Research Articles

Cytogenetic Damage in Female Chilean AgriculturalWorkers Exposed toMixtures of Pesticides

CarolinaMa¤ rquez,1Cecilia Villalobos,2 Susana Poblete,1 Eva Villalobos,2

Mar|¤a de los Angeles Garc|¤a,1 and Soledad Duk1*1Laboratorio de Citogenetica y Genetica Toxicologica, Departamento de

Biologıa Celular, Facultad de Ciencias Biologicas, Universidad deConcepcion, Concepcion, Chile

2Departamento de Obstetricia y Puericultura, Facultad de Medicina,Universidad de Concepcion, Concepcion, Chile

INTRODUCTION

Since 1945, more than 15,000 individual chemicals and

35,000 formulations have been used as agricultural pesti-

cides [Forget et al., 1993]. The chemicals are an impor-

tant tool for controlling agricultural pests, but they also

represent a significant source of occupational and nonoc-

cupational exposure to potentially toxic agents.

Fruit exportation has become an important source of

Chilean income. In 1974, there were 63,000 ha of industrial

orchards; by 1994, that figure had increased to 185,040 ha

(Statistical National Institute of Chile). As a consequence,

the use of chemical pesticides has also significantly

increased, leading to a larger number of exposed workers

and higher levels of environmental exposure. The number of

people working in seasonal agriculture in Chile is estimated

at nearly 250,000, and in the workforce of the VIII Region

of Bıo-Bıo, in central Chile, many of them are women

[Wesseling et al., 1998]. The health risks associated with

this increasing exposure to pesticides among Chilean women

are presently unknown. In order to evaluate the risk, we have

carried out a biomonitoring study on seasonal female work-

ers from the VIII Region. The workers participating in the

study were employed in greenhouses, plant nurseries, and

performed pruning, sorting, harvesting, and packing, rotating

among the different tasks during the season.

Previous studies have associated acute and chronic pes-

ticide exposure with asthma, allergic dermatitis, and

respiratory disease [Zuskin et al., 1993]. Anecdotal

reports on acute poisoning, respiratory problems, vision

distress, headaches, and depression were also collected in

the present study. In addition, pesticides have been stud-

ied for their long-term risk, including their association

with increases in the incidence of cancers, such as leuke-

mia [Daniels et al., 1997], bladder cancer [Viel and

Chalier, 1995], and pancreatic cancer [Ji et al., 2001].

*Correspondence to: Soledad Duk, Casilla 160-C, Facultad de Ciencias

Biologicas, Departamento de Biologıa Celular, Universidad de Concep-

cion, Concepcion, Chile. E-mail: sduk@udec.cl

Grant sponsor: Direccion de Investigacion Universidad de Concepcion;

Grant number: 201.031.089-1.

Received 27 February 2004; provisionally accepted 24 April 2004; and

in final form 23 August 2004

DOI 10.1002/em.20085

Published online 16 December 2004 in Wiley InterScience (www.interscience.

wiley.com).

�c 2004Wiley-Liss, Inc.

Environmental andMolecular Mutagenesis 45:1^7 (2005)

The VIII Region of Bıo-Bıo is a major fruit-growingarea of Chile that makes intensive use of agricul-tural pesticides. The cytogenetic damage asso-ciated with exposure to mixtures of pesticides wasevaluated by comparing peripheral blood lympho-cyte micronucleus (MN) frequencies in a group of64 female agricultural workers and 30 female con-trols. The exposed subjects worked during thespring and summer in thinning and pruning fruittrees and in harvesting and packing different fruits,such as raspberries, grapes, apples, and kiwis.They did not use any protective measures duringtheir work activities. A significant increase in the fre-

quency of binucleated cells with micronuclei(BNMN) was found in the exposed women as com-pared with the controls (36.94 � 14.47 vs. 9.93 �6.17 BNMN/1000 BN cells; P < 0.001). The fre-quency of BNMN varied as a function of age inboth the exposed and control groups, but no corre-lation was found between BNMN frequency andthe duration of exposure. Also, smoking and otherhabits had no effect on MN frequency. Our studyconfirms that occupational exposure to pesticidemixtures results in cytogenetic damage. Environ.Mol. Mutagen. 45:1–7, 2005. �c 2004 Wiley-Liss,Inc.

