( PDF ) Avaliação dos efeitos citogenéticos da exposição ocupacional a agrotóxicos em agentes de saúde pública vinculados à prefeitura de Belo Horizonte

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UNIVERSIDADE FEDERAL DE MINAS GERAIS

INSTITUTO DE CIÊNCIAS BIOLÓGICAS

PÓS-GRADUAÇÃO EM GENÉTICA

AVALIAÇÃO DOS EFEITOS CITOGENÉTICOS DA EXPOSIÇÃO

OCUPACIONAL A AGROTÓXICOS EM AGENTES DE SAÚDE

PÚBLICA VINCULADOS À PREFEITURA DE BELO HORIZONTE

Fernanda de Souza Gomes Kehdy

Belo Horizonte-MG

2005

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Fernanda de Souza Gomes Kehdy

AVALIAÇÃO DOS EFEITOS CITOGENÉTICOS DA EXPOSIÇÃO

OCUPACIONAL A AGROTÓXICOS EM AGENTES DE SAÚDE

PÚBLICA VINCULADOS À PREFEITURA DE BELO HORIZONTE

Dissertação apresentada ao Programa de

Pós-Graduação em Genética do

Departamento de Biologia Geral do

Instituto de Ciências Biológicas de

Universidade Federal de Minas Gerais,

como requisito parcial à obtenção do título de

Mestre em Genética

Orientadora: Profª Maria Cristina Lima de Castro

Belo Horizonte

Universidade Federal de Minas Gerais

Instituto de Ciências Biológicas

2005

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iii

Aos meus pais com imenso amor.

AGRADECIMENTOS

À todos os participantes do projeto que concordaram em contribuir para esta pesquisa.

À minha orientadora Profª Maria Cristina Lima de Castro, por ser essencial não só na

minha formação profissional como pessoal. Também pelo carinho, paciência, conselhos e

confiança.

À Profª Marisa B. Bonjardin por estar sempre presente com bons conselhos e sugestões.

À Profª Miriam T. P. Lopes do Laboratório de Substâncias anti-tumorais do Departamento

de Farmacologia do ICB, UFMG, por disponibilizar seu laboratório.

À amiga Renata Antonaci, por estabelecer meu contato com a Prefeitura de Belo Horizonte.

Ao Carlos e Francisco do Centro de Controle de Zoonoses da Regional de Venda Nova,

pela boa vontade e por sempre facilitarem nosso trabalho.

À Marlene de Miranda pela imprescindível ajuda na coleta das amostras.

À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) pela bolsa de

estudos concedida.

À Marina pelo carinho, boa vontade e paciência com que conduz a secretaria de Pós-

Graduação.

À Juliana e Bruno pela ajuda nas fotografias.

Aos amigos do Departamento Renatinha, Reinaldo, Dani e Lilia, por tornarem minha rotina

divertida.

Aos amigos especiais Savito e Chico, pelo carinho.

Aos amigos Rodrigo e Nessim por tornarem meus dias bem mais engraçados.

Ao grande amigo Miguelito pela amizade, conselhos e contagiante alegria.

Ao querido amigo Ricardo Camelo (Ric) pela amizade e pela imprescindível ajuda durante

toda a realização deste trabalho.

À queridíssima Carolzinha, pela eterna amizade e por estar incondicionalmente presente em

todos os meus momentos.

À minha irmã de cor, Simone (Si), pela amizade e indispensáveis conselhos.

À amiga Erica, companheira desde a graduação.

Ao meu namorado Filipe, pela paciência, companheirismo e amor incondicionais.

Ao meu irmão Rafael e minha super-cunhada Aline por estarem sempre por perto.

À minha mãe, Regina, simplesmente por existir.

Ao meu pai, grande homem, que a cada dia me mostra como a vida pode ser simples e feliz.

SUMÁRIO

LISTA DE FIGURAS............................................................................................................1

RESUMO...............................................................................................................................3

INTRODUÇÃO......................................................................................................................4

ARTIGO: Evaluation of the cytogenetic effects of ocupational exposure to pesticides on

sanitary workers in Belo Horizonte, Brazil..........................................................5

ANEXOS...............................................................................................................................30

Figuras.............................................................................................................................31

Termo de consentimento.................................................................................................40

Questionário....................................................................................................................43

Aprovação do Comitê de Ética em Pesquisa da UFMG.................................................46

LISTA DE FIGURAS

Figura 1 - Fotomicrografia de linfócito mononucleado típico corado por Giemsa (observado

sob aumento de 1000 X).......................................................................................................36

Figura 2 – Fotomicrografia de linfócito binucleado corado por Giemsa (observado sob

aumento de 1000 X)..............................................................................................................36

Figura 3 - Fotomicrografia de linfócito binucleado corado por Giemsa (observado sob

aumento de 1000 X)..............................................................................................................37

Figura 4 – Fotomicrografia de linfócito binucleado (BC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X).....................................................37

Figura 5 - Fotomicrografia de linfócito binucleado (BC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X).....................................................38

Figura 6 - Fotomicrografia de célula (linfócito) binucleada (BC) contendo micronúcleo

(MN) corado por Giemsa (observado sob aumento de 1000 X)...........................................38

