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Cytogenetic Monitoring of Pesticide Sprayers
ENVIRONMENTAL RESEARCH ARTICLE NO.
75, 113–118 (1997)
ER973753
Cytogenetic Monitoring of Pesticide Sprayers Gordana Joksic´,1,* Aleksandar Vidakovic´,† and Vera Spasojevic´-Tis˘ma* *Medical Protection Center, ‘‘Vinca’’ Institute of Nuclear Sciences, PO Box 522, 11001 Belgrade, Yugoslavia, and †Institute of Occupational and Radiological Health, Deligradska 29, 11000 Belgrade, Yugoslavia Received November 12, 1996
clei averaged 39.92 micronuclei per 1000 binucleated cells, with individual means ranging from 21 to 62. The appearance of more than one micronucleus per binucleated cell was related to the results on chromosome aberrations. The frequencies of chromosomal aberrations and micronuclei were significantly higher (P < 0.001, P < 0.000) in the exposed group than in their matched control groups. The yield of micronuclei in pesticide sprayers at the end of the season was higher than expected with respect to chromosomal aberration frequency, which provides some evidence that some of the micronuclei are induced by the spindle-inhibiting effects of pesticides. A statistically significant (P < 0.003) difference in micronuclei in the first control group was observed compared with the reference control group at the end of the spraying season. With respect to the incidence of micronuclei in the control group in the vine-growing area, a poor but positive correlation (r = 0.074, P < 0.104) with duration of the spraying season was found, which is probably due to airborne pesticides in the vine-growing area. SCE frequencies of the workers’ lymphocytes were not significantly changed due to the exposure. The yield of aberrations as well as that of micronuclei in exposed subjects correlated positively (r = 16, P = 0.016) with duration of exposure. ©1997 Academic Press
The induction of chromosome aberrations, micronuclei, and sister-chromatid exchanges (SCEs) was examined in cultured lymphocytes of 27 vineyard growers exposed to pesticides. Cytogenetic examinations were performed during the prespraying period, a month after spraying, and at the end of the spraying season. For comparison purposes, the same cytogenetic monitoring program was applied to two control groups. The first consisted of 15 individuals from a nearby town, and the second consisted of 20 volunteers living 200 km from the vinegrowing area (reference control group). A positive, though low statistically significant (P < 0.017) difference in the yield of unstable chromosomal aberrations in exposed sprayers was observed compared with both control groups during the prespraying period. The mean group value of micronuclei in exposed workers averaged 5.41 per 1000 binucleated cells, with individual means ranging from 0 to 15. In both control groups, the yield of micronuclei averaged 5.09 per 1000 binucleated cells, with individual means ranging from 1 to 10. No statistically significant (P < 0.5) differences in yield of micronuclei were found in exposed subjects compared with both control groups. Significant individual variation (F = 14.09, P < 0.000) in SCE frequency was observed in exposed subjects, as well as in both control groups (F = 14.09, P < 0.000). A month after spraying, the average incidence of unstable aberrations in pesticide sprayers was 0.22%, and the yield of micronuclei averaged 17.78 per 1000 binucleated cells, with individual means ranging from 7 to 28. The incidence of micronuclei a month after spraying in exposed subjects was elevated (statistically significant at P < 0.01) in comparison with the prespraying period, while the difference in the yield of chromosomal aberrations in exposed subjects was insignificant (P < 0.5). At the end of the spraying season, the average incidence of unstable aberrations in exposed subjects was 0.79%, and the yield of micronu-
INTRODUCTION
Contamination of our environment with chemicals is a major concern considering that more than 1000 new synthetic chemicals are introduced each year (Maugh, 1978). A large number of these chemicals are widely used in agriculture as pesticides and represent potential environmental pollutants. Of particular importance are potentially hazardous effects associated with the widespread application of pesticides. With respect to cytogenetic investigation of producers and workers, there are relatively few data in the literature (Crossen et al., 1978; Georgieva,