Key words: cytogenetic; micronucleus; women; pesticides; occupational exposure

Genotoxic potential is a primary risk factor for long-term

effects such as carcinogenic and reproductive disorders.

In a retrospective study, Rojas et al. [2000] made the first

attempt to associate congenital malformations with pesti-

cide exposure in Chile. Genotoxicological biomonitoring

in human populations is a useful tool to estimate the

genetic risk from an integrated exposure to complex mix-

tures of chemicals [Bolognesi, 2003].

The micronucleus (MN) assay is a genotoxicity biomoni-

toring method that has been used widely for evaluating risk

in human populations exposed to carcinogens and/or muta-

gens [Tucker and Preston, 1996; Garcıa et al., 1999;

Fenech, 2000]. Micronuclei are formed from acentric

chromosomal fragments or whole chromosomes left behind

during mitotic cellular division, allowing the detection of

clastogenic and aneugenic events. Micronuclei reflect

chromosomal damage and may thus provide a marker of

early-stage carcinogenesis [Bonassi et al., 2003]. There are

a number of reports with positive findings of cytogenetic

effects in the lymphocytes of people exposed to pesticides

[Dulout et al., 1985; de Ferrari et al., 1991; Bolognesi

et al., 1993; Gomez-Arroyo et al., 2000]. Other studies,

however, have obtained negative results [Carbonell et al.,

1990; Scarpato et al., 1996; Lucero et al., 2000].

Our study group contained seasonal agricultural working

women from the VIII Region of Bıo-Bıo, Chile. Even

though the women in the exposed population were not

directly involved in pesticide handling, there were some

cases of acute exposure. This often was due to early

entrance into the field, fumigation of nearby orchards, and

pesticide drift. Chronic exposure resulted from touching the

fumigated fruit with bare hands, working in their everyday

clothing, eating in the orchard, and having only sporadic

access to fresh running water. For both types of exposure,

the workers wore no protective equipment, not even gloves.

In Chile, genotoxic damage due to pesticide exposure

has been evaluated only in a group of 22 men who

employed protective measures [Venegas et al., 1998].

This is the first monitoring study of women exposed to

mixtures of pesticides in the VIII Region of this country.

We compared chromosomal damage in a group of

exposed agricultural women laborers and a group of con-

trol women. The statistical analysis controlled for poten-

tial confounding factors such as smoking and alcohol

consumption. A significant increase in chromosomal

damage was found in the exposed group.

MATERIALS ANDMETHODS

Study Population

A total of 94 Caucasian women from the VIII Region of Bıo-Bıo, Chile,

were analyzed in this study. Sixty-four temporary women workers com-

prised the exposed group and worked in contact with a complex mixture of

pesticides (Table I); they rotated among different tasks during the season.

The control group had no contact with pesticides or any particular environ-

mental genotoxic agent, since they were mainly homemakers or office

workers in urban Concepcion. The socioeconomic and educational levels

of the control group were higher than those of the exposed. Prior to the

blood sampling, a standard health, family, and work history questionnaire

was completed (questionnaire available upon request), and confounding

factors were considered for exclusion. All individuals contacted agreed to

participate and signed an informed consent agreement. In the case of the

exposed group, further questions related to farming were included, such as

pesticide use and duration of exposure.