Figura 7 - Fotomicrografia de linfócito binucleado (BC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X).....................................................39

Figura 8 - Fotomicrografia de linfócito binucleado (BC) contendo ponte nucleoplasmática

(NB) corado por Giemsa (observado sob aumento de 1000 X)............................................39

Figura 9 - Fotomicrografia de linfócito trinucleado (TC) típico corado por Giemsa

(observado sob aumento de 1000 X)....................................................................................40

Figura 10 - Fotomicrografia de linfócito trinucleado (TC) corado por Giemsa (observado

sob aumento de 1000 X).......................................................................................................40

Figura 11 Fotomicrografia de linfócito trinucleado (TC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X).....................................................41

Figura 12 - Fotomicrografia de linfócito tetranucleado (QC) típico corado por Giemsa

(observado sob aumento de 1000 X).....................................................................................41

Figura 13 - Fotomicrografia de linfócito tetranucleado (QC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X).....................................................42

Figura 14 - Fotomicrografia de linfócito em apoptose corado por Giemsa (observado sob

aumento de 1000 X)..............................................................................................................42

Figura 15 - Fotomicrografia de linfócito em apoptose corado por Giemsa (observado sob

aumento de 1000 X)..............................................................................................................43

Figura 16 - Fotomicrografia de linfócito em necrose corado por Giemsa (observado sob

aumento de 1000 X)..............................................................................................................43

Figura 17 - Fotomicrografia de linfócito em necrose corado por Giemsa (observado sob

aumento de 1000 X)..............................................................................................................44

Figura 18 - Fotomicrografia de linfócito em necrose corado por Giemsa (observado sob

aumento de 1000 X)..............................................................................................................44

RESUMO

Agentes sanitários responsáveis pela aplicação de pesticidas para o controle de vetores de

doenças constituem uma população ocupacionalmente exposta a genotóxicos potenciais.

Sendo assim, o objetivo deste estudo foi determinar a relação entre a exposição ocupacional

a pesticidas e a presença de danos citogenéticos. Foram selecionados 59 homens (29

agentes sanitários e 30 indivíduos controle) com idade entre 18-57 anos que viviam e

trabalhavam na mesma região em Belo Horizonte (Brasil). Através do Teste do

Micronúcleo (MN) em linfócitos periféricos, as freqüências de micronúcleos (MN), células

binucleadas micronucleadas (CBMN), pontes nucleoplasmáticas (PN), células apoptóticas

(APOP), células necróticas (NECR) e índice de divisão nuclear (IDN) foram determinados.

A análise de covariância (ANCOVA) revelou freqüências médias significativamente

maiores (p<0,05) de MN (15,81 ± 1,31 vs. 4,71 ± 0,42), CBMN (15,10 ± 1,22 vs. 4,62 ±

0,44), PN (4,59 ± 0,76 vs. 1,00 ± 0,34), NECR (12,07 ± 1,45 vs. 5,17 ± 0,70) no grupo

exposto, em relação aos indivíduos controle respectivamente. Não houve diferença

significativa entre as freqüências de APOP entre os grupos exposto e controle, enquanto o

IDN foi significativamente menor (p<0,05) nos expostos (1,49 ± 0,02 vs. 1,61 ± 0,02).

Houve relação direta da idade dos indivíduos e as freqüências de MN e CBMN. Não foi

observada influência do tempo de exposição ou dos hábitos de fumar e ingerir bebidas

alcoólicas sobre os parâmetros citogenéticos analisados. De acordo com estes resultados, a

exposição ocupacional à pesticidas mostrou efeito genotóxico e citotóxico nos linfócitos

dos agentes sanitários.

INTRODUÇÃO

Atualmente, muitas ocupações levam os trabalhadores à exposição a substâncias

químicas que muitas vezes são prejudiciais à saúde dos mesmos. Um exemplo são os

agentes sanitários vinculados ao governo, responsáveis pela aplicação de pesticidas para o

controle de doenças transmitidas por animais. O Serviço de Controle de Zoonoses da

Secretaria Municipal de Belo Horizonte mantém, atualmente, um contingente de 1200

agentes sanitários. Como estes trabalhadores estão expostos a longas jornadas de trabalho

com contato direto e/ou indireto com diversos compostos químicos, cujos efeitos crônicos

são pouco conhecidos, constituem uma população de risco.

Um dos possíveis efeitos da exposição à pesticidas é a genotoxicidade, que

representa um fator de risco primário para a carcinogênese. Tendo em vista que a

manifestação clínica do câncer ocorre muito depois de suas causas estarem estabelecidas, é

possível o monitoramento e intervenção de indivíduos que estejam com algumas destas

causas estabelecidas, mas que ainda não tenham desenvolvido o tumor.

Desta forma, com o intuito de estimar o risco genético da exposição a pesticidas em

agentes sanitários vinculados à Prefeitura de Belo Horizonte, este estudo de

biomonitoramento genotoxicológico foi realizado. Já que não existem Comissões Internas

de Prevenção de Acidentes (CIPA) na Prefeitura de Belo Horizonte, os resultados obtidos

com o presente estudo poderão fornecer subsídios para implementação das medidas de

proteção e promoção da saúde destes trabalhadores.