1 To whom correspondence should be addressed. Fax: +381–11– 444–01–95.
113 0013-9351/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
´ , VIDAKOVIC ´ , AND SPASOJEVIC ´ -TIS˘MA JOKSIC
114
1977; Pilinskaya, 1970; Tarner et al., 1990; Yoder et al., 1973) compared with the huge number of results concerning other chemical pollutants. A special problem is the outdoor application of pesticides, considering that during the past 20 years, the accumulated knowledge suggests that many human cancers may be the result of lifestyle and environmental factors (Higginson and Muir, 1979). To estimate potential hazardous effects associated with the application of pesticides in agriculture, a group of 27 vine growers were chosen for cytogenetic examination. They spray large amounts of pesticides, which usually involve mixtures of compounds dissolved in organic solvents (mostly xylene). The proportion of individual compounds for spraying during the fruitgrowing season is changeable, mostly because of resistance evoked in pests and insects, so that the toxicological characteristics of a mixture are usually unknown. Recently, it has been demonstrated that surface water extracted from vineyard runoff, where fungicides had been sprayed, showed remarkable genotoxicity in a DNA synthesis inhibition test (von Aufess et al., 1989). In comparison with the huge number of results concerning in vitro investigations, there are relatively few data on occupational exposure in the literature, although biological monitoring of exposed workers has stimulated considerable interest in the prevention of health hazards. The aim of this study is to fill this gap. On the other hand, we were interested in evaluating potential hazardous effects associated with the outdoor application of pesticides. The endpoints used in this study are analysis of chromosome aberrations, micronuclei, and sister-chromatid exchanges in circulating lymphocytes of exposed workers. The parameters analyzed were correlated with duration of exposure during the heavy spraying season. SUBJECTS
The following groups of subjects were recruited to evaluate the effects of pesticide exposure. Group 1 comprised 27 nonsmoking male vine growers, aged 39 years on average, (SD 4 6.15). These subjects had been employed in agriculture for 12.1 years on average (SD 4 6.02). They applied a wide variety of pesticides during the spraying season (Table 1 lists the pesticides used by sprayers), the insecticide diazinon and fungicide dithiocarbamate being the most commonly used. To estimate the exposure of sprayers, air samples were taken as personal samples with a sampling pump (Casella SP). Gas chromatographic analysis of absorption
tube contents indicated that the active substance from the pesticide diazinon was detected in samples taken from tractor drivers and preparation makers who used basuidine insecticide. In other air samples, no active substances from the applied preparation were detected. The spraying period varied from 3 days to 1 week, with 2–9 working hours per day. On average, each worker sprayed approximately 4 hr per day, 6 months a year. Mostly tractor drivers and preparation makers were chosen for cytogenetic analysis as they were considered heavily exposed workers. Group 2 comprised 15 volunteer, nonsmoking male schoolteachers from a nearby town (agricultural area), aged 42.31 on average (SD 4 7.02); this was the first control group. The second reference control group consisted of 20 nonsmoking males from Belgrade (volunteers, students, and employees of Belgrade’s university), aged 38.92 on average (SD 4 6.24). These subjects were recruited as controls outside the vine-growing area. Exposed subjects were matched to their control subjects on sex, age, and smoking habits. All volunteers in this investigation were interviewed about diseases, drug intake, and exposure to ionizing radiation or compounds with known or suspected mutagenic ability. Those with positive anamnesis were excluded from the study. Only healthy individuals were selected for examination. METHODS
The chromosome aberration analysis was carried out on cultures of phytohemaglutinin-stimulated blood lymphocytes. To 5 ml of RPMI-1640 medium supplemented with 10% of calf serum was added 0.5 ml of whole blood. Lymphocytes were incubated at 37°C for 48 hr with a 3-hr colcemid block (GIBCO), at final concentration of 0.1 mg/ml. FixaTABLE 1 Pesticides Used during Spraying Season Pesticide Nortron Agrobet Monosan herbi
Usage Herbicide Herbicide Herbicide
Active substance
Ethofumesate Phenmedifan, desmediphan (2,4-Dichlorophenoxy)acetic acid (2,4-D) Bajleton VP-25 Fungicide Triadimefon Basudin Insecticide Diazinon Cineb Fungicide Dithiocarbamate Ridomi EZ VP-72 Fungicide Metalaxyl + Cu Ronilan Fungicide Vinclozolin TILT TC 250 Fungicide Propioconazole, xylene
CYTOGENETIC MONITORING OF PESTICIDE SPRAYERS
tion of the cultures and preparation of the slides were carried out according to conventional methods (IAEA, 1986). Giemsa-stained metaphases were scored for unstable-type aberrations, i.e., dicentric, centric ring, and excess acentric chromosomes. For completeness stable chromosomal aberrations, as well as chromatid aberrations, were also recorded. The criteria for scoring were as described by the IAEA (1986). Two hundred well-spread and complete metaphases were analyzed for each person. The yields of dicentric chromosomes, ring chromosomes, and excess acentrics were recorded. Results are presented as a percentage of structural chromosomal aberrations. For sister-chromatid exchange analysis, lymphocytes were incubated at 37°C for 1 hr and 8 mg/ml 5-bromo-2-deoxyuridine (BrdU, Sigma) was added. The cells were fixed after 62 hr of sampling including a 3-hr colchicine treatment. Fixation of the cultures and preparation of the slides were carried out according to a modification of the method of Wolff and Perry (1974). The slides were stained with Hoechst 33258, washed with distilled water, and exposed 2 hr to UV light during which the slides were covered with PBS. After being washed with distilled water, slides were stained in Giemsa (2%) for 8 min. Thirty second-division metaphases were scored for each sample. For micronucleus preparation, cytochalasin B at a final concentration of 4 mg/ml was added after 44 hr of culture incubation, according to the method of Fenech and Morley (1985). The lymphocyte cultures were incubated for another 24 hr. Micronucleus slides were made by fixation in methanol:acetic acid (3:1), after short cold hypotonic (0.56% KCl) treatment. The slides were stained in 2% alkaline Giemsa. At least 1000 binucleated cells per person were analyzed. Results are presented as the incidence of micronuclei per 1000 binucleated cells. All slides were scored blind. Two scorers contributed to the scoring of slides, and each aberration noted was confirmed by a second scorer. All slides were analyzed on Zeiss microscopes. Chromosome aberration analysis, as well as the cytochalasin B micronucleus test, in the exposed group was performed during the prespraying period, a month after spraying, and at the end of the spraying season. Sister-chromatid exchange analysis was done during the prespraying period and at the end of the season. In both control groups, analysis of chromosomal aberrations and micronuclei, as well as sister-chromatid exchange, was performed during the prespraying period and at the end of the spraying season.
