Mutagenesis, Vol. 18, No. 3, 249-258,
May 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Biomonitoring of four European populations occupationally exposed to pesticides: use of micronuclei as biomarkers
Grup de Mutagènesi, Departament de Genètica i de Microbiologia, Facultat de Ciències, Universitat Autònoma de Barcelona, Bellaterra, Spain, 1 Delegación Provincial de la Consejería de Salud de Almería, Spain, 2 Department of Radiation and Environmental Biology, The H.Niewodniczanski Institute of Nuclear Physics, Kraków, Poland, 3 Department of Human Genetics and Teratology, ‘B.Johan’ National Center for Epidemiology, Budapest, Hungary and 4 DNA Repair Laboratory, National Centre for Scientific Research ‘Demokritos’, Aghia Paraskevi, Athens, Greece
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Abstract |
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This paper presents the results obtained within the framework of an EU research project aimed at investigating the relationship between occupational exposure to pesticides and the induction of cytogenetic damage. Populations from Greece, Spain, Poland and Hungary, all of them characterised by intensive agricultural activity, were the subject of the study. A total of 239 agricultural workers and 231 unexposed controls were examined for cytogenetic effects in lymphocytes of peripheral blood and exfoliated cells of the oral mucosa. The frequency of micronuclei (MN) was evaluated in both cell types and their relationship to different confounding factors (e.g. sex, country, smoking habit, etc.) was determined. The cytokinesis block proliferation index (CBPI) was also calculated to detect possible variations in the proliferative kinetics of lymphocytes due to pesticide exposure. The results obtained indicate that there are no increases in MN frequencies in the agricultural workers when compared with the controls for either lymphocytes or buccal cells. However, exposed individuals showed a significant decrease in CBPI when compared with controls. When the effect of the different confounding factors was evaluated, age was positively related with MN in lymphocytes and the Polish population showed a MN frequency significantly higher than those observed in the other populations. For buccal cells, the Spanish population showed a higher MN frequency, attaining significant differences in comparison with the other populations. Finally, the CBPI was found to be inversely influenced by age and Hungarian exposed men were the group that showed the lowest values.
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Introduction |
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As is well known, pesticides are extensively used all over the world and, in recent years, their use has increased spectacularly. Large amounts of these chemicals are released into the environment and many of them affect non-target organisms, being a potential hazard to human health. Pesticide exposure is ubiquitous, due not only to agricultural pesticide use and contamination of foods, but also to the extensive use of these products in and around residences. Individuals occupationally exposed to pesticides (such as field workers, mixers, loaders, applicators, etc.) who are in direct contact with these chemicals may provide a good opportunity to study their adverse health consequences.
At present there are 834 active pesticide substances registered in the European Union (Commission to the European Parliament and the Council, 2001
), some of which have been classified as possible or probable mutagens and/or carcinogens by the International Agency for Research on Cancer (IARC, 1990, 1991).
Thus, exposure to pesticides has been associated with an increase in the incidence of non-Hodgkin’s lymphoma (Hardell and Eriksson, 1999; Zheng et al., 2001), multiple myeloma (Khuder and Mutgi, 1997), soft tissue sarcoma (Kogevinas et al., 1995), lung sarcoma (Blair et al., 1983), pancreatic, stomach, liver, bladder and gall bladder cancer (Ji et al., 2001; Shukla et al., 2001), Parkinson disease (Jenner, 2001; Sherer et al., 2001), Alzheimer disease (Gauthier et al., 2001) and reproductive outcomes (Arbuckle et al., 2001), among others.
In view of these findings, the detection of populations at risk constitutes a very important topic. In this context, it must be pointed out that cytogenetic markers such as chromosomal aberrations (CA), sister chromatid exchange (SCE), micronuclei (MN) and, recently, single cell gel electrophoresis (SCGE) have been extensively used for the detection of early biological effects of DNA-damaging agents. Regarding pesticide exposure, many reports dealing with CA (Amr, 1999; Au et al., 1999; Antonucci and de Styllos Colus, 2000; Zeljezic and Garaj-Vrhovac, 2001), SCE (De Ferrari et al., 1991; Garaj-Vrhovac and Zeljezic, 2001; Shaham et al., 2001) and SCGE (Garaj-Vrhovac and Zeljezic, 2000; Zeljezic and Garaj-Vrhovac, 2001) found significant increases in these biomarkers, providing suggestive evidence of genotoxic effects induced by pesticides.
