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1.

Evaluation of cell types for assessment of cytogenetic damage in arsenic exposed population
Abstract
Background
Cytogenetic biomarkers are essential for assessing environmental exposure, and reflect adverse human health effects such as cellular damage. Arsenic is a potential clastogen and aneugen. In general, the majority of the studies on clastogenic effects of arsenic are based on frequency of micronuclei (MN) study in peripheral lymphocytes, urothelial and oral epithelial cells. To find out the most suitable cell type, here, we compared cytogenetic damage through MN assay in (a) various populations exposed to arsenic through drinking water retrieved from literature review, as also (b) arsenic-induced Bowen's patients from our own survey.

Results
For literature review, we have searched the Pubmed database for English language journal articles using the following keywords: "arsenic", "micronuclei", "drinking water", and "human" in various combinations. We have selected 13 studies consistent with our inclusion criteria that measured micronuclei in either one or more of the above-mentioned three cell types, in human samples. Compared to urothelial and buccal mucosa cells, the median effect sizes measured by the difference between people with exposed and unexposed, lymphocyte based MN counts were found to be stronger. This general pattern pooled from 10 studies was consistent with our own set of three earlier studies. MN counts were also found to be stronger for lymphocytes even in arsenic-induced Bowen's patients (cases) compared to control individuals having arsenic-induced non-cancerous skin lesions.

Conclusion
Overall, it can be concluded that MN in lymphocytes may be superior to other epithelial cells for studying arsenic-induced cytogenetic damage.

Background
Exposure to inorganic arsenic through drinking water results in cancers of skin, urinary bladder, liver and the lungs. Cytogenetic assays play an important role in toxicological hazard evaluation as the first step towards quantification of cancers. Various genetic toxicological end-points have been used as biomarkers to understand the biological effects of arsenic exposure [1]. Biomarkers serve as internal indicators of environmental or occupational exposures and have the potential for prevention of effects of carcinogen exposure by early detection [2]. Micronuclei are the potential biomarkers and traditionally been used for biomonitoring of genotoxic effects in humans [3]. Micronuclei are chromosomal fragments or the whole chromosomes that are not included in to the daughter nuclei during cell division and are incorporated as much smaller nuclei. The formation of MN is therefore induced by substances that cause breakage of chromosomes (clastogens) as well as by agents, which affect the spindle apparatus (aneugens). The speed and ease of MN analysis, and non-requirement of metaphasic cells made this assay very popular.

In West Bengal, India, the ground water in nine out of eighteen districts is heavily contaminated with arsenic. More than 6 million people are endemically exposed to inorganic arsenic in their ground water, which exceed the maximum contamination level of 10 g/l [4,5]. Although only 1520% of the population exposed to arsenic shows clinical features of chronic arsenic poisoning, the remaining individuals are also at risk of developing arsenic-induced health effects [6]. Therefore, genetic monitoring of population exposed to arsenic through drinking water is important. Numerous population-monitoring studies have been carried out in various arseniasis endemic countries such as Mexico, Finland, Taiwan and Argentina to determine genotoxic potential of arsenic [1,7-9]. MN assay are conducted in lymphocytes, urothelial, and buccal mucosal cells. Cytokinesis-block micronucleus test (CBMN) in human peripheral blood lymphocytes is a standard cytogenetic test for genetic toxicology testing [10]. The use of exfoliated cells for MN assays has become well established in epidemiological studies aimed at defining genotoxic effects on target tissue following chronic exposure to epithelial carcinogens [2]. We have been working on the mutagenic and genotoxic effects of different drugs, chemicals and environmental toxicants, including arsenic [11-16]. Last five years we have been working on the assessment of cytogenetic damage as measured by MN assays in three cell types i.e. lymphocytes, urothelial cells and buccal mucosa cells in the population exposed to arsenic through drinking water in West Bengal, India. Peripheral lymphocytes can be used as surrogate target cells [17]. Collection of lymphocyte is socially and ethically acceptable, and involve minimum invasive route. The cytokinesis-block micronucleus (CBMN) technique in lymphocyte culture is widely regarded as a sensitive and reliable method for assessing chromosome damage [18]. It is based on the use of cytochalasin B, an inhibitor of actin polymerization, which blocks mitotic cytokinesis without preventing nuclear division. In this assay MN are scored after a single cell division using binucleated lymphocytes to eliminate the confounding effect of altered cell division kinetics on the MN index [18]. Exfoliated epithelial cells have traditionally been used for cancer screening and bio-monitoring of genotoxic effects in human [3]. Large number of these cells can be rapidly and non-invasively collected from study participants. The frequencies of MN observed in exfoliated cells of buccal mucosa and urinary bladder serve as an appropriate index to monitor the genotoxicity induced by arsenic because these cells are in direct contact with the carcinogen [2]. MN in urothelial cells reflects damage to the bladder epithelial tissue, which occurs approximately 1 3 weeks prior to the exfoliated cells appearing in urine [19]. Although all three-cell types provide important information about cytogenetic damage, selection of an appropriate cell type is important. On one hand, life span of lymphocytes is higher than that of the other two exfoliated cell types, and on the other hand, turn over of exfoliated epithelial cells is variable, therefore, it is reasonable to assume that lymphocytes might yield higher MN count/1000 cells compared to other exfoliated cells. We earlier reported MN counts in three different cell types from the same individual. In this paper, we extend the research based on our systematic literature review and comparing the summary data with findings from our earlier research. We have also conducted a matched case-control study taking arsenic-induced Bowen's patients as cases and age-sex matched individuals with arsenic-induced noncancerous skin lesion as control. The purpose is to study (a) which cell type show stronger effects on arsenic exposure and (b) whether this cell type is also similarly informative for cancer patients.