Lymphocyte Cultures andMNAnalysis

Blood samples were obtained by venipuncture in June–July (2000–

2002), after the women finished their work season, which lasted between

October and March (1999–2001). All blood specimens were collected in

sterile tubes with sodium heparin and were processed within 24 hr. Two

lymphocyte cultures per subject were initiated by adding 0.5 ml of whole

TABLE I. Pesticides to Which Agricultural Women AreExposed in Bıo-Bıo, Chile, Indicating the Type,World Health Organization (WHO) HazardClassification, and CAS Numbers

a

Type Product Class (WHO) CAS number

Insecticides Azinfos metil II 86-50-0

Azomark I 6923-22-4

Belmark III 51630-58-1

Diazinon III 333-41-5

Furadan II 1563-66-2

Imidan III 732-11-6

Metamidofos II 10265-92-6

Mimic IV 112410-23-8

Fungicide Sulphur IV 7704-34-9

Benlate IV 17804-35-2

Captan IV 133-06-2

Cuprodul IV 1317-39-1

Hortyl IV 1897-45-6

Manzate IV 8018-01-7

Metalaxil IV 70630-17-0

Herbicide Afalon IV 330-55-2

Paraquat II 1910-42-5

MCPA III 94-74-6

Simazina IV 112-34-9

Atrazina IV 1912-24-9

Betanal IV 13684-63-4/1368-56-5/

26225-79-6

Linurex IV 330-55-2

aData obtained by the authors through a checklist in field trips to the

orchards to detect compliance with sanitary conditions required by law.

TABLE II. General Characteristics of the Study Groups

Control Exposed P

Number of subjects 30 64

Years of age (mean � SD) 36.6 � 11.4 40.5 � 7.8 0.1156

Years of exposure (mean � SD) 8.0 � 4.8

Smoking 0.4337

Number of nonsmokers 17 (56.7%) 41 (64.1%)

Number of smokers 13 (43.3%) 23 (35.9%)

Alcohol consumption 0.5839

Number of nondrinkers 12 (40%) 30 (46.9)

Number of drinkers 18 (60%) 34 (53.1)

2 Ma¤ rquez et al.

blood to 4.5 ml RPMI-1640 medium supplemented with 10% heat-inacti-

vated fetal calf serum, 1% antibiotics (penicillin and streptomycin), and L-

glutamine, all provided by Gibco-Invitrogen (Carlsbad, CA). Lymphocytes

were stimulated by 1% phytohemagglutinin (Gibco-Invitrogen) and incu-

bated for 72 hr at 378C. Cytochalasin B (Sigma, St. Louis, MO) was added

at a final concentration of 6 mg/ml after 44 hr of culture to arrest cytokin-

esis. At the end of the incubation, the cultures were harvested by centrifu-

gation at 150 g for 8 min and treated 2–3 min at 48C with a hypotonic

solution (0.075 M KCl). After centrifugation, the cells were treated twice

with cold fixative solution (3:1 methanol:acetic acid). The cells were then

resuspended in a small volume of the fixative and dropped onto clean

slides. The slides were stained with 10% Giemsa (Merck, Darmstadt,

Germany) in phosphate buffer (pH 6.8) for 10 min, coded, and

scored without knowledge of their identity.

A single individual carried out the microscopic analysis. Two coded

slides were scored per subject. A total of 1,000 binucleated cells with

well-preserved cytoplasm were scored in order to determine the fre-

quency of binucleated cells with micronuclei (BNMN) and the total

number of micronuclei in binucleated lymphocytes (MNL). In addition,

500 lymphocytes were scored to evaluate the percentage of cells with

one to four nuclei, and the cytokinesis block proliferation index (CBPI)

was calculated.

Statistical Analysis

Nonparametric statistical tests were used. The experimental unit for

statistical evaluations was the study subject. The Mann-Whitney U-test

was used to detect differences between the mean of BNMN, MNL, and

CBPI in the exposed workers and the controls. Statistical analyses were

performed on the mean MN frequencies for each individual for all

cytogenetic variables. The relationship between age and BNMN fre-

quency was evaluated using regression analysis. All data were analyzed

using the CSS Statistica software package (StatSoft, Tulsa, OK). The

level of significance was taken as P < 0.05.