MN are formed by the condensation of acentric chromosomal fragments or by whole chromosomes lagging behind the cell division. This is the only biomarker that allows the evaluation of both clastogenic and aneuploidogenic effects in a vast range of cells, since they are detected in interphase. The sensitivity and reliability of the MN assay in human lymphocytes, by blocking cytokinesis with cytochalasin B (Cyt B), has been shown to be an effective tool to measure cytogenetic damage by pesticides in several populations (Bolognesi et al., 1993; da Silva Augusto et al., 1997; Joksi
et al., 1997; Falck et al., 1999). In addition, this assay also allows the detection of effects on cell proliferation and cytotoxicity. Moreover, MN can be evaluated in different kinds of cells that do not necessarily have to divide in vitro (such as epithelial cells), thus, the analysis of MN in exfoliated buccal cells has been demonstrated to be a sensitive method for monitoring genetic damage in human populations (Sarto et al., 1990; Karahalil et al., 1999). Nevertheless, few studies on pesticide-exposed populations have been carried out using buccal cells and, from the available data, only one has found a positive relationship with exposure (Gómez-Arroyo et al., 2000).
In the present study, to assess whether prolonged exposure to complex mixtures of pesticides leads to an increase in cytogenetic damage, human peripheral lymphocytes and buccal epithelial cells were analysed using the MN assay. This is a large study in which agricultural workers from four different European countries were included. We previously evaluated these populations in separate studies (Lucero et al., 2000; Pastor et al., 2001a,b, 2002) within the frame of a European project. This paper does not try to summarise the results already reported, but aims to present a global analysis of the populations included in the project, the goal of which was to determine if pesticide exposure is reflected in an increase in cytogenetic damage.
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Subjects studied
A total of 478 individuals from four European countries were selected for the study. Of these, 247 were agricultural workers exposed to pesticides and 231 were controls. Their origins were: 50 exposed and 66 controls from an area outside Athens, called Nea Makri, in Greece; 63 exposed and 51 controls from the province of Almería in Spain; 50 exposed and 49 controls from Malopolska, a region of southern Poland; 84 exposed and 65 controls from south east Hungary. With the exception of Greece and Hungary (with 20 and 26 exposed and 25 and 12 control women, respectively), the rest of the population studied was composed of men.
Prior to the study, all the individuals signed an informed consent form and filled in a detailed questionnaire enquiring into information about possible confounding factors such as age, gender, smoking and drinking habits, vaccination, medication, X-ray examinations and diet. In the case of the exposed group, occupational activity, years of agrochemical exposure, main pesticides used, kinds of crops, protective measures used, etc., were also recorded. The main characteristics of the study population are listed in Table I. It is necessary to emphasise that due to extrinsic factors some data were missing, thus the final number of individuals analysed was lower, 457 individuals for MN scoring in lymphocytes and 441 individuals for MN analysis in buccal cells.
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With regard to smoking habit, individuals were classified as non-smokers, when they had never smoked or had quit smoking more than 5 years ago, as ex-smokers, if they had stopped smoking between 1 and 5 years before sampling, and current smokers. A particular characteristic of the Greek population is that it was constituted only of non- and ex-smokers.
All the agricultural workers were regularly exposed to complex mixtures of pesticides that differed depending on region, climate and kind of crop. Nevertheless, carbamates, organophosphates and pyrethroids were the most used families of pesticides (Table II). The farmers worked mainly in greenhouses, although the Polish and Hungarian cohorts also worked in open fields. The principal crops were vegetables and ornamental plants. Pesticide application was usually carried out from above the head in Greece, Spain and Poland and under the head in Hungary. Almost 80% of the pesticide-exposed workers asserted use of some kind of protection during the preparation and application of pesticides; in Spain and Poland they usually used more than one protective measure (gloves, breathing masks, glasses, impermeable boots, etc.). In spite of that, 21.5% of them had suffered recent pesticide intoxication. Most of these intoxications were by dermal contact and inhalation and manifested as dermatitis, eczema and irritability of mucous membranes (eyes and nose).
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The control individuals carried out clerical and health care jobs in the same village or region from which the exposed individuals came. None of them had recent exposure to agrochemicals or other suspected genotoxic agents and they had no previous occupational exposure to genotoxicants.