Search strategy & inclusion/exclusion criteria


We searched the Pubmed literature database using the following keywords in various combinations: "arsenic", "micronuclei", "drinking water", and "human" [20]. We restricted our selection of studies to those published in English language only since we did not have facilities for translations nor did we have access to translation services. Initially, titles and abstracts were scanned to identify whether the study met the minimum inclusion criteria to be retained for review. We selected only case-control studies. If the study met the minimum criteria, then full text of the study was obtained. Data were abstracted from each study to a pre-defined database and the database was prepared for analysis. The reference section of each study was scanned to identify other similar articles. A study was included in our review if it contained the following information as minimum: clearly written methods section, unbiased or minimally biased measurement of arsenic exposure, micronuclei counts per 1000 binucleated cells either in one or more of the three cell types, such as- lymphocyte, urothelial and buccal mucosa cells, and clear presentation of the results in the format of difference in effect sizes of MN per 1000 cells. Where the counts were not reported per 1000 binucleated cells, if the study met the other criteria and was accepted, we converted the figures to reflect changes per 1000 cells and rounded off the decimal points to two places after zero. Studies with non-human samples were also excluded.

Analysis of literature review


Our literature search retrieved initially a list of 20 publications. After filtering through inclusion and exclusion criteria, we found 13 studies to be eligible (Table 1).
Table 1. Micronuclei in three different cell types for all the studies included in the review

Lymphocyte as a cell type for MN assay


To our knowledge, only five studies document lymphocyte as a cell type for arsenic-induced MN formation. About 5 fold increase in MN lymphocytes was observed among native children and women of northwestern Argentina exposed to high level of arsenic via drinking water in contrast to controls by Dulout et al., 1996 [9]. Higher number of MN formation in lymphocyte due to arsenic exposure was observed from the studies of Martinez et al., (2004) in the population of Northern Chile [21]. Since a large number of individuals are exposed to arsenic in West Bengal, India, three separate studies were conducted from different region of West Bengal, India and it was observed that, MN in lymphocyte is much higher in cases compared to control [15,16,22].

Urothelial as a cell type for MN assay


Relatively more studies were performed with urothelial cell for MN assay. Studies by Warner et al (1994) in a population exposed to arsenic in Neveda, USA showed about 1.8 fold increase in the mean frequency of micronucleated urothelial cells compared to control [23]. Using advanced technology for detecting MN by fluorescent method, Moore et al (1996) observed similar result for urothelial cells [24]. Further, they have showed higher prevalence of MN in urothelial cell in cases compared to control, in Chilean population [25]. To find out the relationship of urinary arsenic (an estimate for arsenic intake) and a biomarker of effect, prevalence of MN was found to be 2 fold higher in cases compared to control [26]; while Gonsebatt et al. (1997) have shown 4.65 fold increases [7]; and Tian et al (2001) observed about 3 fold increases in mean MN count for urothelial cells in cases [27]. All the three studies from our group strongly support increase mean MN count in urothelial cell in arsenic exposed cases compared to control [15,16,22].

Buccal mucosa as a cell type for MN assay

Gonsebatt et al. (1997) have shown about 3.8 fold increase in MN in buccal cells in exposed individuals compared to unexposed [7]. Similar inference was also obtained from the studies of Tian et al. [27]. MN frequency in buccal cells of arsenic exposed population from Antofagasta region, North Chile was shown to be higher compared to the referent individuals from Concepcion, Chile; but not statistically significant [28]. Again, a recent study by Chakraborty et al. (2006), on 45 arsenic exposed individuals from West Bengal revealed 3.34 fold increases in MN in buccal mucosa cells compared to 25 controls studied [29].

