometric figure formed after the acid exposure in protocol
II would be different from the expected cylinder, which is formed when the
original technique is used. Again, it must be emphasized that the wear results
obtained in this study apply to bovine teeth and this profile should be
confirmed when using deciduous human enamel.
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The biopsy depths found in this study presented lower means than
those found in other studies (Cleymaet, 1991 a, b, c, d). Despite this, DELL
was almost twice as high in the first layer compared to the second. Observing
that the difference between first and second layers is approximately 0.5 µm,
this fact highlights the steep lead gradient in SDE and the importance of
calculating the biopsy depth with precision. On the other hand, the individual
DELL values were very different comparing the results from both protocols
applied in the same individual. Nonetheless, the mean DELL was not found
to be significantly different. A threshold definition for DELL is a controversial
point to discuss. Almeida et al (2008) suggested the use of deeper biopsies
is more reliable to compare DELL among different populations because these
authors detected a plateau (600 µg/g) in lead content in the biopsies
removed from deciduous teeth in biopsy depths from 3.18-5.9 µm. For this
conclusion, the precision of the biopsy depth is crucial.
In summary, this study demonstrated the risk of comparing DELL
results between studies that used different methodological protocols. In
addition, all alterations performed in analysis techniques should be minutely
studied to verify whether the expected results after the modifications would
be the same as those obtained by the original method, as in the case in
question – the biopsy depth.
FUNDING
This work was supported by Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP) [Grants 01/09641-1 and 06/56530-4]; the
Manuscrito IV – Toxicological Sciences
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq);
and the Projeto Milênio Redoxoma. KPKO is recipient of a fellowship from
the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES).
ACKNOWLEDGEMENTS
The authors acknowledge the valuable collaboration of all directors
and teachers from Projeto Girassol, Projeto Agente Jovem and Centro Irmã
Adelaide, the volunteers and their families involved in this investigation. We
thank Prof. Dr. Thomas Attin for the use of the profilometer (Clinic for
Preventive Dentistry, Periodontology and Cariology, University of Zurich-
Switzerland).
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REFERENCES
Almeida, G.R.C. Saraiva, M.C.P. Barbosa Jr, F. Krug, F.J. Cury, J.A. Sousa,
M.L.R. Buzalaf, M.A.R. Gerlach RF(2007). Lead contents in the surface
enamel of deciduous teeth sampled in vivo from children in uncontaminated
and in lead-contaminated areas. Environ. Res. 104, 337-345.
Almeida, G.R.C. Guerra, C.S. Tanus-Santos, J.E. Barbora Jr, F. Gerlach,
R.F. (2008). A plateau detected in lead accumulation in subsurface
deciduous enamel from individuals exposed to lead may be useful to identify
children and regions exposed to higher levels of lead. Environ. Res. 107,
264-270.
Budd, P. Montgomery, J. Cox, A. Krause, P. Barreiro, B. Thomas, R.G.
(1998). The distribution of lead within ancient and modern human teeth:
implications for long-term and historical exposure monitoring. Sci. Total
Environ. 220, 121-136.
Budd, P. Montgomery, J. Evans, J. Barreiro, B. (2000). Human tooth enamel
as a record of the comparative lead exposure of prehistoric and modern
people. Sci. Total Environ. 263, 1-10.
Manuscrito IV – Toxicological Sciences
.
164
Budd, P. Montgomery, J. Evans, J. Trickett, M. (2004). Human lead exposure
in England from approximately 5500 BP to the 16
th
century AD. Sci. Total
Environ. 318, 45-58.
Brudevold, F. Reda, A. Aasenden, R. Bakhos, Y. (1975). Determination of
trace elements in surface enamel of human teeth by a new biopsy procedure.
Arch. Oral Biol. 20, 667 –673.
Brudevold, F. Reda, A. Aasenden, Srinivasian, B.N. Bakhos, Y. (1977). Lead
in enamel and saliva, dental caries and the use of enamel biopsies for
measuring past exposure to lead. J. Dent. Res. 10, 1165-1171.
Cleymaet, R. Bottenberg, P. Retief, D.H. Slop, D. Michotte, Y. Coomans, D.
(1991a). In vivo use of a dual acid etch biopsy for the evaluation of lead
profiles in human surface enamel. Caries Res. 25, 256-263.