RESULTS

The main characteristics of the study groups, such as age,

smoking habits, and duration of exposure to pesticides, are

presented in Table II. Information about acute poisoning,

respiratory problems, and other health problems were col-

lected during the investigation; the results are presented in

Figure 1. The exposed subjects who had health complaints

did not have a higher frequency of micronuclei than the rest

of the exposed group. To evaluate lifestyle factors, donors

were stratified as smokers or nonsmokers and as alcohol

drinkers or nondrinkers. Smokers were current smokers and

smoked between 1 and 10 cigarettes/week. Drinkers con-

sumed between 50 and 500 ml/week of beer or wine. Age

and lifestyle factors were not significantly different between

the control and exposed group (Table II). No correlation

was observed between smoking habits (Table III) or alcohol

consumption (not shown) and the frequency of BNMN for

either the exposed or the control group. The age of the

exposed workers ranged from 25 to 56 years, with a mean

of 40.5 � 7.8, while the age of nonexposed controls ranged

from 20 to 58 years, with a mean of 36.6 � 11.4. A posi-

tive correlation was found between age and BNMN fre-

quency for both groups. Figure 2 shows the relationship

between these two variables. The Spearman regression

coefficient for the exposed group was 0.321 (P < 0.01);

for the control group, 0.629 (P < 0.01). The slope for the

line in the exposed group is twice the slope of the control.

TABLE III. Effect of Smoking on Median BNMN Frequencyfor Exposed and Control Groups

Group/smoking status n BNMN/1,000 cells (range) P

Exposed

Nonsmokers 41 39 (16–79) 0.1895

Smokers 23 33 (9–62)

Control

Nonsmokers 17 10 (2–26) 0.4144

Smokers 13 9 (2–23)

Fig. 2. Correlation between age and BNMN frequency for the exposed

and the control groups. Open square: control group; Spearman correla-

tion (rs) ¼ 0.629; P < 0.01. Closed circle: exposed group; rs ¼ 0.321;

P < 0.01.

Fig.1. Major health complaints of exposed group.

Cytogenetic Damage 3

Tables IV and V present individual BNMN and MNL

frequencies for the exposed and control group, and Figure 3

presents the distribution of these frequencies. The data indi-

cate that there was no obvious subpopulation of people with

very high BNMN levels, which led to an increase in the

mean of the exposed subjects. Table IV also includes years

of exposure; the mean duration of pesticide exposure was

8.0 � 4.8 years. A dose-response relationship between

duration of exposure (measured in years) and BNMN fre-

quency was not observed in our study, although the specific

concentrations of pesticides to which the seasonal agricul-

tural women were exposed is uncertain.

Table VI summarizes the cytogenetic variables for both

control and exposed subjects. The mean BNMN frequen-

cies for the control and exposed groups were 9.93 and

36.93, respectively, and the frequencies of MNL were

10.90 and 42.89. Statistical analysis indicated that these

differences were significant (P < 0.01, Mann-Whitney

U-test). A significant decrease was observed in the CBPI

of the exposed women (P ¼ 0.005).