Finding best suitable cell type


From the reported data in each study, first, effect sizes were calculated as follows. Effect size was defined as the difference in the mean value of the cases versus controls. The mean value of micronuclei count per 1000 for controls were deducted from the mean value of the micronuclei per 1000 for cases, and the difference was identified as the effect size for the specific study. Effect sizes were calculated for all studies identified by the previous search strategy. All studies did not calculate the values for micronuclei counts for all three-cell types in the same individual. In that case, we treated non-reported data as missing for that study. Since this review was not a meta-analysis, but a structured mini-review of observational studies, we did not conduct formal tests of heterogeneity or homogeneity of studies for this review. We plotted the medians of the effect sizes of each cell type as individual bar charts to show the visual differences in effect sizes among the three cell types. We reported further pairwise comparisons for the three combinations: lymphocyte versus urothelial cells, lymphocyte versus buccal cells, and urothelial cells versus buccal cells. We used the nonparametric Wilcoxon rank sum tests for the pairwise comparisons with a pre-specified significance level of 0.05. We conducted the tests for two sets of studies: (i) all studies taken together and (ii) studies that were reported from our research group. Wilcoxon rank sum test for the pairwise comparison for median effect size (based on MN counts) in three cell types for the full set of studies (Table 2) as also studies conducted by our group (Table 3) was calculated. Reviewing all the studies, it was observed that, lymphocyte is a better candidate compared to other two (Fig. 1). Similar inference was observed from the studies of our group, where MN counts were taken for all the 3 cell types for each individual. The data also supports that lymphocyte is a better cell type compared to other two (Fig. 2).
Table 2. Wilcoxon rank sum test for the pairwise comparison for median effect size (based on MN counts) in three cell types for the full set of studies Table 3. Wilcoxon rank sum test for the pairwise comparison for median effect size (based on MN counts) in three cell types for the studies conducted by our group

Figure 1. Comparison of median effect size (with error bar) based on micronuclei counts among lymphocytes, urothelial, and buccal mucosa for all the studies in the review.

Figure 2. Comparison of median effect size (with error bar) based on micronuclei counts among lymphocytes, urothelial, and buccal mucosa for the subset data (set of studies from our group).

Is the observation also true for arsenic-induced Bowen's (in situ carcinoma) patients?

To evaluate whether the best suitable cell type suggested from literature review, also applicable in arsenic-induced Bowen's cases, we considered 25 cases of histopathologically confirmed arsenicinduced Bowen's patients from our epidemiological survey [30] and conducted a matched case-control study taking non-cancerous skin lesions individuals as control. These histologically confirmed 25 cases of Bowen's disease (designated as "case") were pair wise matched with 25 cases of non-cancerous arsenicosis individuals (designated as "control") on age, gender and smoking status (Table 4). The selection of control individuals was done via the generation of random numbers using MS-Excel spreadsheet program. Micronuclei results for all three cell types of these Bowen's cases were not reported elsewhere.
Table 4. Comparison of Micronuclei in three different cell types in arsenic exposed cases (Bowen's patients) and control (individuals with non- cancerous arsenic induced skin lesions)

Comparing the mean MN count for three cell types between cases and controls, we found MN count was significantly increased in cases for all the cell types, i.e. in lymphocyte, urothelial and oral mucosa cells (Table 4). To our surprise, MN count in lymphocyte was 1.9 fold higher compared to oral mucosa cells and 1.6 fold higher compared to urothelial cells, in both cases as well as control (Table 4).

Conclusion
All the studies on arsenic exposed populations clearly demonstrated a significant genotoxic effect in lymphocytes as well as exfoliated epithelial cells. We have found that overall, compared to urothelial cells and buccal mucosal cells, the median effect sizes for lymphocyte based MN counts were stronger. This inference was further supported by the general pattern pooled from other 10 studies conducted by different groups. Lymphocyte based MN count was also stronger for arsenic-induced Bowen's patients. Therefore, it can be inferred that lymphocyte is the most suitable cell type for studying cytogenetic damage. The findings from our review need to be interpreted in the light of its several limitations. We considered here only studies in English language. There may also be additional unexplained variabilities, including scoring variability among different study groups. Exposure level of the study groups also varies considerably, which might influence in outcome. It has been known from in vitro studies that, MN prevalence increase along with exposure but again return to baseline when, exposure is highest, possibly, due to inhibition of MN formation at high doses due to cytotoxicity/cytostasis [31,32]. Arsenic generates reactive oxygen species (ROS) including peroxy radical, superoxide radical, and hydroxyl radical and these in turn cause DNA damage [33]. Moreover, arsenic is a known clastogen and an aneugen, which could give rise to chromosomal mal-segregation leading to MN formation. Significant chromosomal aberrations are observed in peripheral lymphocytes when exposed to arsenic [1,7,8,16,34]. Except for a few negative results, the majority of the cytogenetic studies clearly indicated a positive clastogenic effect in population exposed to arsenic. Since, chromosomal aberration primarily lead to MN formation, lymphocyte can be used as suitable biomarker to study genotoxic effects of arsenic. In conclusion, lymphocyte is an excellent as also the most suitable cell type for the analysis of cytogenetic damage as measured by micronuclei formation in the individuals exposed to arsenic through drinking water, and is also applicable in arsenic-induced Bowen's patients.