EVALUATION OF THE CYTOGENETIC EFFECTS OF OCUPATIONAL

EXPOSURE TO PESTICIDES ON SANITARY WORKERS IN BELO

HORIZONTE, BRAZIL

Fernanda S. G. Kehdy

, Maria Cristina Lima de Castro

Laboratório de Genética de Neoplasias e Mutagênese, Departamento de Biologia Geral, Instituto de Ciências

Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.

ABSTRACT

Sanitary workers responsible for the application of pesticides in the control desease

vectors constitute a population that is exposed to possible genotoxic substances while at

work. Thus, the aim of this study was to determine the relation between the occupational

exposure to these pesticides and the presence of cytogenetic damages. Fifty-nine men were

selected (29 sanitary workers and 30 control individuals) with ages varying between 18-57

years who lived and worked at the same area in Belo Horizonte (Brazil). Through the

cytokinesis-block micronucleus assay (CBMN) in peripheral blood lymphocytes the

frequencies of micronuclei/1000 binucleaded cells (MN/1000 BC), binucleated cells with

micronuclei (BCMN)/1000 BC, nucleoplasmic bridges (NB)/1000 BC, apoptotic (APOP)

and necrotic (NECR) cells/ 500 cells and nuclear division index (NDI) were determined for

all individuals. The analysis of covariance (ANCOVA) showed significantly higher (p <

0,05) mean frequencies of MN (15,81 ± 1,31 vs. 4,71 ± 0,42), BCMN (15,10 ± 1,22 vs. 4,62

± 0,44), NB (4,59 ± 0,76 vs. 1,00 ± 0,34), NECR (12,07 ± 1,45 vs. 5,17 ± 0,70) in the

exposed group when compared to the control group. There was no significant difference in

the APOP frequencies beteween both groups, while the NDI was significantly higher (1,49

± 0,02 vs. 1,61 ± 0,02) in the control group. Neither the time exposure nor the smoking or

alcohol drinking habits influenced the cytogenetic parameters evaluated. According to these

results, the occupational exposure to pesticides showed genotoxic and cytotoxic effects on

sanitary workers.

Keywords: Pesticide exposure; Biomonitoring; Micronucleus test; Sanitary workers

1. INTRODUCTION

Pesticides compose a group of natural or synthetic chemical substances assigned to

combat plagues that generally attack, harm or transmit illness to living organisms including

humans [1]. Although they may be selective against specific organisms (as bacteria, fungi,

undergrowth and rodent), most of them do not have an absolute selectivity, becoming a

potential risk to the human health [2].

Many studies showed an association between the exposure to pesticides and the

increase of the incidence of some cancers including the non-Hodgkin’s lymphoma [3,4],

the multiple myeloma [5], sarcomas [6,7], the pancreatic [8] and the bladder cancer [9].

This is why lots of scientists have evaluated the genetic risk associated with the exposure.

The genotoxic effects of a pesticide is a primary factor for carcinogenesis, so the

genotoxicologic biomonitoring will become useful in human populations exposed to it [2].

Meanwhile, the results of this kind of biomonitoring obtained until now are ambiguous [10-

14], probably due to different conditions of the populations studied, the specific genotoxic

effect of the different pesticides employed and due to interindividual variability [2].

In tropical countries illnesses such as dengue, malaria, yellow fever, leishmaniosis

and leptospirosis, are health care problems once the ambient conditions favour the

development and the proliferation of insects and other vectors [15]. The struggle to

eliminate these animals is a task performed by the government at different levels, with the

promotion of several control measures, including the application of pesticides in areas with

high concentration of cases. The sanitary workers engaged in the application of pesticides

are occupationally exposed to possible potential genotoxics, becoming a cancer risk

population [2].

This work aimed to evaluate the genotoxic effect of the occupational exposure to

pesticides on the population of sanitary workers of Belo Horizonte, Brazil. The

micronucleus (MN) lymphocyte culture test was employed. It consists in a cytogenetic

method that measures breaks (clastogenic effect) and chromosome losses (aneugenic effect)

in binucleated cells [16]. Other cytogenetic parameters such as the nucleoplasmic bridges

(NB), apoptotic (APOP) and necrotic (NECR) cells, and the nuclear division index (NDI)

were analysed [17]. Factors such as age, smoking and alcohol drinking habits were taken

into account because they may influence the expression of the evaluated cytogenetic

parameters [18].

2. MATERIAL AND METHODS

2.1 Subjects

Fifty nine males between 18 and 57 years old agreed to take part in the research,

between August and October 2004. All the participants lived and worked in Belo Horizonte

(Brazil) and they did not present any serious morbidities at the time of the sample

collection. The exposed group was composed of 29 sanitary workers working for the City

Hall of Belo Horizonte who were occupationally exposed to several pesticides (Table 1).

The control group was selected from the population which worked next to the exposed

individuals and was composed of 30 volunteers who had never been occupationally

exposed to pesticides.