Taking into account the heterogeneity of the populations studied, in this work we have carefully considered a wide range of external confounding factors that might influence the results. It should be noted that in the previous studies some variables were not included, in spite of their relevance (e.g. X-rays, miscarriages, etc.), due to a lack of data for some of the populations.
The data reported here correspond to blood and buccal samples collected during 1998.
Lymphocyte cultures, staining and binucleated cells with micronuclei (BNMN) scoring
Blood samples were obtained from each subject by venipuncture in heparinized vacutainers. Samples from Nea Makri (Greece) and Almería (Spain) were sent to the Universitat Autònoma de Barcelona (Spain) within 24 h, where they were immediately processed. Lymphocyte cultures from samples collected in Malopolska (Poland) were set up in the laboratory of the Department of Environmental and Radiation Biology (DERB) of the H.Niewodniczanski Institute of Nuclear Physics (Kraków), and the blood samples from Hungary were processed in the B.Johan National Center for Epidemiology in Budapest. The same standardised protocol was used in all participating laboratories.
Lymphocyte cultures were set up by adding 0.5 ml of whole blood to 4.5 ml of RPMI 1640 medium supplemented with 15% heat-inactivated fetal calf serum, 1% antibiotics (penicillin and streptomycin) and L-glutamine. Lymphocytes were stimulated with 1% of phytohaemagglutinin and incubated for 72 h at 37°C. Two cultures per subject were established. A final concentration of 6 µg/ml Cyt B (Surrallés et al., 1994) was added to the cultures 44 h later to arrest cytokinesis. At 72 h incubation, the cultures were harvested by centrifugation at 800 r.p.m. for 8 min and treated with a hypotonic solution (2–3 min in 0.075 M KCl at 4°C). The cells were then centrifuged and a methanol:acetic acid (3:1 v/v) solution was gently added. This fixation step was repeated twice and the resulting cells were resuspended in a small volume of fixative and dropped onto clean slides.
The slides of all samples were stained and scored in the Laboratory of Mutagenesis, Department of Genetics and Microbiology, Universitat Autònoma de Barcelona. They were stained with 10% Giemsa in phosphate buffer (pH 6.8) for 10 min. Following the criteria proposed by Fenech (1993) to determine the frequency of BNMN and the total number of MN in lymphocytes (MNL), a total of 1000 binucleated cells with well-preserved cytoplasm (500 per replicate) were scored per subject on coded slides. In addition, 500 lymphocytes were scored to determine the percentage of cells with one to four nuclei and the cytokinesis block proliferation index (CBPI) was calculated according to Surrallés et al.(1995). To avoid differences between observers, the same individual carried out all the microscopic analyses.
Buccal cell procedure, staining and MN scoring
Buccal cell samples were obtained by rubbing the inside of the cheeks with a toothbrush. The cells were collected in a conical tube containing 20 ml of buffer solution (0.1 M EDTA, 0.01 Tris–HCl and 0.02 M NaCl, pH 7) and immediately transported to the respective laboratory for further processing (Athens, Barcelona, Kraków or Budapest). After three washes in buffer solution followed by centrifugation at 1500 r.p.m. for 10 min, 50 µl of adequate cell suspension density was dropped onto preheated (55°C) slides and allowed to air dry for 15 min on a slide warmer. The slides were fixed in 80% cold methanol for 30 min and air dried overnight at room temperature. Next, the slides were sent to Barcelona where they were stored at -20°C until use. They were stained with a DNA-specific stain, namely 1 µg/ml 4',6-diamidino-2-phenylindole dihydrochloride (DAPI), that avoids possible scoring artefacts. A total of 2000 cells/donor were scored, on coded slides, by one scorer under an Olympus BX50 fluorescence microscope. The criteria for MN evaluation were those suggested by Titenko-Holland et al. (1998). The frequency of mononucleated buccal cells with micronuclei (BCMN) and the total number of micronuclei in buccal cells (MNBC) were determined for each subject studied.
Statistical method
The statistical computations were performed using the SPSS v.10.0 software (SPSS, Chicago, IL) and the SAS system for Windows, v.8.0 (SAS, Cary, NC). Student’s t-test, ANOVA and the 
2 test were used to compare means and frequencies for demographic, dietary and habit factors, between populations, exposures and sexes.