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Statistical analysis. To evaluate statistical differences between mean values of the yield of chromosomal aberrations and micronuclei, the Wilcoxon rank-sum test was used. For sister-chromatid exchange, analysis of variance (F test) was used. RESULTS
The basic results for scoring chromosome-type aberrations, micronuclear frequencies, and incidence of sister-chromatid exchanges are presented in Table 2. Dicentric and ring chromosomes were almost always accompanied by an acentric fragment, but only excess acentric fragments are listed in Table 2. Prespraying Period The percentage of unstable aberrations in exposed subjects was 0.13%. Including stable aberrations, the percentage of aberrations was 0.19%. A positive, though low statistically significant (P < 0.017) difference in the yield of unstable chromosomal aberrations in exposed workers compared with both control groups was observed (footnote b, Table 2). The average yield of micronuclei in exposed workers was 5.41 per 1000 binucleated cells, with individual means ranging from 0 to 15. In both control groups, the yield of micronuclei averaged 5.09 per 1000 binucleated cells, with individual means ranging from 1 to 10. Binucleated cells with one micronucleus were most commonly observed. No statistically significant (P < 0.5) differences in yield of micronuclei was found in exposed subjects compared with both control groups (footnote c). Intercontrol differences were insignificant (footnote j, P < 0.5). Significant individual variation (F 4 15.09, P < 0.000) in SCE frequency was observed in exposed subjects, as well as in both control groups (F 4 14.09, P < 0.000). One Month after Spraying The incidence of unstable aberrations in exposed workers a month after spraying was 0.22%; the yield of micronuclei averaged 17.78 per 1000 binucleated cells, with individual means ranging from 7 to 28. Although the incidence of unstable chromosomal aberrations a month after spraying was elevated in comparison to the prespraying period, the difference was statistically insignificant compared (footnote d, P < 0.5). The incidence of micronuclei in exposed subjects was elevated (footnote e, statistically significant at P < 0.01) in comparison with the prespraying period.
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TABLE 2 Frequency of Chromosomal Aberrations (CAs), Micronuclei (MNs), and Sister-Chromatid Exchanges (SCEs) in Lymphocytes of Pesticide-Exposed Workers and Control Subjects
0 0
0.067 0.064
First control group 5.90 ± 3.44 3.05i 0.36j 9.63 ± 5.69
0 0.72
0.05 0.055
Reference control group 5.09 ± 2.53 3.23l 5.20 ± 2.01 0.65m
0 0.52
0.13 0.22 0.79
3000 3010
— —
— 1
1 1
4023 3980
1 —
— 1
2 1
1 3
0 0
0 0 2.56
2 9 30
1 3
3.93–6.43
0 1.31 2.96
3 2 10
5237 5305 5296
5.41 ± 1.26
Exposed workers 2.36b 5.41 ± 3.67 17.78 ± 5.68 0.55d 3.23g 39.92 ± 12.31
2 1 1
1 2 3
0.65c 4.54e 4.54h
Z (Wilcoxon rank sum test)
No. of rings
No. of dicentric
Range of individual means of SCE frequency
% CB cells with more than 3 MNs
% Unstable CAs
Time of analysisa
Average No. of SCEs per cell
% CB cells with 3 MNs
No. of accesses acentrics
No. of cells analyzed
Z (Wilcoxon rank sum test)
Average No. MNs/1000 CB cells
f
f
5.25 ± 1.42
4.53–6.26
0.65c 2.92k
4.96 ± 0.62 5.25 ± 1.42
4.01–6.62 4.53–6.26
0.65c 3.43n
4.83 ± 0.62 5.28 ± 0.80
3.88–6.12 3.44–6.66
a (1) Prespraying period (first time of analysis); (2) month after spraying (second time of analysis); (3) end of spraying season (third time of analysis). b P 4 0.017 compared with both control groups, statistically significant difference. c P 4 0.5, compared with both control groups, no statistically significant difference. d P 4 0.576, compared with the first time of analysis, no statistically significant difference. e P 4 0.000, compared with the first time of analysis, highly statistically significant difference. f Analysis not performed. g P 4 0.001, compared with the second time of analysis, statistically significant difference. h P 4 0.000, compared with the second time of analysis, highly statistically significant difference. i P 4 0.000, incidence of chromosomal aberrations examined in exposures at the third time of analysis compared with first control group, highly statistically significant. j P 4 0.5, compared with both control groups at the first time of analysis, no statistically significant difference. k P 4 0.003, compared with reference control group at the first time of analysis, statistically significant difference. l P 4 0.001, incidence of chromosomal aberrations examined in exposures at the third time of analysis compared with second control group, highly statistically significant. m P 4 0.577, compared with the first time of analysis, no statistically significant difference. n P 4 0.000, incidence of micronuclei in first control group compared with reference controls at the third time of analysis, highly statistically significant.