All the individuals answered a questionnaire, supplying information related to age,

smoking and alcohol drinking habits and, in the case of the exposed group, the duration and

the frequency of exposure and the personal protection equipment (PPE) employed. People

were considered smokers when they usually smoked 15 or more cigarettes per day through

at least one year and people who often drinked any alcoholic beverage two or more times

per week were considered alcohol drinkers. The main characteristics of both groups are

shown in Table 2.

The project was approved by the Committee on Ethics and Research of the Federal

University of Minas Gerais (ETIC 373/04) and all the subjects signed the agreement term.

2.2 Blood sample collection and cell culture

Ten millilitres of peripheral blood was obtained from each subject by vein puncture

using heparinized vacutainers. Mononuclear cells were fractionated in a density gradient

Histopaque

(Sigma) and added (10

cells/mL) to a RPMI 1640 (Gibco) medium

supplemented with 10% (v/v) foetal bovine serum (Gibco), 2mmol/L of ℓ-glutamine

(Gibco), antibiotics (penicillin, 100 U/mL, streptomycin, 100 µg/mL and amphotericine B,

25 µg/mL; Gibco) and 2% (v/v) of phytohaemagglutinin A (Gibco). The cultures were

maintained at 37°C in a 5% CO

incubator. After 44 hours cytochalasin B was added to the

culture (6 µg/mL; Sigma) [17].

At the end of the cultures (72 hours) the cells were centrifuged at room temperature

and gently dissolved in a cold methanol 70% (v/v): acetic acid (3:1) fixation solution. This

procedure was performed twice. The cellular suspension was dropped in three previously

identified clean slides. The slides were air dried and stained with Giemsa solution (4%

(v/v); Gibco) in Dulbecco`s Phosphate Buffered Saline pH 7,1 (Gibco) for 15 minutes.

2.3 Slides analysis

The slides were blindly analysed in an optic microscope with 1000X lens. For each

individual, 500 lymphocytes were analysed in order to determine the apoptotic cells

(APOP) frequency, the necrotic cells (NECR) frequency and the number of cells with one

to four nuclei, employed in the calculus of nuclear division index (NDI). This index is

determined by the formula NDI = MC+2BC+3TC+4QC/total viable cells, where MC-QC

represents the number of cells with one to four nuclei, respectively. The frequency of the

micronuclei (MN), the micronucleated binucleated cells (BCMN) and the nucleoplasmic

bridges (NB) were determined by counting 1000 binucleated viable cells (BC) with

preserved cytoplasm. BC, MN, NB, APOP and NECR were determined in agreement with

the previously described criteria [19].

2.4 Statistical analysis

In order to evaluate the possible differences between the control group and the

exposed one in relation to age, an unpaired Student’s t test between two means was

performed. In relation to smoking and alcohol drinking habits, the possible differences

between both groups were analysed by a Z test for two independent proportions. The effects

of the exposure, smoking and alcohol drinking habits on the cytogenetic variables (MN,

BCMN, NB, APOP and NECR) and on the NDI were evaluated by the analysis of

covariance (ANCOVA) including age and time of exposure as covariates. The statistic

analysis were performed with the STATISTICA software (5.0 for Windows). Differences

were considered statistically significant when p values were under 0.05.

3. RESULTS

The sanitary workers included in this study were exposed to several pesticides. As it

is shown in Table 1, some of these pesticides are mutagenic and/or possibly carcinogenic

composites and belong to the organophosphorate and pyrethroid insecticides and

hidroxicumarinic rodenticides groups. The application of the composites was performed by

spraying (pyrethroids), powder, pelleting bait, paraffin bait (hidroxicumarinic and

indandione) and ultra-low volume nebulization or sand mixed granulated

(organophosphorate). The composites were applied separately and the PPE used was

specific for each composite, except for malathion and temephos whose application was

performed without PPE. The majority of the pesticides are used sporadically. All the

exposed individuals worked for 40 hours a week.

The main characteristics of the population are described on Table 2. The age and the

alcohol drinking habit were similar in both groups. The exposed group had a larger number

of smokers (p < 0.05, Z test). The average pesticide exposure time of the workers was 5.28

+ 0.60 years.

Table 3 summarizes mean values of cytogenetic variables and nuclear division

indexes studied in the groups. The ANCOVA results and the regression coefficients are

presented in Table 4 and 5 respectively. The exposed group showed MN frequencies,

BCMN, NB and NECR significantly higher than the control group (p < 0.01) (Table 4).

The Figure 1 shows the MN frequencies in both groups. Although the APOP frequency

difference was not significant, it was higher in the exposed group (Table 4). The exposed

group had significantly lower NDI values (p < 0.01) (Table 4). The regression coefficients

(Table 5) indicated that, from the covariates introduced in the analysis, only the age of the

individuals had a significant influence over the MN and BCMN frequencies (p < 0.01). The

pesticides exposure time did not have an influence over the parameters analysed (Table 5).

Neither the smoking habits nor the alcohol drinking ones influenced the analysed

cytogenetic variables (Table 4).

4. DISCUSSION

With the increase of the global population, new areas are being rough-hewed and

the humans are more exposed to illnesses transmitted by wild vectors [15]. The control of

the increase of infected transmitter animals becomes a central issue in the sanitary

surveillance kept by governments. The use of pesticides has become routine, mainly in

underdeveloped countries, but the genotoxic potential of these substances is yet unknown

[2]. Most of the population that lives in the affected areas and the sanitary workers

responsible for the application are at cytotoxic and genotoxic risks.