Cleymaet, R. Bottenberg, P. Slop, D. Clara, R. Coomans, D. (1991b). Study
of lead and cadmium content of surface enamel of schoolchildren from a
industrial area in Belgium. Community Dent. Oral Epidemol. 19, 107-111.
Cleymaet, R. Quartier, E. Slop, D. Retief, D.H. Smeyers-Verbek, J.
Coomans, D. (1991c). Model for assessment of lead content in human
surface enamel. J. Toxicol. Environ. Health 32, 111-127.
Manuscrito IV – Toxicological Sciences
.
165
Cleymaet, R. Retief, D.H. Quartier, E. Slop, D. Coomans, D. Michotte, Y.
(1991d). A comparative study of the lead and cadmium content of surface
enamel of Belgium and Kenyan children. Sci Total Environ. 104, 175-189.
Gomes, V.E. Souza, M.L.R. Barbosa Jr, F. Krug, F.J. Saraiva, M.C.P. Cury,
J.A. Gerlach, R.F. (2004). In vivo studies on lead content of deciduous teeth
superficial enamel of preschool children. Sci. Total Environ. 320, 25-35.
Magalhães, A.C. Kato, M.T. Rios D. Wiegand, A. Attin, T. Buzalaf, M.A.
(2008). The effect of an experimental 4% Tif4 varnish compared to NaF
varnishes and 4% TiF4 solution on dental erosion in vitro. Caries Res. 42,
269-274.
Olympio, K.P.K. Gonçalves, C. Günther, W.M.R. Bechara, E.J.H. (2009a).
Neurotoxicity and aggressiveness triggered by low lead-levels in children.
Pan Am J Publ Health [in press].
Olympio, K.P.K. Oliveira, P.V. Naozuka, J., Cardoso, M.R.A. Günther W.M.R.
Bechara, E.J.H. (2009b). Surface dental enamel lead levels and antisocial
behavior in Brazilian adolescents. Toxicol. Sci. (Suppl.), in press, (abstract).
Rabinowitz, M.B. (1995). Relating tooth and blood lead levels in children.
Bull. Environ. Contam. Toxicol. 55, 853-857.
Manuscrito IV – Toxicological Sciences
.
166
White, D.J. (1987). Reactivity of fluoride dentifrices with artificial caries 1.
Effects on early lesions: F uptake, surface hardening and remineralisation.
Caries. Res. 21, 126-140.
Wilson, P.R. Beynon, A.D. (1989). Mineralization differences between human
deciduous and permanent enamel measured by quantitative
microradiography. Arch. Oral. Biol. 34, 85-88.
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Table 1. Means ± SD and medians of biopsy depth [µm] and dental enamel
lead levels (DELL) [ppm] found in both maxillary central incisors by protocol I
(4 mm in diameter; 35 s, 10 µL HCl) in the microbiopsies samples for first
and second removed layers.
Layer Biopsy Depth DELL
Right Incisor Left Incisor Right Incisor Left Incisor
First
Layer
1.05±0.18
(n=79)
Median= 1.06
1.04±0.21
(n=79)
Median= 1.09
217.04±356.88
(n=72)
Median= 122.24
219.03±364.34
(n=72)
Median= 111.00
Second
Layer
1.12±0.16
(n=70)
Median= 1.11
1.15±0.19
(n=70)
Median= 1.14
110.99±154.24
(n=61)
Median= 76.92
102.03±153.66
(n=61)
Median= 60.00
There were no significant differences between right and left incisors both for
depth and DELL, in each layer (Wilcoxon matched-pairs tests, p>0.05).
Manuscrito IV – Toxicological Sciences
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Table 2. Means ± SD and medians of dental enamel lead levels (DELL)
[ppm] and biopsy depth [µm] found by protocols I (4 mm in diameter; 35 s, 10
µL HCl) and II (1.6 mm in diameter, 20 s, 5 µL HCl) in the microbiopsy
samples for the first and second layer in each protocol.