At the End of the Spraying Season The incidence of unstable aberrations in exposed subjects at the end of the spraying season was 0.79%; the mean group value of micronuclei was 39.92 per 1000 binucleated cells, with individual means ranging from 21 to 62. The frequencies of chromosomal aberrations (footnote i, P < 0.002; footnote l, P < 0.001) and micronuclei (footnote h, P < 0.000) were significantly higher in exposed workers than in their matched controls. The frequency of chromosomal aberrations in exposed sprayers was also highly (footnote g, P < 0.001) statistically significant in comparison with the prespraying period. The appearance of more than one micronucleus per binucleated cell was related to the results of chromosome aberrations. A significant percentage of cells (2.96) carried more than three micronuclei. A statistically significant increase in micronuclei in the first control group was observed compared with the reference control group at the end of the spraying season (footnotes k and n, P 4 0.000). SCE frequencies of the workers’ lymphocytes were not
changed by the exposure. The yields of aberrations and micronuclei correlated positively (r 4 0.16, P 4 0.016) with duration of exposure. DISCUSSION
Although evidence of the ability of pesticides to induce cytogenetic damage in humans is known (Yoder et al., 1973; Crossen et al., 1978; Dulout et al., 1985), some of these positive findings have not been substantiated by other investigators (Hogstedt et al., 1980; de Cassia Stocco et al., 1982). Variations in the degree of exposure or the use of different pesticides may explain the differences in findings. The applicants of pesticides in this study were considered to be heavily exposed since each individual was at a focal point of the spraying site. All individuals in this study were intentionally selected to be nonsmokers, to eliminate confounding factors such as cigarette smoking (Obe and Herha, 1978), when trying to demonstrate occupational as well as environmental exposure. The subjects in our study showed an increased yield of chromosome aberrations (par-
CYTOGENETIC MONITORING OF PESTICIDE SPRAYERS
ticularly rings and excess acentrics) and micronuclei at the end of spraying. Compared with the control groups, all results were statistically highly significant (P 4 0.000). A statistically significant (P < 0.003) difference in yield of micronuclei in the first control group compared with the reference control group was observed at the end of the spraying season. With respect to the incidence of micronuclei in the control group in the vine-growing area, a poor but positive correlation (r 4 0.074, P < 0.104) with duration of the spraying season was found. Obviously, the environments of nearby villages and towns were affected by airborne pesticides, which caused an elevated yield of micronuclei in occupationally unexposed citizens. Comparing SCE frequencies and incidence of chromosomal aberrations, as well as yield of micronuclei, it was observed that the values differed. This phenomen had already been described (Madle et al., 1986), and it is explained by different mechanisms in the formation of genetic alterations. There was no correlation between SCE frequency and duration of exposure during the heavy spraying period, but a good correlation between incidence of unstable chromosomal aberrations, especially micronuclei, and duration of exposure was found. Although the results of micronucleus and chromosome aberration assays did correlate, the yield of micronuclei at the end of the season was higher than expected. Since micronuclei can be induced by agents damaging either the chromosomes directly or the spindle apparatus, we suppose that some of the micronuclei are formed by entire chromosomes due to failure of the mitotic spindle. Our present data, particularly the percentage of binucleated cells carrying more than three micronuclei (even up to seven micronuclei per cell) (Table 2) provide some evidence that a portion of the micronuclei were induced by the spindle-inhibiting effects of pesticides. The influence of organic solvents in the applied pesticide preparations also cannot be ruled out. Little further insight into quantifying the portion of micronuclei caused by failure of the mitotic spindle can be gained using in situ hybridization with whole centromere DNA probes in cytochalasin-induced binucleated cells. Improvements in the micronucleus assay in vitro, particularly a method for chromosome painting in micronuclei, developed more recently are expected to elucidate the interrelationship between micronucleus formation and mutagenesis and to evaluate the relative hazards of low dose response to aneugenic chemicals (Parry et al., 1996). This will lead to a better understanding and interpretation of results obtained by examination of in vivo exposed humans.
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Further prospective monitoring of persons with chromosomal and micronuclear findings will make it possible to determine the full significance of exposure to pesticides and to evaluate whether they are prognostically associated with an increased risk of adverse health consequences. CONCLUSION
In summary, from the data presented here, we conclude that environmental exposure to a mixture of pesticides caused genetic hazards in circulating lymphocytes of exposed sprayers. Genetic hazards correlated positively with duration of exposure. In occupationally unexposed subjects, living in an agricultural area, an elevated incidence of spontaneously occurring micronuclei was observed at the end of the spraying season, which is probably due to pollution of the air by airborne pesticides. The positive correlation between the results of the micronucleus and chromosome aberration assays documents that the micronucleus assay, which is a cheaper, simpler, and more sensitive method, can be successfully used for screening purposes. This work illustrates the need to implement more rigid guidelines to minimize or prevent further exposure. ACKNOWLEDGMENTS This study was carried out in cooperation with the Occupational Health Department in Aleksandrovac and the Institute of Occupational and Radiological Health in Belgrade. The authors are grateful for the skillful assistance of the medical staff in both institutions. We are grateful to Dr. Dus˘an Djuric´ for his enthusiastic cooperation and helpful suggestions during this study.