The aim of this study was to evaluate if the occupational exposure to pesticides

might cause cytogenetic damage compared to a control group that had never been exposed

to these chemicals. Fifty-nine males (29 occupationally exposed and 30 control subjects)

were included in the study. The age of the subjects and alcohol drinking habits were similar

in both groups, but there was a larger number of smokers in the exposed group. In spite of

that, this difference did not influence the results, since through the analysis of covariance

the smoking habit did not have a significant effect on the cytogenetic parameters evaluated.

To evaluate the genetic damages that took place in these subjects the cytokinesis-

block micronucleus assay in human lymphocyte cultures (CBMN assay) was used [20,21].

Through this assay the clastogenic and aneugenic effects were detected since the MN are

originated from chromosomal fragments or from an entire chromosome non-included in the

main nucleus of the descent cell during cellular division [21,22]. Recently, it was proposed

the inclusion of other cytogenetic parameters in the CBMN assay [23]. These parameters

are the presence of nucleoplasmic bridges (indicators of chromosomic rearrangement),

apoptotic and necrotic cells (indicators of cellular viability), and cellular division index

[24,25]. Because this test offers simultaneous information on DNA damage and

cytotoxic/cytostatic effects caused by possible aggressive agents, nowadays it is a simple

and important tool for the monitoring of human population [25].

In this study, the group exposed to pesticides showed a significant higher frequency

of chromosome damage (MN, BCMN and NB) compared with the control group. Some

studies also showed a positive association between the genotoxicity and the occupational

exposure to pesticides [12,26-33], although other studies did not conclude the same

[10,13,14,34,39]. Such disagreement may be explained both by different exposure

conditions (protection measure used and specific genotoxic potential of the substances

used) or by demographic factors and individual habits and genetic features associated

[2,18]. By this way each biomonitoring study is unique and, in order to estimate the effects

of an occupational exposition, each population should be studied separately and the results

should not be generalised.

The presence of chromosome damage in the exposed group can be explained by the

genotoxic power of the substance they were exposed to. The pyrethroid group is the most

frequently used in solution through spraying. When applying it the workers make use of

PPE. Of the pyrethroid used by the workers the U. S. Environmental Protection Agency

(EPA) classified only cypermethrin as a possible carcinogen, while the other pyrethroids do

not have mutagenic and/or carcinogenic activity [40]. This reduces the possibility of the

remaining pyrethroids (except cypermethrin) of being responsible for the damage detected.

The organophosphorate group is applied daily in the solid form without using PPE. In

agreement with the EPA classification [40], malathion showed mutagenic activity in the

experimental system, but carcinogenic activity was not observed. Other substances

(hidroxicumarinic and indandione) are sporadically applied with the use of PPE. So far,

most of them haven’t been tested for their mutagenic and carcinogenic effects, and so it is

not possible to say if the exposure to this substances could be considered safe. This

suggests that malathion (organophosphorate) and cypermethrin (pyrethroid) should be the

main pesticides responsible for the chromosome damage found in the exposed workers.

However, it is important to notice that an exposure to a great number of different

compounds makes it difficult to know which agent could be responsible for the observed

cytogenetic damages.

Another aspect that could contribute to the positive association between the

observed cytogenetic damage and pesticide exposure is that most of the biomonitoring

studies in occupational exposed populations are done with individuals that used a mix of

several chemical compounds [14,38,39,41-43]. In this study, the pesticides are applied

separately and probably in higher concentrations than those found in the mixtures and these

concentrations may present a higher genotoxic potential.

In spite of the use of the protection equipment in the application by of most

products, it was observed an induction of chromosome damage caused by the pesticide

exposure. These findings can be explained by the ineffectiveness or inappropriate use of the

protection measures. Another explanation could be the fact that malathion

organophosphorate may be the main responsible substance for the chromosome damage

found, because it was applied without PPE.

The cytotoxicity was higher in the sanitary workers than in the control group, since

the frequency of necrotic cells was significantly higher in the exposed group. One

possibility is the existence of two substance groups: a genotoxic group that causes

chromosome damage without killing the cells and a cytotoxic group that causes cell

necrosis. The fact is that the surviving cells are perpetuating important mutations proved by

the increased frequencies of MN, BCMN and NB.

There was not a significant difference in the APOP frequencies between the

exposed and the control groups. This suggests that the damages to DNA caused by the

exposure were not sufficient to cause apoptosis, but they caused MN and NB [24].

The reduction in the NDI found in the exposed group, according to other studies

[39,41], corroborates the hypothesis that cells with DNA damage delay the cell cycle in

order to repair the damage and avoid the fixation of mutations during replication [25].

However, the fact that MN, BCMN and NB frequencies having been higher in the exposed

group, show that the repair may not be efficient to correct the induced mutations caused by

exposure. Another hypothesis is that the chemicals have cytotoxic properties that affect the

cell proliferation kinetics [35,44].