Variable Protocol I Protocol II
First Layer Second Layer
First Layer Second Layer
Biopsy
Depth
0.56±0.13
a,A
(n=59)
Median= 0.55
0.61±0.15
b,A
(n=58)
Median= 0.61
1.04±0.63
a,B
(n=59)
Median= 0.98
1.31±0.76
b,B
(n=57)
Median= 1.19
DELL
124.96±153.15
a,A
(n=59)
Median= 49.54
60.99±84.27
b,A
(n=58)
Median= 23.73
126.94±225.24
a,A
(n=59)
Median= 28.81
54.69±94.73
b,A
(n=57)
Median= 18.09
Wilcoxon matched-pairs test: different lowercase letters indicate significant
differences (p< 0.05) between first and second layers for protocol I and II;
different uppercase letters indicate significant differences in the biopsy depth
and DELL between protocols I and II for both layers.
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Table 3. Individual and total means (µm) of dental enamel wear, measured
by profilometry, after in vitro surface dental enamel etch-acid microbiopsies in
bovine enamel blocks.
Enamel
Block
First Layer Second Layer
Protocol I Protocol II Protocol I Protocol II
1 1.909 0.649 3.698 2.252
2 2.674 1.021 4.385 2.898
3 2.182 0.745 3.635 2.439
4 1.771 1.327 3.517 2.791
5 1.665 0.856 3.373 2.276
6 1.905 0.905 3.523 2.730
7 1.969 1.466 3.298 2.824
8 1.403 0.882 3.203 2.208
Mean±SD 1.935±0.377
A,a
0.981±0.281
B,b
3.579±0.366
A,a
2.552±0.288
B,b
Paired t test: different uppercase letters indicate significant differences
between first and second layers in each protocol; t test: different lowercase
letters indicate significant differences between protocols I and II for each
layer.
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Figure 1 Profilometry drawing (A. Protocol I: 4 mm in diameter, 35 s, 10 uL
HCl; B. Protocol II: 1.6 mm in diameter, 20 s, 5 uL HCl). In blue: considered
area; In yellow: eroded area.
A
B
Conclusões
.
171
5
CONCLUSÕES e RECOMENDAÇÕES
1. Mesmo com a presença de tantos problemas familiares e sociais, a
exposição ao chumbo foi o fator de risco mais fortemente associado a
comportamento anti-social, avaliado pelo CBCL, tendo sido analisado
o ponto de vista dos pais ou responsáveis pelos adolescentes
brasileiros estudados. Maiores escores alcançados pelo auto-relato
de delinqüência não foram associados à maior exposição ao chumbo;
2. A população foi exposta ao chumbo por algumas fontes estudadas.
Os fatores de risco domiciliares mais associados à exposição ao
chumbo foram residir no entorno ou em região próxima de indústrias
que utilizam chumbo em seu processo produtivo e ter alguma pessoa
que mora ou morou com o adolescente trabalhando em empresas que
utilizam chumbo. Essas empresas devem evitar o arraste externo
deste metal, efetuado pelos funcionários por meio das roupas,
sapatos e corpos. Sugere-se reforço na vigilância ambiental destas
indústrias, visando minimizar a exposição da população circunvizinha
e os conseqüentes efeitos à saúde;
3. O esmalte dentário mostrou-se um marcador biológico fidedigno para
avaliação da carga corporal de chumbo. No entanto, comparações da
concentração de chumbo entre estudos diferentes não deveriam ser
realizadas sem cautela, uma vez que variações metodológicas são
encontradas entre as diversas pesquisas, com conseqüências de
alterações importantes para interpretação dos resultados. Assim, uma
padronização metodológica da técnica de microbiópsia ácida de
Conclusões
.
172
esmalte dentário superficial faz-se necessária para tornar viável a
utilização deste biomarcador como recurso a ser utilizado em Saúde
Pública.
Referências
*Estas referências são complementares àquelas anteriormente relacionadas nos
manuscritos.
173
REFERÊNCIAS*
1. Achenbach TM, Rescorla LA. Manual for the ASEBA school-age forms
& profiles, ASEBA, Burlington, VT, 2001.
2. Bordin IAS, Mari JJ, Caeiro MF. Validação da versão brasileira do
“Child Behavior Checklist” (CBCL) (Inventário de comportamentos da
infância e adolescência): dados preliminares. ABP-APAL 1995; 17:
55-66.
3. Bordin IAS. Fatores de risco para comportamento anti-social na
adolescência e início da vida adulta [Tese de Doutorado]. São Paulo:
Escola Paulista de Medicina, Universidade Federal de São Paulo;
1996.