2.Cytogenetic analysis of Greek farmers using the micronucleus assay in peripheral lymphocytes and buccal cells
Top Abstract Introduction Materials and methods Results Discussion References

Abstract
The potential cytogenetic damage associated with pesticide use in Greek agricultural workers was evaluated using micronuclei (MN) as biomarkers in lymphocytes of peripheral blood and exfoliated cells of the buccal mucosa. In addition, the effects of pesticide exposure and other variables on the cytokinesis block proliferation index (CBPI) in lymphocytes were also evaluated. Both the exposed and control individuals were selected from Nea Makri, a village near Athens (Greece). This location was selected for its high greenhouse density. Micronuclei were analysed in 50 agricultural workers exposed to pesticides (30 men and 20 women) and in 66 nonexposed individuals that constituted the control group (41 men and 25 women). The comparison between workers and controls did not reveal any statistical significant difference in the MN frequency for either lymphocytes or buccal cells. Nevertheless, the multiple regression analysis revealed that the age and the interaction between gender and the number of X-ray examinations during the last 3 years preceding the sampling increased the number of MN in lymphocytes. Moreover, the results of the negative binomial regression analysis suggested that the level of MN in buccal cells could be reduced by the intake of fish, whilst being increased by olive oil consumption. Regarding CBPI, the value found in the exposed group was lower than in controls, the difference being statistically significant. On the other hand, CBPI was inversely associated with both age and X-ray exposure.

Introduction

Although pesticides are useful in enhancing crop productivity, their Abstract extensive use may have adverse health effects in humans. Some studies Introduction Materials and methods have found a relationship between exposure to pesticides and an Results extensive number of symptoms and diseases, including increase in the Discussion incidence of some cancers. In this context, it has been reported that References exposure to pesticides can enhance the incidence of leukaemia and nonHodgkin lymphoma (Hardell and Eriksson, 1999 ; Meinert et al., 2000 ), bladder and pancreatic cancer (Viel and Chalier, 1995 ; Ji et al.., 2001 ), reproductive problems (Petrelli et al., 2000 ; Rojas et al., 2000 ) and, more recently, the incidence of Parkinson disease (Lockwood, 2000 ; Woodward, 2001 ). It must be pointed out that pesticides not only have a negative effect on human health, but also on other organisms and systems. Populations occupationally exposed to pesticides, which are in direct contact almost daily with these chemicals, constitute one of the human groups at genotoxic risk. Many biomonitoring studies have evaluated cytogenetic effects in pesticide-exposed workers from different countries. Although some papers have found increases of cytogenetic damage in the exposed groups (Dulout et al., 1985 ; De Ferrari et al., 1991 ; Amr, 1999 ; Antonucci and De Syllos, 2000 ; Garaj-Vrhorac and Zeljezic, 2000 ; Gmez-Arroyo et al., 2000 ; Lander et al., 2000 ), others did not detect any effects (Carbonell et al., 1990 ; Hoyos et al., 1996 ; Scarpato et al., 1996a ; Gregorio d'Arce and Colus, 2000). In this respect, it must be noted that the results from these kind of studies are difficult to extrapolate and generalize, because different pesticide formulations are used and complex combinations are applied depending on the regions, crops, seasons, etc. Taking that into account, in general, farmers mix different pesticides, the information on the particular adverse effects of a defined compound is not enough to adequately evaluate the real genotoxic risk related to complex mixtures. In biomonitoring studies, the use of the cytokinesis-block micronucleus assay in peripheral lymphocytes is increasing as a useful technique to evaluate cytogenetic damage. The analysis of micronuclei (MN) may be considered a useful biomarker of genotoxic effects in populations occupationally exposed to genotoxicants. Micronuclei can be formed both from whole and fragmented chromosomes lagging behind the cell division; thus, the MN assay, in principle, allows the detection of both clastogenic and aneugenic agents. In addition, and in comparison with other cytogenetic techniques, the MN assay, when using the cytokinesis-block method, is a relatively rapid and simple test that permits the identification of cells that have divided once by adding cytochalasin-B (Fenech, 1993 ). For more detailed information on the advantages/disadvantages of the human lymphocytes MN assay, see Surralls and Natarajan (1997). Several studies in populations exposed to pesticides have showed that the MN assay is a good method of detecting increases of cytogenetic damage in the exposed individuals (Bolognesi et al., 1993 ; Scarpato et al., 1996a ,b ; da Silva Augusto et al., 1997 ; Joksic et al., 1997 ; Meng and Zhang, 1997 ; Calvert et al., 1998 ; Gmez-Arroyo et al., 2000 ), although a lack of effect in the same assay was found in other investigations (Titenko-Holland et al., 1997 ; Davies et al., 1998 ; Venegas et al., 1998 ; Lucero et al., 2000 ; Pastor et al., 2001 ). To add further knowledge to the genetic risk related to pesticide exposure, we applied the MN assay in

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peripheral blood lymphocytes and epithelial buccal cells to evaluate possible cytogenetic damage in a group of Greek farmers. Exfoliated epithelial cells, which are continuously in contact with the environment, can be easily collected and rapidly analysed and are therefore a very appropriate cell system for the study of the effects of mutagenic pollutants (Titenko-Holland et al., 1996 ; Moore et al., 1997 ). This group was occupationally exposed to complex mixtures of pesticides and the observed results were compared with those from a control group from the same area and with similar general characteristics. Cytogenetic data were statistically analysed with relation to some confounding factors such as age, diet, alcohol, etc., that may influence the expression of the cytogenetic parameters evaluated.