REFERENCES Crossen, P. E., Morgan, W. F., Horan, J. J., and Stewart, W. L. (1978). Cytogenetic studies of pesticide and herbicide sprayers. N.Z. Med. J. 88, 192–195. DeCassia Stocco, Becak, R. W., Gaeta, R., and Rabello-Gay, M. N. (1982). Cytogenetic study of workers exposed to methylparathion. Mutat. Res. 103, 71–76. Dulout, F. N., Pastori, O. A., Olivero, M., Gonzales Cid, Loria, D., Matos, F., Sobel, U., de Bujan, E. C., and Albiano, N. (1985). Sister-chromatid exchanges and chromosomal aberration in population exposed to pesticides. Mutat. Res. 147, 179–244. Fenech, M., and Morley, A. A. (1985). Measurement of micronuclei in lymphocytes. Mutat. Res. 147, 29–36. Georgieva, V. L. (1977). Cytogenetic investigations in agricultural workers in occupational contact with pesticides. Med. Genet., 279–300. Higginson, J., and Muir, C. S. (1979). Environmental carcinogenesis: Misconceptions and limitations to cancer control. J. Natl. Cancer Inst. 63, 1291. Hogstedt, B., Koling, A. M., Mitelman, F., and Skerfing, S. (1980).
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Cytogenetic studies of pesticides in agricultural work. Hereditas 92, 177–178.
Pilinskaya, M. A. (1970). Chromosome aberrations in the persons contacted with Ziram. Genetika 6, 157–163. [Russian]
IAEA (1986). ‘‘Biological Dosimetry: Chromosomal Aberration Analysis for Dose Assessment,’’ Tech. Rep. No. 260. International Atomic Agency, Vienna.
Tarner, B., Schoneich, J., Huttner, E., and Steinbicker, V. (1990). Cytogenetic monitoring of chemicals workers in production of the drug 1-propoxy-2 acetamino-4 nitrobenzol with reference to their smoking habits. Mutat. Res. 241, 291–295.
Madle, E., Korte, and Beech, B. (1986). Species differences in mutagenic testing. II. Sister-chromatid exchange and micronucleus induction in rats, mice and Chinese hamsters treated with cyclophosphamide. Mutagenesis 1, 419–422. Maugh, T. H. (1978). Chemicals: How many are there? Science 199, 162. Obe, G., and Herha, J. (1978). Chromosome aberrations in heavy smokers. Hum. Genet. 41, 259–263. Parry, J. M., Parry, E. M., Bourner, R., Doherty, A., Ellard, S., Odonovan, J., Hoebee, B., Destoppelaar, J. M., Mohn, G. R., Onfelt, A., Reuglin, A., et al. (1996). The detection and evaluation of aneugenic chemicals. Mutat. Res. Fundam. Mol. Mechanisms Mutagen. 353 (1,2), 11–46.
Von Aufess, G., Beicht, W., Borquin, H. D., Hantage, E., Heil, J., Muller, M. J., Opfermann, H., Reimer, J., Zahn, R. K., and Zimmer, K. H. (1989). Uniter-Suchungen zum Austrag von Pflauzenschutzmitteln und Nahr-stroffen aus Rebachen des Moseltals. Dtsch. Verb. Wasserwirtsch. Kulturbay Schriftenr. 88, 1–78. Wolff, S., and Perry, P. (1974). Differential Giemsa staining of sister chromatids and the study of SCE without autoradiography. Chromosoma 48, 344–358. Yoder, J., Watson, M., and Benson, W. W. (1973). Lymphocyte chromosome analysis of agricultural workers during extensive occupational exposure to pesticides. Mutat. Res. 21, 335–340.
75, 113–118 (1997)
ER973753
Cytogenetic Monitoring of Pesticide Sprayers Gordana Joksic´,1,* Aleksandar Vidakovic´,† and Vera Spasojevic´-Tis˘ma* *Medical Protection Center, ‘‘Vinca’’ Institute of Nuclear Sciences, PO Box 522, 11001 Belgrade, Yugoslavia, and †Institute of Occupational and Radiological Health, Deligradska 29, 11000 Belgrade, Yugoslavia Received November 12, 1996
clei averaged 39.92 micronuclei per 1000 binucleated cells, with individual means ranging from 21 to 62. The appearance of more than one micronucleus per binucleated cell was related to the results on chromosome aberrations. The frequencies of chromosomal aberrations and micronuclei were significantly higher (P < 0.001, P < 0.000) in the exposed group than in their matched control groups. The yield of micronuclei in pesticide sprayers at the end of the season was higher than expected with respect to chromosomal aberration frequency, which provides some evidence that some of the micronuclei are induced by the spindle-inhibiting effects of pesticides. A statistically significant (P < 0.003) difference in micronuclei in the first control group was observed compared with the reference control group at the end of the spraying season. With respect to the incidence of micronuclei in the control group in the vine-growing area, a poor but positive correlation (r = 0.074, P < 0.104) with duration of the spraying season was found, which is probably due to airborne pesticides in the vine-growing area. SCE frequencies of the workers’ lymphocytes were not significantly changed due to the exposure. The yield of aberrations as well as that of micronuclei in exposed subjects correlated positively (r = 16, P = 0.016) with duration of exposure. ©1997 Academic Press
The induction of chromosome aberrations, micronuclei, and sister-chromatid exchanges (SCEs) was examined in cultured lymphocytes of 27 vineyard growers exposed to pesticides. Cytogenetic examinations were performed during the prespraying period, a month after spraying, and at the end of the spraying season. For comparison purposes, the same cytogenetic monitoring program was applied to two control groups. The first consisted of 15 individuals from a nearby town, and the second consisted of 20 volunteers living 200 km from the vinegrowing area (reference control group). A positive, though low statistically significant (P < 0.017) difference in the yield of unstable chromosomal aberrations in exposed sprayers was observed compared with both control groups during the prespraying period. The mean group value of micronuclei in exposed workers averaged 5.41 per 1000 binucleated cells, with individual means ranging from 0 to 15. In both control