4. Freitas CU. Vigilância de população exposta a chumbo no município
de Bauru – São Paulo: investigação de fatores de risco de exposição
e avaliação da dinâmica institucional [Tese de Doutorado]. São Paulo:
Faculdade de Saúde Pública, Universidade de São Paulo; 2004.
5. Laboratório de Terapia Comportamental do Instituto de Psicologia da
Universidade de São Paulo. (Trad.). (2006). Guia para profissionais
Referências
*Estas referências são complementares àquelas anteriormente relacionadas nos
manuscritos.
174
da saúde mental sobre o Sistema de Avaliação Empiricamente
Baseado do Achenbach (ASEBA). São Paulo. Tradução da obra:
Achenbach TM & Rescorla LA (2004). Mental health practionaires’
guide for the Achenbach System of Empirically Based Assessment
(ASEBA) (4th Ed.). Burlington, VT: University of Vermont, Research
Center for Children, Youth & Families, Burlington, VT. Tiragem de
circulação interna.
Anexos
175
ANEXO 1
Anexos
176
UNIVERSIDADE DE SÃO PAULO
Faculdade de Saúde Pública
D
EPARTAMENTO DE
S
AÚDE
A
MBIENTAL
Av. Dr. Arnaldo, 715 - CEP: 01246-904 –São Paulo–SP
Fone: 3082-3842/3066-7712 - Fax: 3066-7732
e-mail: [email protected]r
Modelo dirigido aos pais ou responsáveis que responderão ao questionário
TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO
O chumbo está presente em muitos locais como na tinta de casas antigas, pintura de cerâmicas,
no solo, água e ar de regiões industrializadas. Existem relatos publicados de que a contaminação
pelo chumbo pode causar diversos problemas de comportamento social. Assim, o objetivo desta
pesquisa é verificar a concentração de chumbo presente no esmalte do dente e sua relação com
desvios de comportamento social. Para isto, será realizada a aplicação de uma gota de ácido
clorídrico sobre o dente do adolescente e esta gota será analisada para a verificação da
quantidade de chumbo. Além disso, será aplicado um questionário ao adolescente sobre o seu
próprio comportamento e um questionário ao pai ou responsável sobre o comportamento de seu
filho ou adolescente que esteja sob sua tutela. Todos os adolescentes receberão limpezas nos
dentes e aplicação de flúor. Esta pesquisa não trará riscos, desconforto ou gastos financeiros para
os participantes. Ao final da pesquisa, os dados obtidos poderão ser publicados em revistas
especializadas com fins científicos. As informações obtidas com a pesquisa não identificam o
participante, mantendo a privacidade do indivíduo. Os resultados desta pesquisa ajudarão na
formulação de políticas públicas de prevenção da contaminação por chumbo pela população e na
compreensão dos efeitos da contaminação por chumbo à saúde humana. Esperando contar com o
seu apoio, desde já agradecemos. Em caso de dúvida, você poderá entrar em contato com as
pesquisadoras pelo e-mail kellypko@usp.br (Kelly), wgunther@usp.br (Profa. Wanda).
Assim, pelo presente instrumento que atende às exigências legais, o Sr. (a)
_________________________________________________________________________________,
portador (a) da cédula de identidade __________________________, residente à
_________________________________________________________, responsável legal pelo
adolescente ___________________________________________ declara ter tomado conhecimento
dos objetivos e procedimentos da pesquisa, devidamente explicada e detalhada pelos pesquisadores,
não restando quaisquer dúvidas a respeito do lido e explicado, firma seu CONSENTIMENTO LIVRE E
ESCLARECIDO para responder ao questionário sobre o comportamento social do adolescente em
questão.
Por estarem de acordo, assinam o presente termo.
Local (Bauru ou Lins), ____ de _______________ de __________.