The cytogenetic variables BNMN and CBPI were analysed using a generalized linear model (GLZ). All variables that could have any influence on the results were included in the analyses (age, sex, exposure, country, diet, cigarettes, etc.). Post hoc comparisons using Tukey’s correction were also done. The BNMN data were square root transformed to achieve all the requirements of the method. The cytological variable BCMN, scored in buccal cells, was first studied by Poisson regression, but due to the high dispersion found, a negative binomial regression analysis was finally carried out. A backward selection method was used in all cases (BNMN, CBPI and BCMN) as an exploratory method. The most important variables as well as the main interactions were taken into account.
The type III sum of squares method was used because it is a test of effects after controlling for all other factors and, in addition, it is easily interpreted. P values correspond to two-sided tests and an 
error < 0.05 was considered the significance level.
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Results |
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The main characteristics of the farm workers and controls from the four populations studied are presented in Table I
. This table also indicates those dietary characteristics that can act as potential confounding factors on the MN frequency analysis.
Regarding average age, a slight statistically significant difference (P = 0.004) can be observed between controls and exposed, nevertheless, the small average difference does not represent biological significance. Differences between sexes were only appreciable in the exposed group, in which the women were older than the men (P = 0.002). Significant age differences between countries were also observed in the analysis of exposed men, the Spanish agricultural workers being the youngest (P 
0.001).
Two different groups can be established with regard to years of occupational exposure to pesticides. On the one hand, the Mediterranean subjects (Greece and Spain) and, on the other, those from Middle Europe (Poland and Hungary). The average period working in agriculture for the farmers in the first group was ~9 years and, therefore, they were exposed for fewer years than those in the second group, who had been occupationally exposed to chemical pesticides for 16–18 years. Such a difference is highly statistically significant (P 0.001).
In relation to alcohol consumption (g/week), the values obtained for the controls and exposed indicated similar behaviour, nevertheless, differences were clearly detected between populations and between males and females. Thus, in the Greek population alcohol consumption in controls was higher than in exposed farmers, whilst in the Hungarian population the tendency was the opposite. Differences were observed between men and women (P < 0.001), men drinking more.
Tobacco is a well-known factor that can influence the level of genotoxic damage. The Greek population did not include current smokers, thus an elevated number of ex-smokers was studied. This led to statistically significant differences when compared with the other populations for both controls and exposed. All smokers consumed similar numbers of cigarettes per day. Among the smokers, there are more men than women, in both the control (P = 0.0003) and exposed (P = 0.002) groups.
Taking into account that the studied populations came from different countries, which can be expected to have different dietary habits, the effect of several dietary factors on MN frequency has been studied. Thus, the frequency of ingestion of red meat per week could be differentiated into two groups, the Mediterranean population (Greece and Spain) and the Middle European (Poland and Hungary). This second group was a greater consumer of red meat. A significantly increased consumption of red meat was found in exposed versus controls (P = 0.007). On the other hand, fish intake was the reverse, the Greek and Spanish populations being those with a high fish intake. The low level of fish consumption in the Hungarian population was remarkable. Concerning the ingestion of raw vegetables per week, a significant difference was only found between the Spanish and Polish controls (P = 0.005), while the consumption of cooked vegetables was very heterogeneous and significant differences were found between populations and between controls and exposed (P = 0.015). Finally, fruit consumption per week did not differ between the four populations, including men and women, with the exception of exposed men, Hungarians having a significantly greater consumption of fruit than Spaniards (P < 0.0001) and Poles (P = 0.029).
As can be observed in Table II, each population used their own pesticides. Different factors have influenced the choice of each product (kind of crop, weather conditions, pests, etc.) and although slight differences can be seen in the compounds used, generally the main chemical families that the pesticides belong to are the same. Carbamates are used at approximately the same frequency in each population; pyrethroids differed especially between Mediterranean and Middle European countries, the latter with a high percentage of use. Regarding organophosphates, the only difference found was in Hungary, where the percentage use was higher. The use of antibiotics in Spain should be mentioned.
Table III shows the means of the cytogenetic variables evaluated. All statistical analyses take into account dietary and demographic factors and tobacco and alcohol habits. However, due to the lack of significance of some variables, they were not taken into account (backward method), although others were retained in the study for their apparent interest. The results of the GLZ final models for BNMN and CBPI are summarised in Table IV. It is observed that exposure to pesticides does not induce any significant increase in the frequency of BNMN (Figure 1), nevertheless, age shows a strong, positive significant effect over BNMN (P < 0.0001, B = 0.014), which means that the frequency of MN increases with the age of individuals. It must be mentioned that all figures show the least squares means (ls means), corresponding to the mean adjusted for the other terms in the model.