Top Abstract Introduction Materials and methods Results Discussion References

Materials and methods


Population A total of 116 individuals (50 exposed to pesticides and 66 controls) were analysed in this study. All of them came from an area outside Athens (Greece), called Nea Makri. This is a village surrounded by cultivated land, with many greenhouses. The exposed group was selected from the village farmers and was composed of 30 men and 20 women who were regularly exposed to complex mixtures of pesticides. Both the women and the men did the same work in the greenhouses. Table I gives the main pesticides used, with an indication of their frequency of use and hazard classification. Most of them belong to the carbamate, nicotinoid and organophosphorus families. Forty-one men and 25 women who carried out clerical jobs in the same village composed the control group. They had no previous occupational exposure to pesticides or any particular environmental agent.

View this Table I. . Pesticides used by the studied group, with indication of their frequency of use, WHO classification by hazard and mutagenicity (M) and table: [in this carcinogenicity (C) experimental data window]

[in a new window]

At the time of drawing samples for cytogenetic determination, a personal history questionnaire was filled-in. The questionnaire covered standard demographic questions (age, gender, etc.), as well as medical (genetic disorders, number of X-ray diagnoses, vaccinations, medication, etc.), lifestyle (smoking, coffee, alcohol, diet, etc.) and occupational questions (working hours/day, years of exposure, etc.). For the exposed group, a further questionnaire was completed including specific questions related with farming: kind of crops, pesticide application, use of protective measures, etc. All the individuals included in the study were non- or ex-smokers. Table II shows the main characteristics of both groups.

View this table: Table II. . Characteristics of the population studied [in this window] [in a new window]

Previous to the study, all individuals gave informed consent, and blood and buccal cell samples were obtained following the procedure described below and manipulated according to the ethical standards. The samples were collected in late winter/early spring of 1997. Lymphocyte cultures and MN analysis Blood samples were obtained from each subject by venipuncture in heparinized vacutainers, coded and sent within 24 h to the laboratory where they were processed (Universitat Autnoma de Barcelona, Spain). Lymphocyte cultures were set up by adding 0.5 ml whole blood to 4.5 ml RPMI 1640 medium supplemented with 15% heat-inactivated fetal calf serum, 1% antibiotics (penicillin and streptomycin) and L-glutamine (all obtained from Gibco, Paisley, UK). Lymphocytes were stimulated by 1% phytohaemagglutinin (PHA; Gibco) and incubated for 72 h at 37C. Two cultures per subject were established. A final concentration of 6 g/ml cytochalasin B (Sigma, St Louis, MO) was added to the cultures 44 h later to arrest cytokinesis (Surralls et al., 1994 ). At 72 h of incubation, the cultures were harvested by centrifugation at 800 r.p.m. for 8 min and treated with an hypotonic solution (23 min in 0.075 M KCl at 4C). Cells were centrifuged thereafter and a 3:1 (v/v) methanol:acetic acid solution was gently added. This fixation step was repeated twice and the resulting cells were resuspended in a small volume of fixative solution and dropped onto clean slides. Finally the slides were stained with 10% Giemsa (Merck, Darmstadt, Germany) in phosphate buffer (pH 6.8) for 10 min and scored. To determine the frequency of binucleated cells with micronuclei (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. This is the number of cells usually scored in most laboratories (Surralls and Natarajan, 1997 ). 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 Surralls et al. (1995). To minimize variability, the same expert carried out all the microscopic analysis. MN analysis in buccal cells Buccal cell samples were obtained by rubbing the inside of the cheeks with a toothbrush. Cells were collected in a conical tube containing 20 ml buffer solution (0.1 M EDTA, 0.01 M Tris HCl and 0.02 M NaCl, pH 7). After three steps of washes in this solution followed by centrifugation at 1500 r.p.m. for 10 min, 50 l of an adequate cell suspension density was dropped onto preheated (55C) slides and allowed to air-dry for 15 min on a slide-warmer. The slides were fixed in 80% cold methanol for 30 min, air-dried overnight at room temperature and stored at 20C until use. Then coded slides were sent to the laboratory of mutagenesis, Department of Genetics and Microbiology, University of Barcelona, where they were stained with a final concentration of 1 g/ml 4',6-di-amidino-2-phenylindole dihydrochloride (DAPI) solution (Sigma), a DNA-specific fluorochrome that avoids possible artefacts. According to the criteria of Tolbert et al. (1992), a total of 2000 cells/donor were scored by one observer under an Olympus BX50 fluorescent 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 studied subject. Statistical analysis The statistical computations were performed using the SPSS, version 10.0 (SPSS, Chicago, IL) and the SAS system for windows, version 8.0 (SAS, Cary, NC). To detect differences between groups with regard to the mean value of confounding factors (age, alcohol, etc.), the MannWhitney U-test was applied due to the observed departure from normality. The models for main variable adjustment took into account all the continuous and dichotomized variables, as well as the interactions between the most important variables studied. Cytogenetic variables BNMN, MNL and CBPI, scored in lymphocytes, were first investigated by using multiple linear regression analysis. Due to the lack of adjustment to the model requirements, MNL and BNMN were suitably transformed to normalize distributions and homogenize variances. Thus, the square root transformation was performed to analyse the MNL data and a BoxCox transformation ( = 0.166) was used for BNMN. The CBPI values did not require transformation. Different methods for variable selection (stepwise, backward and forward) were used. After these analyses, all relevant variables were analysed by a final multiple regression analysis. Each model was checked for adequacy of the fit by the analysis of residuals, tolerance limits and homogeneity of variances. The cytological variables BCMN and MNBC were studied first by Poisson regression. Due to the high over-dispersion found, a binomial regression analysis was performed. A backward selection method was used. P values correspond to two-sided tests, with a type I error < 0.05 as the significance level.