DISCUSSION

We have studied a group of agricultural women work-

ers from the VIII Region of Bıo-Bıo, Chile, in order to

TABLE IV. Individual MN Frequencies for the Exposed Groupand Years of Exposure

Subjects Years of exposure BNMN/1,000 cells MNL/1,000 cells

E-1 6 25 30

E-2 16 31 39

E-3 9 31 35

E-4 3 28 31

E-5 7 21 23

E-6 11 25 25

E-7 2 44 53

E-8 4 34 37

E-9 10 13 13

E-10 3 39 43

E-11 4 25 26

E-12 9 55 64

E-13 7 36 39

E-14 8 30 36

E-15 12 43 49

E-16 7 39 42

E-17 7 62 69

E-18 10 24 27

E-19 9 33 41

E-20 6 24 26

E-21 8 56 64

E-22 6 44 59

E-23 7 9 9

E-24 12 37 46

E-25 7 20 21

E-26 16 31 38

E-27 5 30 33

E-28 2 46 56

E-29 15 27 33

E-30 5 40 45

E-31 3 16 22

E-32 12 47 51

E-33 10 40 44

E-34 7 59 76

E-35 7 19 22

E-36 7 42 55

E-37 19 45 57

E-38 7 26 33

E-39 13 11 12

E-40 6 43 50

E-41 12 53 61

E-42 7 28 32

E-43 18 79 101

E-44 6 61 74

E-45 1 35 43

E-46 18 37 38

E-47 5 59 74

E-48 5 31 38

E-49 2 26 28

E-50 8 16 17

E-51 4 65 76

E-52 2 23 25

E-53 9 46 50

E-54 2 48 54

E-55 18 45 55

E-56 3 33 39

E-57 3 45 55

E-58 13 68 89

E-59 3 37 45

E-60 20 50 60

E-61 4 49 61

E-62 10 68 80

E-63 2 36 44

E-64 10 65 71

TABLE V. Individual MN Frequencies for the Control Group

Subjects BNMN/1,000 cells MNL/1,000 cells

C-1 6 7

C-2 7 7

C-3 2 2

C-4 10 10

C-5 7 8

C-6 21 25

C-7 10 10

C-8 9 10

C-9 13 14

C-10 4 4

C-11 5 5

C-12 2 2

C-13 7 8

C-14 5 7

C-15 13 14

C-16 9 11

C-17 5 5

C-18 10 10

C-19 4 4

C-20 8 8

C-21 15 15

C-22 7 7

C-23 19 23

C-24 6 7

C-25 23 25

C-26 6 6

C-27 14 15

C-28 17 18

C-29 26 29

C-30 8 11

4 Ma¤ rquez et al.

evaluate associations between pesticide exposure and

cytogenetic damage. The exposed group of agricultural

workers was potentially exposed during the 5-month

season to a complex mixture of different pesticides,

including insecticides, fungicides, and herbicides. These

chemicals include compounds with known genotoxic

properties. For instance, the pesticides most often used

were carbamates, organophosphates, and pyrethroids,

which are genotoxic in both bacterial and mammalian

systems [Bolognesi, 2003]. Given the complex exposure

to a large number of compounds, it was not possible to

predict whether the overall effect was clastogenic or

aneugenic without carrying out fluorescent in situ hybridi-

zation (FISH) with centromeric probes. It has been postu-

lated that exposure to mixtures of pesticides induces

chromosomal aberrations such as acentric fragments and

dicentrics, as well as aneuploidy [Garaj-Vrhovac and

Zeljezic, 2001].

In the present study, age influenced BNMN frequency

in the exposed as well as the control group. These results

confirm the findings of other authors who identified age

as an important variable that influences the baseline MN

frequency [Fenech and Morley, 1991; Migliore et al.,

1991; Barale et al., 1998]. When we analyzed the slope

of the lines in Figure 2, the difference in slopes for the

two groups confirmed the presence of an additive effect

of exposure and age in the exposed group.

No correlation was found between smoking habits and

BNMN frequency in our population, which is also in

agreement with other published reports [Pitarque et al.,

1996; Bukvic et al., 2001; Bonassi et al., 2003].

Our findings indicate that the exposed worker popula-

tion from Bıo-Bıo, Chile, had a significant increase in the

cytogenetic damage in their peripheral blood lympho-

cytes. There was a significant increase in the frequency

of BNMN. There was also a decrease in the CBPI of the

exposed group, suggesting that the agricultural women

workers were exposed to chemicals with cytotoxic prop-

erties, which affected the cell proliferation kinetics

[Pastor et al., 2002]. The fact that these positive

responses in the exposed workers were detected several

weeks after the last known exposure to pesticides sug-

gests that the damage produced in the lymphocytes was

relatively persistent.