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There were differences between populations due to the fact that two of them included women, so we created a new variable that includes both the country and the sex of individuals, termed CS (country–sex). CS was introduced into the model as a random factor (like the other variables). CS seemed to have a significant influence on BNMN. The Polish population (all men) was the group with highest levels of BNMN, showing significant differences with respect to Greeks, Spaniards and Hungarians (men and women) (Figure 2
). Greek women showed higher levels of cytogenetic damage than Spanish (P = 0.01); Hungarian men had the lowest damage level, being significantly lower than the levels found in Greek men and women (P = 0.02 and 0.001). There were no significant differences between men and women in the Greek and Hungarian populations and also between women.
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Other variables were the interactions that we considered of interest because they could contribute to a better understanding of the results. When the interaction CSxExposure was taken into account, the Polish population continued to show significant BNMN differences regarding all possible combinations (control, exposed, men and women) with the exception of Greek and Hungarian control women (Figure 3
). No differences were found between controls and agricultural workers from Poland. The CSxAge interaction in the BNMN model indicated that the age effect was accentuated in women from Greece.
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Table IV
also shows the results for CBPI. Thus, the group occupationally exposed to pesticides showed a significantly lower CBPI compared with the controls (Figure 4). On the other hand, CBPI was inversely correlated with age. In the final model selected for analysis of CBPI, CS and the interaction CSxExposure were included. Two blocks were clearly differentiated according to sex and geographic area; on one side, Greeks and Spaniards, on the other, Poles and Hungarians. Differences between men were found for the different populations, and also for women. Mediterranean people showed significantly greater levels of CBPI than Middle European people (P < 0.0001, for all possible combinations) (Figure 5), although the Hungarian CBPI levels were significantly lower than those of Poles (men, P < 0.0001; women, P = 0.0017). No differences between sexes were found in the Greek and Hungarian populations. The results for the interaction CSxExposure showed, as indicated before, significant differences between countries. Greeks and Spaniards had higher CBPI values than Poles and Hungarians, independent of sex (P < 0.0001) (Figure 6). Greek control men showed the higher CBPI values, being significantly different from Greek exposed men (P = 0.0005) and Spanish control men (P = 0.02). Hungarian exposed men, who had the lowest CBPI levels, showed significant differences compared with Polish (controls and exposed, P < 0.0001) and Hungarian controls (men, P < 0.0001; women, P = 0.001) and Hungarian exposed women (P = 0.01). Hungarian exposed women also showed significant differences compared with Polish (exposed, P = 0.009; controls, P = 0.0001).
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Regarding BCMN, no differences were found between the agricultural workers and controls (Table V
and Figure 7). The CS variable revealed that the Spanish population differs significantly from the other populations (Figure 8). Differences were also observed between Hungarian control men and women (P = 0.049) (Table V). Similar differences were found when exposure and CS were studied together, confirming that Spaniards, independently of their exposure, showed significantly higher levels of MN in buccal cells than the other populations studied (Figure 9).
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Alcohol did not influence the frequency of BCMN, even when included as an interaction with gender (data not shown). No differences were obtained when the other variables were introduced.
It must be recalled that to study the possible effect of tobacco, the data from Greece had to be removed from the analysis since this population lacked smokers. Thus, when smoking habit is included in the GLZ analysis of BNMN, BCMN and CBPI, the results did not change in significance. Consequently, smoking habit did not affect the parameters evaluated (BNMN, P = 0.513; BCMN, P = 0.180; CBPI, P = 0.303).
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Discussion |
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In summary, the results of this study indicate that the four populations of agricultural workers occupationally exposed to pesticides do not reveal a significant induction of cytogenetic damage, as measured by the MN assay in both lymphocytes and buccal epithelial cells. Although several studies have also reported a lack of cytogenetic effects as a consequence of occupational exposure to pesticides (Hoyos et al., 1996
; Davies et al., 1998; Venegas et al., 1998; Lander et al., 2000; Pastor et al., 2001a,b, 2002), many others have demonstrated the induction of cytogenetic damage, indicating that MN frequency is a highly effective biomarker in revealing the association between chromosome damage and pesticide exposure (da Silva Augusto et al., 1997; Figgs et al., 2000; Garaj-Vrhovac and Zeljezic, 2000, 2001; Gómez-Arroyo et al., 2000; Shaham et al., 2001).