groups, the yield of micronuclei averaged 5.09 per 1000 binucleated cells, with individual means ranging from 1 to 10. No statistically significant (P < 0.5) differences in yield of micronuclei were found in exposed subjects compared with both control groups. Significant individual variation (F = 14.09, P < 0.000) in SCE frequency was observed in exposed subjects, as well as in both control groups (F = 14.09, P < 0.000). A month after spraying, the average incidence of unstable aberrations in pesticide sprayers was 0.22%, and the yield of micronuclei averaged 17.78 per 1000 binucleated cells, with individual means ranging from 7 to 28. The incidence of micronuclei a month after spraying in exposed subjects was elevated (statistically significant at P < 0.01) in comparison with the prespraying period, while the difference in the yield of chromosomal aberrations in exposed subjects was insignificant (P < 0.5). At the end of the spraying season, the average incidence of unstable aberrations in exposed subjects was 0.79%, and the yield of micronu-
INTRODUCTION
Contamination of our environment with chemicals is a major concern considering that more than 1000 new synthetic chemicals are introduced each year (Maugh, 1978). A large number of these chemicals are widely used in agriculture as pesticides and represent potential environmental pollutants. Of particular importance are potentially hazardous effects associated with the widespread application of pesticides. With respect to cytogenetic investigation of producers and workers, there are relatively few data in the literature (Crossen et al., 1978; Georgieva,
1 To whom correspondence should be addressed. Fax: +381–11– 444–01–95.
113 0013-9351/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
´ , VIDAKOVIC ´ , AND SPASOJEVIC ´ -TIS˘MA JOKSIC
114
1977; Pilinskaya, 1970; Tarner et al., 1990; Yoder et al., 1973) compared with the huge number of results concerning other chemical pollutants. A special problem is the outdoor application of pesticides, considering that during the past 20 years, the accumulated knowledge suggests that many human cancers may be the result of lifestyle and environmental factors (Higginson and Muir, 1979). To estimate potential hazardous effects associated with the application of pesticides in agriculture, a group of 27 vine growers were chosen for cytogenetic examination. They spray large amounts of pesticides, which usually involve mixtures of compounds dissolved in organic solvents (mostly xylene). The proportion of individual compounds for spraying during the fruitgrowing season is changeable, mostly because of resistance evoked in pests and insects, so that the toxicological characteristics of a mixture are usually unknown. Recently, it has been demonstrated that surface water extracted from vineyard runoff, where fungicides had been sprayed, showed remarkable genotoxicity in a DNA synthesis inhibition test (von Aufess et al., 1989). In comparison with the huge number of results concerning in vitro investigations, there are relatively few data on occupational exposure in the literature, although biological monitoring of exposed workers has stimulated considerable interest in the prevention of health hazards. The aim of this study is to fill this gap. On the other hand, we were interested in evaluating potential hazardous effects associated with the outdoor application of pesticides. The endpoints used in this study are analysis of chromosome aberrations, micronuclei, and sister-chromatid exchanges in circulating lymphocytes of exposed workers. The parameters analyzed were correlated with duration of exposure during the heavy spraying season. SUBJECTS
The following groups of subjects were recruited to evaluate the effects of pesticide exposure. Group 1 comprised 27 nonsmoking male vine growers, aged 39 years on average, (SD 4 6.15). These subjects had been employed in agriculture for 12.1 years on average (SD 4 6.02). They applied a wide variety of pesticides during the spraying season (Table 1 lists the pesticides used by sprayers), the insecticide diazinon and fungicide dithiocarbamate being the most commonly used. To estimate the exposure of sprayers, air samples were taken as personal samples with a sampling pump (Casella SP). Gas chromatographic analysis of absorption
tube contents indicated that the active substance from the pesticide diazinon was detected in samples taken from tractor drivers and preparation makers who used basuidine insecticide. In other air samples, no active substances from the applied preparation were detected. The spraying period varied from 3 days to 1 week, with 2–9 working hours per day. On average, each worker sprayed approximately 4 hr per day, 6 months a year. Mostly tractor drivers and preparation makers were chosen for cytogenetic analysis as they were considered heavily exposed workers. Group 2 comprised 15 volunteer, nonsmoking male schoolteachers from a nearby town (agricultural area), aged 42.31 on average (SD 4 7.02); this was the first control group. The second reference control group consisted of 20 nonsmoking males from Belgrade (volunteers, students, and employees of Belgrade’s university), aged 38.92 on average (SD 4 6.24). These subjects were recruited as controls outside the vine-growing area. Exposed subjects were matched to their control subjects on sex, age, and smoking habits. All volunteers in this investigation were interviewed about diseases, drug intake, and exposure to ionizing radiation or compounds with known or suspected mutagenic ability. Those with positive anamnesis were excluded from the study. Only healthy individuals were selected for examination. METHODS