Of the analysed variables that could influence the cytogenetic parameters evaluated,

only the age was positively related to MN and BCMN frequencies. These results agree with

many other studies that showed an increase of spontaneous MN frequency with the age

[18,45-47]. This effect has been attributed to an increase of aneuploidy mainly of the X and

Y chromosomes [48].

It was not observed an association between the cytogenetic parameters frequency

and the time of exposure of the workers to the pesticides. This observation differs from the

results found by other authors. Bolognesi (2002) [42] found a positive relation between the

MN incidence and the pesticide exposure duration when individuals were exposed for more

than 10 years, suggesting that chromosome damage is accumulated during continued

exposure to pesticides. The lack of association between the cytogenetic parameter evaluated

and time of exposure in the present study could be explained by the slight variation in the

exposure time. In this study, exposure time was from 1,5 to 18 years and only one

individual was exposed for more than 10 years.

The smoking habit did not influence the cytogenetic parameters evaluated. In the

biomonitoring studies of populations occupationally exposed to genotoxic agents, the

smoking habit influence on the MN frequency is controversial. Few studies showed an

association between those variables [49,50], while most of the studies did not find any

association at all [14,38,39,41,42,51]. A possible explanation is that the damage caused by

tobacco could kill the cells in culture or delay the cell cycle making it impossible the

analysis of the MN [52]. However, it was not found an influence of smoking habit on

APOP/NECR frequncies and neither on NDI. Another possibility is that the number of

smokers in the control group was small (6 individuals) and could constitute a non-

representative sample.

Like the smoking habit, the alcohol drinking habit did not influence the parameters

evaluated. Data in literature showed positive, preventive or no effects [47,49,53,54] of

alcohol drinking habit on MN frequency. Normally these effects happen as a consequence

of the relation between alcohol drinking habit and other variables, such as age [38].

Concluding, the occupational exposure to pesticides by sanitary workers in Belo

Horizonte showed a genotoxic and cytotoxic effects, measured by higher frequencies of

MN, BCMN, NB and necrotic cells when compared with the control group. The reduced

NDI in the exposed group suggests a possible adaptive response to a chronic exposure to

pesticides. The age factor presented a direct relation with the MN frequency while the

smoking and drinking habits did not influence the cytogenetic parameters evaluated.

These results evidence a genetic hazard related to occupational pesticide exposure

and therefore the need for educational programs to stimulate the correct use of the PPE

and/or implement new protection measures for the sanitary workers.

ACKNOWLEDGEMENTS

We are very thankful to the volunteers who participated in the study, to Laboratório

de Substâncias Antitumorais and to Marlene de Miranda for collecting blood samples. This

study was supported by CAPES.

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Table 1

Pesticides used by the sanitary workers, with indication of their frequency of use, mutagenicity (M) and

carcinogenicity (C) experimental data

, personal protection equipment (PPE) used and application way.

I, inseticide; R, rodenticide; PYR, pyrethroid; OP, organophosphorate; HC, hidroxicumarinic; IND, indandione.

Dly, daily; Ep, during epidemics; Sly, sporadically.

Environmental Protection Agency (EPA) classification: -, no observed effect; +, positive effect; Pos., possibly

carcinogenic; NA, not available.

Type

Product Group Freq. use CAS # M C PPE Application

α-cypermethrin

PYR Dly 69865-74-0 - Pos.

Mask, gloves and overalls Spraying

I cypermethrin PYR Dly 52315-07-8 - Pos.

Mask, gloves and overalls Spraying

I deltamethrin PYR Dly 52918-63-5 - - Mask, gloves and overalls Spraying

I temephos OP Dly 3383-96-8 - - No protection Sand mixed granulated

malathion

OP Dly 121-75-5 + - No protection Sand mixed granulated

I fenithrothion OP Ep 122-14-5 - - Impermeable overalls,

mask and gloves

Ultra- low nebulization

R brodifacum HC Sly 56073-10-0 NA NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R coumachlor HC Sly 81-82-3 NA NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R coumafuryl HC Sly 117-52-2 NA NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R coumatetralyl HC Sly 5836-29-3 NA NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R difethialone HC Sly 104653-34-1 NA NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R flocoumafen HC Sly 90035-08-8 - - Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R difenacoum HC Sly 56073-07-5 NA NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R bromadiolone HC Sly 28772-56-7 - NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R diphacinone IND Sly 82-66-6 NA NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

R pindone IND Sly 83-26-1 NA NA Rubber gloves and surgical

mask

Powder, pelleting bait,

paraphine bait

Table 2

Characteristics of the groups studied

No statistical difference between both groups (t-test).

p< 0,05 (Z-test), compared with control group.

No statistical difference between both groups (Z-test).

Characteristics Control Exposed

Number of subjects 30 29

Age (years) (mean ± SE)

Range (years)

29.17 ± 1.64

18-49

30.31 ± 1.48

21-57

Smoking habit

Smokers (n, %)

Non-smokers (n, %)

6 (20.00)

24 (80.00)

14 (48.28)

15 (51.72)

Drinking habit

Yes (n, %)

No (n, %)

19 (63.33)

11 (36.67)

15 (51.72)

14 (48.28)

Years of pesticide exposure (mean ± SE)

Range (years)

- 5.28 ± 0.60

1.50-18

Table 3

Mean values (±SE) of the cytogenetic variables analysed in the groups studied

MN, micronuclei in 1000 binucleated cells; BCMN, binucleated cells

with micronuclei in 1000 binucleated cells; NB, nucleoplasmic bridges

in 1000 binucleated cells; APOP, apoptotic cells in 500 viable cells;

NECR, necrotic cells in 500 viable cells; NDI, nuclear division index.