Top Abstract Introduction Materials and methods Results Discussion References

Results
Table II shows the main characteristics of the population studied. Age and sex ratio were similar in both groups. Regarding nutritional habits, it was observed that the controls drank more wine than the exposed. As indicated above, only non- and ex-smokers were included in this study to avoid any possible interference of tobacco in the results. Donors were classified as non-smokers when they had never smoked or had quit smoking for more than 5 years, and as ex-smokers when they had quit smoking between 1 and 5 years ago. An elevated number of individuals received X-rays (as diagnostic) in the past 3 years: 40% of the controls and 28% of the exposed. With regard to the working activity of the farmer group, the majority carried out more than one kind of activity (farming, applying, harvesting, packing, etc.); the crop types were mainly ornamental plants (78%), vegetables (8%) or both (8%). The pesticide application was usually carried out from above the head with motor and manual sprayers, which increased the probability of both inhalation and dermal contact. The hours of pesticide application were similar between seasons. Relating to the protection measures used, 62% of the farmers asserted to use some kind of protection during the preparation and application of pesticides (52% used gloves, 38% impermeable boots, 42% breathing masks). Three individuals (6%) had suffered pesticide intoxication, two of which required hospitalization. Table III shows some of the characteristics of the farmers with reference to the exposure levels, measured as the average of hours directly involved in the use of pesticides.

Table III. . Characteristics of the group of greenhouse farmers Mean SE Years at current job 8.62 1.13

Last pesticide application (days) Pesticides application (h/week)a Spring Summer Autumn Winter Average Data from 50 individuals.
a

7.29 1.05

2.50 0.31 3.06 0.31 2.42 0.32 1.84 0.36 2.45 0.31

Hours of mixing and spraying pesticides.

A summary of the mean data of the cytogenetic variables studied and the cell proliferation index (CBPI) are indicated in Table IV . The data are expressed separately for both men and women to highlight possible gender-related differences. Although control women presented higher MN values, the differences were not statistically significant. Table IV. . Summary of the cytogenetic variablesa Controls n BNMN Men Women MNL Men Women BCMN Men 66 41 25 66 41 25 56 34 Mean SE 14.42 1.29 12.90 1.20 16.92 2.77 16.38 1.50 14.68 1.44 19.16 3.16 1.73 0.19 1.56 0.23 n 50 30 20 50 30 20 47 28 Exposed Mean SE 11.12 0.82 10.90 0.98 11.45 1.45 12.22 0.93 12.33 1.20 12.05 1.51 1.45 0.23 1.39 0.33

Women MNBC Men Women CBPI Men Women

22 56 34 22 66 41 25

2.00 0.34 2.00 0.25 1.74 0.29 2.41 0.43 1.88 0.02 1.92 0.02 1.84 0.03

19 47 28 19 50 30 20

1.53 0.33 1.55 0.26 1.50 0.36 1.63 0.36 1.76 0.02 1.75 0.03 1.78 0.03

Abbreviations: BNMN, binucleated lymphocytes with micronucleus; MNL, total number of micronuclei in binucleated lymphocytes; BCMN, mononucleated buccal cells with MN; MNBC, total number of micronuclei in mononucleated exfoliated buccal cells; CBPI, cell blocking proliferation index.
a

Data corresponding to the scoring of 1000 cells per donor for BNMN and MNL, and 2000 for BCMN and MNBC.

The results obtained in the multiple linear regression analysis do not show any effect of exposure on the lymphocyte cytogenetic variables studied (BNMN and MNL). However, from the list of potential confounding factors included in the analysis (age, gender, X-irradiation, coffee, alcohol, diet, etc.), it can be observed that age and the interaction genderX-irradiation have a direct and significant effect on the lymphocyte micronuclei frequency. In the case of ageing effect, the significant association indicated that BNMN and MNL values also increased with increasing age (Table V ). In the case of gender-X-rays interaction, women showed higher BNMN and MNL frequencies than men, when both were equally exposed to X-rays (Table V ). In the multiple linear regression analysis of the CBPI, the exposure as well as the age and the number of X-rays received in the last 3 years affects the proliferation index. Thus, the group exposed to pesticides showed lower CBPI levels than the control group and the increase of both age and number of X-rays taken produced a decrease in the CBPI values (Table V ).