The chromosome aberration analysis was carried out on cultures of phytohemaglutinin-stimulated blood lymphocytes. To 5 ml of RPMI-1640 medium supplemented with 10% of calf serum was added 0.5 ml of whole blood. Lymphocytes were incubated at 37°C for 48 hr with a 3-hr colcemid block (GIBCO), at final concentration of 0.1 mg/ml. FixaTABLE 1 Pesticides Used during Spraying Season Pesticide Nortron Agrobet Monosan herbi
Usage Herbicide Herbicide Herbicide
Active substance
Ethofumesate Phenmedifan, desmediphan (2,4-Dichlorophenoxy)acetic acid (2,4-D) Bajleton VP-25 Fungicide Triadimefon Basudin Insecticide Diazinon Cineb Fungicide Dithiocarbamate Ridomi EZ VP-72 Fungicide Metalaxyl + Cu Ronilan Fungicide Vinclozolin TILT TC 250 Fungicide Propioconazole, xylene
CYTOGENETIC MONITORING OF PESTICIDE SPRAYERS
tion of the cultures and preparation of the slides were carried out according to conventional methods (IAEA, 1986). Giemsa-stained metaphases were scored for unstable-type aberrations, i.e., dicentric, centric ring, and excess acentric chromosomes. For completeness stable chromosomal aberrations, as well as chromatid aberrations, were also recorded. The criteria for scoring were as described by the IAEA (1986). Two hundred well-spread and complete metaphases were analyzed for each person. The yields of dicentric chromosomes, ring chromosomes, and excess acentrics were recorded. Results are presented as a percentage of structural chromosomal aberrations. For sister-chromatid exchange analysis, lymphocytes were incubated at 37°C for 1 hr and 8 mg/ml 5-bromo-2-deoxyuridine (BrdU, Sigma) was added. The cells were fixed after 62 hr of sampling including a 3-hr colchicine treatment. Fixation of the cultures and preparation of the slides were carried out according to a modification of the method of Wolff and Perry (1974). The slides were stained with Hoechst 33258, washed with distilled water, and exposed 2 hr to UV light during which the slides were covered with PBS. After being washed with distilled water, slides were stained in Giemsa (2%) for 8 min. Thirty second-division metaphases were scored for each sample. For micronucleus preparation, cytochalasin B at a final concentration of 4 mg/ml was added after 44 hr of culture incubation, according to the method of Fenech and Morley (1985). The lymphocyte cultures were incubated for another 24 hr. Micronucleus slides were made by fixation in methanol:acetic acid (3:1), after short cold hypotonic (0.56% KCl) treatment. The slides were stained in 2% alkaline Giemsa. At least 1000 binucleated cells per person were analyzed. Results are presented as the incidence of micronuclei per 1000 binucleated cells. All slides were scored blind. Two scorers contributed to the scoring of slides, and each aberration noted was confirmed by a second scorer. All slides were analyzed on Zeiss microscopes. Chromosome aberration analysis, as well as the cytochalasin B micronucleus test, in the exposed group was performed during the prespraying period, a month after spraying, and at the end of the spraying season. Sister-chromatid exchange analysis was done during the prespraying period and at the end of the season. In both control groups, analysis of chromosomal aberrations and micronuclei, as well as sister-chromatid exchange, was performed during the prespraying period and at the end of the spraying season.
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Statistical analysis. To evaluate statistical differences between mean values of the yield of chromosomal aberrations and micronuclei, the Wilcoxon rank-sum test was used. For sister-chromatid exchange, analysis of variance (F test) was used. RESULTS
The basic results for scoring chromosome-type aberrations, micronuclear frequencies, and incidence of sister-chromatid exchanges are presented in Table 2. Dicentric and ring chromosomes were almost always accompanied by an acentric fragment, but only excess acentric fragments are listed in Table 2. Prespraying Period The percentage of unstable aberrations in exposed subjects was 0.13%. Including stable aberrations, the percentage of aberrations was 0.19%. A positive, though low statistically significant (P < 0.017) difference in the yield of unstable chromosomal aberrations in exposed workers compared with both control groups was observed (footnote b, Table 2). The average yield of micronuclei in exposed workers was 5.41 per 1000 binucleated cells, with individual means ranging from 0 to 15. In both control groups, the yield of micronuclei averaged 5.09 per 1000 binucleated cells, with individual means ranging from 1 to 10. Binucleated cells with one micronucleus were most commonly observed. No statistically significant (P < 0.5) differences in yield of micronuclei was found in exposed subjects compared with both control groups (footnote c). Intercontrol differences were insignificant (footnote j, P < 0.5). Significant individual variation (F 4 15.09, P < 0.000) in SCE frequency was observed in exposed subjects, as well as in both control groups (F 4 14.09, P < 0.000). One Month after Spraying The incidence of unstable aberrations in exposed workers a month after spraying was 0.22%; the yield of micronuclei averaged 17.78 per 1000 binucleated cells, with individual means ranging from 7 to 28. Although the incidence of unstable chromosomal aberrations a month after spraying was elevated in comparison to the prespraying period, the difference was statistically insignificant compared (footnote d, P < 0.5). The incidence of micronuclei in exposed subjects was elevated (footnote e, statistically significant at P < 0.01) in comparison with the prespraying period.