Variables Control Exposed

MN 4.71 ± 0.42 15.81 ± 1.31

BCMN 4.62 ± 0.44 15.10 ± 1.22

NB 1.00 ± 0.34 4.59 ± 0.76

APOP 11.81 ± 1.20 18.40 ± 2.60

NECR 5.17 ± 0.70 12.07 ± 1.45

NDI 1.61 ± 0.02 1.49 ± 0.02

Table 4

Summary of the effects for each cytogenetic variable analysed (ANCOVA)

Variables MS Effect MS Error F (df 1.0) p

Exposure

Smoking habit

Drinking habit

615.38

58.94

0.00

17.19

35.81

3.43

0.00

<0.01

0.07

0.99

BCMN

Exposure

Smoking habit

Drinking habit

513.52

47.33

0.03

16.30

31.50

2.90

0.00

<0.01

0.09

0.96

Exposure

Smoking habit

Drinking habit

30.20

4.74

1.96

9.82

3.08

0.48

0.20

0.04

0.49

0.66

APOP

Exposure

Smoking habit

Drinking habit

187.65

0.01

0.55

113.31

1.66

0.00

0.20

0.99

0.94

NECR

Exposure

Smoking habit

Drinking habit

348.12

109.88

6.12

33.33

10.44

3.30

0.18

<0.01

0.07

0.67

NDI

Exposure

Smoking habit

Drinking habit

0.11

0.00

0.04

0.01

8.89

0.11

3.18

<0.01

0.74

0.08

MN, micronuclei; BCMN, binucleated cells with micronuclei; NB, nucleoplasmic bridges; APOP,

apoptotic cells; NECR, necrotic cells; NDI, nuclear division index.

Table 5

Regression coefficients for the covariates introduced in the ANCOVA analysis

Covariates

β ± SE

t p

Age

Years of exposure

0.58 ± 0.08

-0.19 ± 0.26

4.86

-1.58

<0.01

0.12

BCMN

Age

Years of exposure

0.57 ± 0.07

-0.15 ± 0.26

4.77

-1.27

<0.01

0.21

Age

Years of exposure

-0.04 ± 0.06

0.18 ± 0.20

-0.28

1.29

0.78

0.20

APOP

Age

Years of exposure

-0.23 ± 0.20

-0.07 ± 0.68

-1.61

-0.51

0.11

0.61

NECR

Age

Years of exposure

-0.06 ± 0.11

-0.25 ± 0.37

-0.40

-1.75

0.69

0.09

NDI

Age

Years of exposure

0.22 ± 0.00

0.06 ± 0.00

1.54

0.45

0.13

0.65

MN, micronuclei; BCMN, binucleated cells with micronuclei; NB, nucleoplasmic

bridges; APOP, apoptotic cells; NECR, necrotic cells; NDI, nuclear division index.

Fig. 1. Micronuclei frequencies in 1000 binucleated cells (MN/1000 BC) in exposed

and control groups.

MN/1000 BC

Exposed Control

Mean+SD

Mean-SD

Mean+SE

Mean-SE

Mean

ANEXOS

Figuras

Figura 1 - Fotomicrografia de linfócito mononucleado típico corado por Giemsa (observado

sob aumento de 1000 X).

Figura 2 – Fotomicrografia de linfócito binucleado corado por Giemsa (observado sob

aumento de 1000 X).

Figura 3 - Fotomicrografia de linfócito binucleado corado por Giemsa (observado sob

aumento de 1000 X).

Figura 4 – Fotomicrografia de linfócito binucleado (BC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X). A seta indica o micronúcleo.

Figura 5 - Fotomicrografia de linfócito binucleado (BC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X). A seta indica o micronúcleo.

Figura 6 - Fotomicrografia de célula (linfócito) binucleada (BC) contendo micronúcleo

(MN) corado por Giemsa (observado sob aumento de 1000 X). A seta indica o

micronúcleo.

Figura 7 - Fotomicrografia de linfócito binucleado (BC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X). A seta indica o micronúcleo.

Figura 8 - Fotomicrografia de linfócito binucleado (BC) contendo ponte nucleoplasmática

(NB) corado por Giemsa (observado sob aumento de 1000 X). A seta indica a ponte

nucleoplasmática.

Figura 9 - Fotomicrografia de linfócito trinucleado (TC) típico corado por Giemsa

(observado sob aumento de 1000 X).

Figura 10 - Fotomicrografia de linfócito trinucleado (TC) corado por Giemsa (observado

sob aumento de 1000 X).

Figura 11 Fotomicrografia de linfócito trinucleado (TC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X).

Figura 12 - Fotomicrografia de linfócito tetranucleado (QC) típico corado por Giemsa

(observado sob aumento de 1000 X).