Table V. . Summary of the results obtained in the multiple linear regression analysis n B Beta Significance Tolerance R2 model

BNMN Intercept Age Gender*X-rays MNL Intercept Age Gender*X-rays CBPI Intercept Exposure Age X-Rays 116 116 116 2.212 -0.138 -0.003 -0.028 -0.422 -0.226 -0.278 0.000 0.000 0.007 0.001 0.982 0.982 0.982 116 116 1.220 0.052 0.106 0.428 0.252 0.008 0.000 0.003 0.997 0.997 116 116 1.196 0.006 0.013 0.405 0.235 0.000 0.000 0.006 0.997 0.997

0.209

0.235

0.260

Abbreviations: B, no standardized coefficient; Beta, standardized coefficient

The negative binomial analysis of buccal cells, evaluating both BCMN and MNBC parameters, also shows a lack of increase of micronuclei in the exposed group. Regarding the role of the different confounding variables, the analysis indicates that buccal cell parameters were inversely influenced by fish consumption and directly affected by olive oil intake (Table VI ).

Table VI. . Summary of the results obtained in the negative binomial analysis n MNBC Intercept Fish consumption Olive oil intake 103 103 B -0.967 -0.303 0.663 P 0.0001 0.0177 0.0117 Scale deviance 115.045 Value/DF 1.150

BCMN Intercept Fish consumption Olive oil intake

103 103

-0.954 -0.265 0.605

0.2136 0.0275 0.0186

117.462

1.174

Abbreviations: B, no standardized coefficient; DF, degrees of freedom

Discussion The main objective of this study was to evaluate if the exposure to complex mixtures of pesticides, primarily in greenhouses, induced increases in the levels of cytogenetic damage. The study was carried out in parallel with an exposed and a control group, both from the same area and with similar individual characteristics. To evaluate the chromosome damage, two different types of cells were chosen covering a wide range of exposure routes: peripheral lymphocytes and epithelial buccal cells, which are the most common cell targets used for human biomonitoring purposes. Peripheral lymphocytes have been classically used for detecting genotoxic effects in a great number of studies, since they are considered to be adequate for detecting general exposure. In addition, these cells are in a non-proliferative stage (G0) and have a long half-life (about 3 years) (Murray and Edwards, 1999 ; Amorin et al., 2000 ; Thierens et al., 2000 ). Exfoliated cells from the buccal mucosa are representative epithelial cells. It must be recalled that epithelial cells are highly proliferative and they are the origin of more than 90% of cancers (Rosin and Gilbert, 1990 ), for which their use in biomonitoring studies is increasing (Casartelli et al., 2000 ; Dietz et al., 2000 ). The results obtained indicate that, under the particular conditions of this study, there is no exposure-related induction of chromosome damage, as measured by the MN assay, in neither lymphocytes (BNMN, MNL) nor buccal epithelial cells (BCMN, MNBC). However, these results are of interest when considering that the exposed group had a decreasing CBPI value, which could be related to the exposure to pesticides, mainly carried out in greenhouses. The negative results, indicating lack of chromosome damage related to pesticide exposure agree with recent studies (Hoyos et al., 1996 ; Scarpato et al., 1996b ; Gregorio d'Arce and Colus, 2000; Lucero et al., 2000 ). In contrast, other studies have revealed the induction of cytogenetic damage after pesticide exposure (Carbonell et al., 1993 ; Falck et al., 1999 ; Antonucci and De Syllos, 2000 ; Garaj-Vrhorac and Zeljezic, 2000 ; Gmez-Arroyo et al., 2000 ). In this context, it must be recalled that every biomonitoring study on populations exposed to chemical pesticides is different from the other(s), since in each area different groups of pesticides are used, depending on the crop type and on environmental factors. In addition, working conditions are generally different, the weather can influence chemical absorption, etc. Furthermore, it is obvious that real