There are conflicting reports in the literature about the

effect of pesticide exposure on human populations, which

may reflect the exposure conditions of the particular

study population. Both positive associations between

chromosome damage and pesticide exposure [Falck et al.,

1991; Bolognesi et al., 1993, 2002; Gomez-Arroyo et al.,

2000; Shaham et al., 2001] and negative associations

[Davies et al., 1998; Pastor et al., 2001, 2002] have been

reported. These conflicting responses to pesticide expo-

sure may at least in part be due to the use of protective

clothing and devices, especially during reentry activities,

in the studies with negative associations. This conclusion

is consistent with data indicating increased cytogenetic

damage in work environments where no protective mea-

sures were taken [Rupa et al., 1989; Kourakis et al.,

1992; Lander and Ronne, 1995] and no increase in geno-

toxicity where protective equipment was used [Pastor

et al., 2002]. De Cock et al. [1996] found that application

and reentry activities in the fruit-growing industry

resulted in significant dermal exposure. We consider der-

mal exposure to be the main route in our study popula-

tion, and, apart from their regular clothing, our

population did not use protective equipment (e.g., gloves)

to prevent dermal exposure.

Some studies have reported that continuous exposure to

pesticide mixtures results in cumulative increases in chro-

mosomal damage [Carbonell et al., 1993; Davies et al.,

1998]. A major problem in interpreting biomonitoring

studies is estimating the degree of exposure. The varia-

tion in the degree of exposure and the use of different

chemical mixtures may be a reason for conflicting results

between studies. Exposure to pesticides can be quite spe-

cific for a particular population because each population

has different lifestyles, climatic conditions, and employs

different cultivation methods and uses different mixtures

of pesticides [Bolognesi et al., 2002]. The fact that a

length of exposure-response relationship was not found in

our study could be due to the practice in Chile of chang-

ing the type of job performed by agricultural women

workers each season that they work.

Exposure to pesticides may also have a role in the

induction of congenital malformations [Bolognesi, 2003].

Fig. 3. BNMN frequencies of exposed workers and controls.

Cytogenetic Damage 5

This is of particular interest since our study population

consisted mostly of women of reproductive age. A recent

study in Rancagua, Chile, showed a positive association

between pesticide exposure and congenital malformations

[Rojas et al., 2000].

In summary, our results confirm an association between

cytogenetic damage and occupational exposure to pesti-

cides. Cytogenetic damage may be viewed as an early

biological effect of a chemical insult; consequently, it

could be an indicator for future development of diseases

such as cancer and congenital malformations [Wesseling,

2003]. Our analysis suggests that the creation of educa-

tional programs that instruct seasonal agricultural workers

on the use of protective measures and safe agricultural

practices may decrease the risks associated with pesticide

exposure in the farming populations of developing

countries.

ACKNOWLEDGMENTS

The authors thank E. Spano for her expert technical

assistance in the preparation of samples and Professor

R. Naveas for her statistical advice. They also thank the

Health Service of Nuble and Los Angeles and the

National Women’s Service.

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TABLE VI. Cytogenetic Variables for the Exposed and Control Populationsa

Control Exposed

Endpoint n Mean � SD Median (range) n Mean � SD Median (range)

BNMN 30 9.93 � 6.17 8 (2–25) 64 36.94 � 14.47 37 (9–79)

MNL 10.90 � 7.01 9 (2–29) 42.89 � 17.72 43 (9–101)

CBPI 2.01 � 0.31 2.02 (1.08–2.38) 1.84 � 0.12 1.83 (1.62–2.09)

aData from scoring 1,000 binucleated cells per donor for BNMN and MNL; CBPI calculated from 500 cells.

6 Ma¤ rquez et al.

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Accepted by—S. Galloway

Cytogenetic Damage 7