Figura 13 - Fotomicrografia de linfócito tetranucleado (QC) contendo micronúcleo (MN)

corado por Giemsa (observado sob aumento de 1000 X).

Figura 14 - Fotomicrografia de linfócito em apoptose corado por Giemsa (observado sob

aumento de 1000 X).

Figura 15 - Fotomicrografia de linfócito em apoptose corado por Giemsa (observado sob

aumento de 1000 X).

Figura 16 - Fotomicrografia de linfócito em necrose corado por Giemsa (observado sob

aumento de 1000 X).

Figura 17 - Fotomicrografia de linfócito em necrose corado por Giemsa (observado sob

aumento de 1000 X).

Figura 18 - Fotomicrografia de linfócito em necrose corado por Giemsa (observado sob

aumento de 1000 X).

Termo de Consentimento

UNIVERSIDADE FEDERAL DE MINAS GERAIS

INSTITUTO DE CIÊNCIAS BIOLÓGICAS

TERMO DE CONSENTIMENTO

Aos funcionários da FUNASA e do Centro de Controle de Zoonozes da Regional

Venda Nova da Prefeitura Municipal de Belo Horizonte

O projeto “Avaliação dos efeitos genotóxicos da exposição ocupacional a pesticidas

sobre agentes sanitários da Fundação Nacional de Saúde do Estado de Minas Gerais e do

Centro de Controle de Zoonoses da Regional Venda Nova da Prefeitura Municipal de Belo

Horizonte” tem como objetivo avaliar os efeitos prejudiciais da exposição ocupacional a

pesticidas em agentes sanitários vinculados a FUNASA e ao Centro de Controle de Zoonoses

da Regional Venda Nova da Prefeitura de Belo Horizonte. Esta avaliação será feita através da

análise da frequência de danos genéticos em células de sangue destes trabalhadores. Nossos

esforços se concentram no sentido de fornecer aos serviços de vigilância a saúde subsídios

para implementação das medidas de proteção a estes trabalhadores.

Para a realização deste estudo contamos com a colaboração de funcionários da

FUNASA e do Centro de Controle de Zoonoses que têm ou tiveram exposição ocupacional

a pesticidas. Para as análises a serem realizadas, serão coletados 10 ml de sangue dos

colaboradores com seringa descartável e estéril, NÃO ACARRETANDO RISCOS À

SAÚDE DO COLABORADOR.

O material coletado será transportado pelos responsáveis pela pesquisa para o

Laboratório de Genética de Neoplasias e Mutagênese do Departamento de Biologia Geral

do Instituto de Ciências Biológicas da UFMG, onde será efetuada a manipulação do

mesmo, sob responsabilidade da Profª Maria Cristina Lima de Castro.

Lembramos ainda que os colaboradores não terão suas identidades reveladas e que

todas as informações fornecidas e os resultados obtidos serão mantidos em absoluto sigilo,

sendo utilizados apenas para divulgação em reuniões e revistas científicas.

As pessoas que concordarem em colaborar com a pesquisa deverão assinar este

termo de consentimento e fornecer, através de um questionário a ser aplicado, algumas

informações a respeito de seu trabalho e de alguns hábitos.

Maria Cristina Lima de Castro (orientadora) Comitê de Ética em Pesquisa da UFMG

Av. Antônio Carlos, 6627 – Pampulha COEP

CEP 31270-901- Belo Horizonte- MG Av. Antônio Carlos, 6627 – Pampulha

Instituto de Ciências Biológicas - Diretoria Reitoria 7° andar

Tel: (31) 3499-2527 Tel: (31) 3499-4027

Fernanda de Souza Gomes Kehdy (pesquisador responsável)

Instituto de Ciências Biológicas - Bloco E3 sala 179 Tel: (31) 3499-2596

UNIVERSIDADE FEDERAL DE MINAS GERAIS

INSTITUTO DE CIÊNCIAS BIOLÓGICAS

TERMO DE CONSENTIMENTO

Aos colaboradores voluntários do projeto

O projeto “Avaliação dos efeitos genotóxicos da exposição ocupacional a pesticidas

sobre agentes sanitários da Fundação Nacional de Saúde do Estado de Minas Gerais e do

Centro de Controle de Zoonoses da Regional Venda Nova da Prefeitura Municipal de Belo

Horizonte” tem como objetivo avaliar os efeitos prejudiciais da exposição ocupacional a

pesticidas em agentes sanitários vinculados a FUNASA e ao Centro de Controle de

Zoonoses da Regional Venda Nova da Prefeitura de Belo Horizonte. Esta avaliação será

feita através da análise da frequência de danos genéticos em células de sangue destes

trabalhadores. Nossos esforços se concentram no sentido de fornecer aos serviços de

vigilância a saúde subsídios para implementação das medidas de proteção a estes

trabalhadores.

No entanto, para a realização deste estudo precisamos, além da colaboração dos

agentes sanitários, da colaboração de doadores voluntários sem história de exposição a

pesticidas.para constituírem o grupo controle. Os danos genéticos encontrados no grupo

controle servirão como referência para serem comparados com os encontrados nos agentes

sanitários ocupacionalmente expostos a pesticidas.

Para as análises a serem realizadas, serão coletados 10 ml de sangue dos