pesticide exposure is highly influenced by the protective measures used by the agricultural workers. Thus, in the population from Nea Makri, 62% of the farmers indicated the use of some kind of protective measure, which would reduce the exposure level. Nevertheless, the results for those farmers who used protection are similar to the findings for those who did not use protective measures. The multiple linear regression analysis reveals that some of the confounding factors have a significant relationship with the cytogenetic variables analysed. Thus, regarding lymphocytes, BNMN and MNL show a significant positive relationship with age. This strengthens the results obtained by other authors who established that spontaneous frequencies of MN in lymphocytes grow in a linear way with age (Fenech and Morley, 1986 ; Ramsey et al., 1995 ; Barale et al., 1998 ; Vaglenov and Carbonell, 1998 ). This effect could be attributed to the increase of aneuploidy with age, mainly in women. In fact, generally higher levels of MN were observed in women when compared with men, as described in Table III . BNMN and MNL also show a significant positive relationship with the interaction gender radiographs. The positive effect of this interaction is due to women exhibiting higher values of BNMN and MNL, when they received the same amount of X-ray radiographs as men. The literature regarding gender and MN shows that the frequencies of MN are greater in females than in males (Barale et al., 1998 ; Fenech, 1998 ; Thierens et al., 2000 ) and this gender effect was attributed to the high X-chromosome micronucleation (Surralls et al., 1996 ; Cataln et al., 1998 ). The increase of cytogenetic damage in lymphocytes due to the exposure to ionizing radiation is also well-known (da Cruz et al., 1994 ; He et al., 2000 ; Thierens et al., 2000 ). Consequently, the finding in our study of X-ray-related increase of micronuclei in women would agree with previous data indicating that exposure to ionizing radiation induces age-dependent aneugenic effects in thyroid cancer women treated with radioactive iodine (Ramrez et al., 1997 ). Nevertheless, the observed aneugenic effect of ionizing radiation indicated that the Xchromosome was not preferentially involved in the effects of radioactive iodine (Ramrez et al., 1997 ) and that this X-independent aneugenic activity is mainly induced in older women. On the other hand, although women showed more micronuclei than men when they were exposed to the same number of X-radiographs, the absorbed dose by women could have also been higher than that absorbed by men. With respect to the buccal cells results, although no effects related to exposure were found, some dietary factors such as olive oil and fish intake seem to influence the frequency of micronuclei. Olive oil and fish consumption was determined by the questionnaire in a semiquantitative way and the results indicate a decrease in MN with increasing fish intake. Some studies have found evidence that the omega-3 polyunsaturated fatty acids (derived from fish) may play a protective role in coronary diseases through a variety of actions, including effects on lipids, blood pressure, cardiac and vascular function, coagulation and immune response (Cho et al., 2001 ;Iso et al., 2001 ;Mori and Beilin, 2001 ). These protective effects of polyunsaturated acids might also exert cell protection against genetic damage. The results from consumption of olive oil indicated a direct relationship between its intake and MN level. This finding is difficult to explain since a positive and protective effect has been generally reported in relation to cancer and other diseases, being attributed to the antioxidant role of monounsaturated fatty acids (Yaqoob, 1998 ; Norrish et al., 2000 ; Stoneham et al., 2000 ). It is possible that the way in which the olive oil is consumed (pure or refined, raw or fried, etc.)

affects the results, for which a specifically well-designed study would be needed to elucidate the eventual role of olive oil intake in modulating genetic damage. Concerning the proliferation index (CBPI), a reduction related to age, exposure and Xirradiation was observed. The decrease observed in CBPI induced by age can be interpreted as an indicator of cell cycle delay due to physiological reasons. In this way, studies on cell proliferation kinetics have also found a negative correlation of the replication index and cell proliferation rate with age (Lazutka et al., 1994 ), which would reflect an age-related decline in the rate of blastogenesis (Lucivero et al., 1988 ). In addition, the observed reduction in the proliferation index of lymphocytes of the group exposed to pesticides suggests that the studied farmers were exposed to chemicals with cytotoxic properties, which would affect the cell proliferation kinetics (Rupa et al., 1991 ; Pasquini et al., 1996 ). The fact that the exposed group shows a decrease in CBPI and a lack of increase in cytogenetic damage could be because the exposure level is not high enough to induce chromosome breakage and/or due to the weakness of the biomarker used for detecting this kind of genetic damage. Another explanation could be that chronic low level exposure to pesticides induces an adaptive response related to an increase in apoptosis sensitivity, or a more extended cell cycle delay that enables appropriate repair (Kirsch-Volders et al., 2001 ). In summary, apart from the CBPI, no differences in cytogenetic damage were observed between the control and the exposed group when using the micronucleus assay. This indicates that, under the particular conditions of exposure, the agricultural tasks related to the use of several pesticides (mainly carbamates, nicotinoids and organophosphorus) was not associated with detectable chromosomal damage when measured by the incidence of micronuclei. Nevertheless, it must be pointed out that the age of the individuals, as well as gender and medical X-ray exposure, are factors affecting MN expression and therefore must be taken into account in biomonitoring studies, as they can influence the results.

3.A cytogenetic study of nuclear power plant workers using the micronucleus-centromere assay
H. Thierensa, *, A. Vralb, M. Barbc, B. Aousalaha and L. De Ridder
Abstract

A cytogenetic study was performed in 215 nuclear power plant workers occupationally exposed to radiation using the micronucleus-centromere assay for peripheral blood lymphocytes. As control population served administrative staff with yearly doses below 1 mSv. The increase of the micronucleus frequency with age, observed in the non-smoking control population, is mainly due to an enhanced number of centromere-positive micronuclei, pointing to an increased chromosome loss. No differences in the number of micronuclei, centromere-positive and centromere-negative micronuclei between smokers and non-smokers are observed. An analysis of the micronucleus data vs. the dose accumulated over the 10 years preceding the venepuncture shows no significant clastogenic or aneuploidogenic effects of the exposure in the studied population which is representative for workers in the nuclear industry at present. According to the linear fits to our data an increase of the micronucleus frequency pro rata 0.5 per 1000 binucleated cells per year, related to the centromere-negative micronuclei, may be expected for workers with the maximal tolerable dose of 20 mSv/year