In our study, a high degree of heterogeneity was observed between the different populations studied, for the frequencies of both BNMN and MNL (Table III). This heterogeneity is in agreement with the results of other studies, which also showed differences in MN frequencies. Although the reported average baseline frequency of MN in human lymphocytes was 7.8 ± 5.2 per 1000 cells (ranging from 3 to 23), with age and sex but not smoking as main confounding factors (Surrallés and Natarajan, 1997), the average in our control group was higher (12.25 ± 0.60). However, this value does not differ by much from that reported by Venegas et al.(1998) (BNMN, 10.69 ± 2.08) and it is clearly lower than the values found by Titenko-Holland et al. (1997) (BNMN, 18.7 ± 7.6) and by Davies et al. (1998) (BNMN, 21.76 ± 1.50).
In principle, these differences can be attributed to methodological aspects, sample manipulation and/or scoring. However, in our study the samples from Greece and Spain, which are the ones that showed the biggest differences in BNMN frequency, were both cultured in Barcelona and scored by the same person in a blind study. In other biomonitoring studies carried out in Barcelona the levels of BNMN in controls were 20.81 (Pitarque et al., 1996), 22.14 (Gutiérrez et al., 1997) and 10.55 (Pitarque et al., 1999), again corroborating the existence of inter-individual and inter-population differences.
As previously reported (Fenech and Morley, 1986; Migliore et al., 1991; Fenech et al., 1994; Davies et al., 1998; Fenech, 1998; Falck et al., 1999), age was strongly associated in a positive way with MN frequency (Table IV). Our overall data show a significant increase of 0.014 MN/1000 binucleated lymphocytes/year. In our study the controls were not age matched to the exposed group, being a little bit older, and this could influence the results. On the other hand, the Spanish group, which was the youngest, showed lower levels of BNMN, which supports the previous findings.
Polish individuals, whether controls or exposed, showed significantly higher levels of BNMN. It can be seen (Table III) that the BNMN and MNL levels of Poles were higher than the rest, with the only exception being Greek and Hungarian control women, who also showed high BNMN and MNL levels. This could be associated with age, but the Poles did not stand out with regard to this characteristic (Table I). This hypothesis would only explain the case of Hungarian women, because they were the oldest; nevertheless, this assumption is not congruent, since Greek control women had more cytogenetic damage but were younger than the Hungarians. Control women did not show any other remarkable characteristics, all of them having common occupations, basically housewives and administrators, and none of them having been chronically exposed to genotoxic agents.
Why did Polish people have higher BNMN values? This higher frequency does not apply only to the individuals occupationally exposed to pesticides, but also to the controls, thus, intrinsic factors may be acting. The Polish group did not show remarkable differences concerning alcohol consumption, pesticide used and smoking habit with respect to the other groups, although differences regarding Mediterranean people were observed with regard to red meat consumption, Poles being the ones who consumed more red meat. Other genetic and/or environmental factors may account for the observed BNMN values in the Polish group.
With regard to the buccal cell study, a lack of an increase in BCMN in the agricultural workers occupationally exposed to pesticides was found. To our knowledge, there is only one study in which a significant increase in MN in buccal cells was found (Gómez-Arroyo et al., 2000), however, those authors found high levels of cytogenetic damage in both controls and exposed (3.8 and 10 MN/1000 cells, respectively). A wide variation in the number of buccal cells with MN has been reported elsewhere. Thus, a wide variation has been found in different control populations, the values ranging from 0.3–0.4
(Sarto et al., 1987, 1990; Tolbert et al., 1992; Rosin et al., 1994; Karahalil et al., 1999) to 2.7 (Livingston et al., 1990), 4.7 (Stich and Rosin, 1983) and 8.4 cells with MN (Özkul et al., 1997).
If we look at the differences between countries, taking into account sex (CS), the Spanish men (both controls and exposed) appeared to have greater levels of BCMN. Why? The only remarkable aspects that could affect the frequency of BCMN which differed from the other populations studied were alcohol and cigarette consumption. Some studies have found a relationship between alcohol consumption and alterations in the normal oral mucosa (apoptosis, reduction in area, keratinization, etc.) as well as increases in MN in epithelial buccal cells (Kassie et al., 2001). On the other hand, some studies (Surrallés et al., 1997; Bloching et al., 2000) found that alcohol did not influence the frequencies of MN.