__________________________ ____________________________
Assinatura do participante Assinatura do autor
1ª via
Anexos
177
UNIVERSIDADE DE SÃO PAULO
Faculdade de Saúde Pública
D
EPARTAMENTO DE
S
AÚDE
A
MBIENTAL
Av. Dr. Arnaldo, 715 - CEP: 01246-904 –São Paulo–SP
Fone: 3082-3842/3066-7712 - Fax: 3066-7732
e-mail: [email protected]r
Modelo dirigido ao responsável, autorizando a participação do adolescente na pesquisa
TERMO DE CONSENTIMENTO LIVRE E ESCLARECIDO
O chumbo está presente em muitos locais como na tinta de casas antigas, pintura de cerâmicas,
no solo, água e ar de regiões industrializadas. Existem relatos publicados de que a contaminação
pelo chumbo pode causar diversos problemas de comportamento social. Assim, o objetivo desta
pesquisa é verificar a concentração de chumbo presente no esmalte do dente e sua relação com
desvios de comportamento social. Para isto, será realizada a aplicação de uma gota de ácido
clorídrico sobre o dente do adolescente e esta gota será analisada para a verificação da
quantidade de chumbo. Além disso, será aplicado um questionário ao adolescente sobre o seu
próprio comportamento. Todos os adolescentes receberão limpezas nos dentes e aplicação de
flúor. Esta pesquisa não trará riscos, desconforto ou gastos financeiros para os participantes. Ao
final da pesquisa, os dados obtidos poderão ser publicados em revistas especializadas com fins
científicos. As informações obtidas com a pesquisa não identificam o participante, mantendo a
privacidade do indivíduo. Os resultados desta pesquisa ajudarão na formulação de políticas
públicas de prevenção da contaminação por chumbo pela população e na compreensão dos
efeitos da contaminação por chumbo à saúde humana. Esperando contar com o seu apoio, desde
já agradecemos. Em caso de dúvida, você poderá entrar em contato com a pesquisadora pelos e-
mails kellypko@usp.br (Kelly), wgunther@usp.br (Profa. Wanda).
Assim, pelo presente instrumento que atende às exigências legais, o Sr. (a)
_________________________________________________________________________________,
portador (a) da cédula de identidade __________________________, residente à
_________________________________________________________, responsável legal pelo
adolescente declara ter tomado conhecimento dos objetivos e procedimentos da pesquisa,
devidamente explicada e detalhada pelos pesquisadores, não restando quaisquer dúvidas a respeito
do lido e explicado, firma seu CONSENTIMENTO LIVRE E ESCLARECIDO, para que o adolescente
____________________________________ ____________________________________________,
portador da cédula de identidade ________________________________________ possa participar
da pesquisa proposta.
Por estarem de acordo, assinam o presente termo.
___________________, ____ de _______________ de __________.
__________________________ ____________________________
Assinatura do responsável Assinatura do autor
________________________
Assinatura do adolescente
1ª via
Anexos
178
ANEXO 2
Disponível em http://www.aseba.org (Copyright 2001 T.M. Achenbach).
Anexos
179
ANEXO 3
Anexos
180
Auto-relato de comportamento
Código:
Estas perguntas lidam com seu próprio comportamento. Não se preocupe com as respostas, seja honesto porque
assim você estará ajudando a pesquisa. TODAS AS RESPOSTAS SÃO CONFIDENCIAIS E NÃO SERÃO
RELACIONADAS À SUA IDENTIDADE. Eu vou ler uma série de comportamentos para você. Por favor, fale
o número certinho de vezes que você fez cada uma destas coisas nos últimos 6 meses e a idade quando você fez
isso pela primeira vez.