Smoking is reported to increase the MN frequency in buccal cells (Sarto et al., 1987; Piyathilake et al., 1995; Kiilunen et al., 1997). However, other studies found that smoking is not reflected in an increase in MN in buccal cells (Machado-Santelli et al., 1994; Torres-Bugarín et al., 1998; Burgaz et al., 1999). From our data it is clear that the Spanish group had the highest percentage of smokers (60.7% of the controls, 55.5% of the exposed); the Polish group had 57% and 36% and the Hungarians 20% and 35.7%, for controls and exposed, respectively. Thus, although in our study smoking is a suggestive factor to explain the high frequency of BCMN in the Spanish group, the statistical analysis does not indicate that MN formation is influenced by cigarettes smoked (P = 0.18, taking into account only smokers; data not shown) and/or alcohol consumption.
An interesting finding in the overall population studied is that pesticide exposure seems to be capable of inducing alterations in the cell proliferation kinetics, suggesting that such exposure induces both a cell cycle delay and a reduction in the proliferation of lymphocytes (CBPI). As indicated by the interactions, the exposed Hungarian men, having the lowest proliferation index, differed significantly from the rest. Other studies (Amorim et al., 2000) have also found a decrease in the mitotic index in men, although in our case this decrease was related to pesticide exposure. Cell proliferation delay due to pesticide exposure has also been previously reported (Rupa et al., 1991; Pasquini et al., 1996), however, other authors observed no such delay or even an acceleration of the cell cycle. The fact that it was the exposed men who showed lower CBPI levels may be related to the different types of activities they carried out (mainly application of pesticides) when compared with women (mainly harvesters). It must also be noted that the samples were not balanced for sex.
On the other hand, a relevant aspect found in this study is the significant difference between the Mediterranean and the Middle European countries. The first group (Greece and Spain) have similar CBPI values, being greater than in the second (Poland and Hungary). Furthermore, the Hungarian agricultural workers had been exposed to pesticides for more years.
To explain the cell cycle delay, it has been hypothesised that chronic low level exposure to toxins, such as pesticides, may induce an adaptive response related to an increase in apoptosis sensitivity and/or a more extended cell cycle delay that enables appropriate repair (Kirsch-Volders and Fenech, 2001). Another factor to be considered is the negative effect of tobacco on lymphocyte proliferation (Amorim et al., 2000; McCue et al., 2000), however, our results showed that smokers did not differ with regard to CBPI when compared with non-smokers or ex-smokers. Finally, we found that age was significantly and negatively associated with CBPI, showing a decrease in cell proliferation index with age. Studies on cell proliferation kinetics have also found a negative correlation of replication index and cell proliferation rate with age (Lazutka et al., 1994).
From the present study, based on four European populations, we can conclude that occupational exposure to pesticides, related to the particular agricultural activities of these areas, does not increase the level of cytogenetic damage when evaluated by the MN assay using peripheral blood lymphocytes and buccal epithelial cells. These results might be surprising taking into account the fact that the four agricultural groups were selected for their high and continued exposure to pesticides, most of them working in greenhouses. It is important to emphasise the working conditions of the individuals studied: 80% of the agricultural workers reported the used of protective measures. This fact, together with the relatively low genotoxic potency of the pesticides used (Table II), might be the reason for the lack of a detectable increase in MN frequency in the agricultural workers. However, an effect of exposure was observed for CBPI, indicating some cytotoxicity due to exposure. Perhaps the mode of action of the chemicals does not involve DNA, but other targets.
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Acknowledgments |
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We are grateful to Laboratorios Lacer S.A. (Barcelona) for kindly giving us the toothbrushes. We thank G.Umbert and A.Corral for their expert technical assistance. The secretarial skills of M.McCarthy and the advice of the Servei d’Estadística (UAB) in the data analysis are much appreciated. This investigation was supported in part by the European Union (CT96-0300, INCO-COPERNICUS).
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Notes |
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5 To whom correspondence should be addressed. Tel: +34 93 581 20 52; Fax: +34 93 581 23 87; Email: ricard.marcos{at}uab.es
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References |
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