Nunca = 0, Uma vez = 1, 2-5 vezes = 2, 6-10 vezes = 3, Mais de 10 vezes = 4
Quantas vezes nos últimos 6 meses você... (repetir esta frase de vez em quando)
____1. Fugiu de casa. _____ (idade da primeira vez que fez isso)
____2. Matou aula ou não foi para a escola sem motivo.____
____3. Mentiu a sua idade para poder entrar em algum lugar ou comprar alguma coisa, por exemplo para assistir
um filme proibido para menores ou para comprar bebida alcoólica?____
____4.Andou de carona do lado de fora de ônibus, trens ou carros quando era proibido fazer isto?____
____5.Carregou arma escondido?____
____6.Gritou, fez bagunça ou não obedeceu as regras de um lugar público e as pessoas reclamaram ou você teve
problemas?____
____7.Pediu dinheiro ou coisas para estranhos?____
____8. Ficou bêbado em lugar público?____
____9.Estragou ou destruiu de propósito coisas que não eram suas, por exemplo pixou, quebrou alguma coisa
que não era sua?____
____10.Colocou fogo em uma casa, prédio, carro ou outra coisa de propósito ou tentou fazer isso?____
____11.Tentou não pagar coisas que você comprou ou utilizou como cinema, ônibus e comida?____
____12.Entrou ou tentou entrar em um lugar para roubar coisas?____
____13.Roubou ou tentou roubar coisas de menos de R$5,00?____
____14.Roubou ou tentou roubar coisas com valor entre R$5,00 e R$50,00?____
____15.Roubou ou tentou roubar coisas com valor entre R$50,00 e R$100,00?____
____16.Roubou ou tentou roubar coisas de R$100,00 ou mais?____
____17.Pegou alguma coisa de uma loja sem pagar?____
____18.Roubou a carteira ou bolsa de alguém?____
____19.Pegou alguma coisa de um carro que não era seu?____
____20.Comprou, vendeu ou pegou alguma coisa que você sabia que era roubada ou tentou fazer isso?____
____21.Pegou para dirigir um carro ou moto sem que o dono soubesse e tivesse deixado?____
____22.Roubou ou tentou roubar uma moto ou carro?____
____23.Usou cheque ilegalmente ou dinheiro falso para comprar alguma coisa?____
____24.Usou ou tentou usar um cartão de crédito ou do banco sem a permissão do dono?____
____25.Tentou enganar alguém, vendendo-lhe alguma coisa que não tinha valor ou que você disse que tinha e
não tinha?____
____26. Atacou alguém com uma arma com a idéia de machucar ou matar a pessoa de verdade?____
____27.Bateu em alguém com a idéia de machucar?____ (outra situação que você ainda não tenha dito)
____28.Usou arma ou força para conseguir dinheiro ou coisas das pessoas?____
____29.Atirou objetos como pedras ou garrafas nas pessoas? (sem contar o que você já falou) ____
____30.Envolveu-se em brigas de gangues?____
____31.Foi pago (a) para fazer sexo com alguém?____
____32.Machucou ou tentou machucar alguém para fazer sexo com você?____
____33.Fez sexo ou tentou fazer sexo com alguém contra a vontade da pessoa?____
____34.Vendeu maconha ou “haxixe”?____
____35.Vendeu drogas pesadas como heroína, cocaína, LSD ou outras drogas pesadas?____
____36.Foi levado (a) pela polícia por algum outro motivo, além de uma leve infração de trânsito?____
Loeber et., 1989
Anexos
181
ANEXO 4
Anexos
Adaptado de Freitas, 2004.
182
Questionário de Identificação e fatores de confusão
Endereço: __________________________________________________________________
Telefone para contato: _______________________________
Nome do pai, mãe ou responsável que respondeu o questionário:
___________________________________________________________________________
Há quanto tempo vocês moram neste endereço?
Em que lugares vocês moraram antes? Escrever rua e bairro.
Onde vocês moraram até os 5 anos de idade do adolescente?
Quantas pessoas moram na sua casa? ______ 0 a 12 anos de idade
______ 13 anos ou mais
Os pais moram juntos? ( ) sim ( ) não
Até que série o pai estudou?
Até que série a mãe estudou?
( ) analfabeto
( ) analfabeto
( ) primário incompleto
( ) primário incompleto
( ) primário completo
( ) primário completo
( ) 1º grau incompleto
( ) 1º grau incompleto
( ) 1º grau completo
( ) 1º grau completo
( ) Ensino médio incompleto
( ) Ensino médio incompleto
( ) Ensino médio completo
( ) Ensino médio
completo
( ) Superior incompleto
( ) Superior incompleto
( ) Superior completo
( ) Superior completo
Você fuma cigarro? (...) Sim (...) Não
Seu filho fuma cigarro ou masca fumo? (...) Sim (...) Não
Você trabalha ou trabalhou? Onde?
Alguém que mora com vocês trabalha ou trabalhou em fábrica:
( ) baterias ( ) pigmentos ( ) tintas ( ) cerâmica
Por quanto tempo?
Na sua casa, você faz uso de:
( ) Cerâmica vitrificada para alimentos quentes
( ) Brinquedos importados
( ) Baterias de carros ao redor ou na sua casa
( ) Zarcão nos portões da casa
Informações Curriculares
183
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