´ , VIDAKOVIC ´ , AND SPASOJEVIC ´ -TIS˘MA JOKSIC
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TABLE 2 Frequency of Chromosomal Aberrations (CAs), Micronuclei (MNs), and Sister-Chromatid Exchanges (SCEs) in Lymphocytes of Pesticide-Exposed Workers and Control Subjects
0 0
0.067 0.064
First control group 5.90 ± 3.44 3.05i 0.36j 9.63 ± 5.69
0 0.72
0.05 0.055
Reference control group 5.09 ± 2.53 3.23l 5.20 ± 2.01 0.65m
0 0.52
0.13 0.22 0.79
3000 3010
— —
— 1
1 1
4023 3980
1 —
— 1
2 1
1 3
0 0
0 0 2.56
2 9 30
1 3
3.93–6.43
0 1.31 2.96
3 2 10
5237 5305 5296
5.41 ± 1.26
Exposed workers 2.36b 5.41 ± 3.67 17.78 ± 5.68 0.55d 3.23g 39.92 ± 12.31
2 1 1
1 2 3
0.65c 4.54e 4.54h
Z (Wilcoxon rank sum test)
No. of rings
No. of dicentric
Range of individual means of SCE frequency
% CB cells with more than 3 MNs
% Unstable CAs
Time of analysisa
Average No. of SCEs per cell
% CB cells with 3 MNs
No. of accesses acentrics
No. of cells analyzed
Z (Wilcoxon rank sum test)
Average No. MNs/1000 CB cells
f
f
5.25 ± 1.42
4.53–6.26
0.65c 2.92k
4.96 ± 0.62 5.25 ± 1.42
4.01–6.62 4.53–6.26
0.65c 3.43n
4.83 ± 0.62 5.28 ± 0.80
3.88–6.12 3.44–6.66
a (1) Prespraying period (first time of analysis); (2) month after spraying (second time of analysis); (3) end of spraying season (third time of analysis). b P 4 0.017 compared with both control groups, statistically significant difference. c P 4 0.5, compared with both control groups, no statistically significant difference. d P 4 0.576, compared with the first time of analysis, no statistically significant difference. e P 4 0.000, compared with the first time of analysis, highly statistically significant difference. f Analysis not performed. g P 4 0.001, compared with the second time of analysis, statistically significant difference. h P 4 0.000, compared with the second time of analysis, highly statistically significant difference. i P 4 0.000, incidence of chromosomal aberrations examined in exposures at the third time of analysis compared with first control group, highly statistically significant. j P 4 0.5, compared with both control groups at the first time of analysis, no statistically significant difference. k P 4 0.003, compared with reference control group at the first time of analysis, statistically significant difference. l P 4 0.001, incidence of chromosomal aberrations examined in exposures at the third time of analysis compared with second control group, highly statistically significant. m P 4 0.577, compared with the first time of analysis, no statistically significant difference. n P 4 0.000, incidence of micronuclei in first control group compared with reference controls at the third time of analysis, highly statistically significant.
At the End of the Spraying Season The incidence of unstable aberrations in exposed subjects at the end of the spraying season was 0.79%; the mean group value of micronuclei was 39.92 per 1000 binucleated cells, with individual means ranging from 21 to 62. The frequencies of chromosomal aberrations (footnote i, P < 0.002; footnote l, P < 0.001) and micronuclei (footnote h, P < 0.000) were significantly higher in exposed workers than in their matched controls. The frequency of chromosomal aberrations in exposed sprayers was also highly (footnote g, P < 0.001) statistically significant in comparison with the prespraying period. The appearance of more than one micronucleus per binucleated cell was related to the results of chromosome aberrations. A significant percentage of cells (2.96) carried more than three micronuclei. A statistically significant increase in micronuclei in the first control group was observed compared with the reference control group at the end of the spraying season (footnotes k and n, P 4 0.000). SCE frequencies of the workers’ lymphocytes were not
changed by the exposure. The yields of aberrations and micronuclei correlated positively (r 4 0.16, P 4 0.016) with duration of exposure. DISCUSSION
Although evidence of the ability of pesticides to induce cytogenetic damage in humans is known (Yoder et al., 1973; Crossen et al., 1978; Dulout et al., 1985), some of these positive findings have not been substantiated by other investigators (Hogstedt et al., 1980; de Cassia Stocco et al., 1982). Variations in the degree of exposure or the use of different pesticides may explain the differences in findings. The applicants of pesticides in this study were considered to be heavily exposed since each individual was at a focal point of the spraying site. All individuals in this study were intentionally selected to be nonsmokers, to eliminate confounding factors such as cigarette smoking (Obe and Herha, 1978), when trying to demonstrate occupational as well as environmental exposure. The subjects in our study showed an increased yield of chromosome aberrations (par-
CYTOGENETIC MONITORING OF PESTICIDE SPRAYERS
ticularly rings and excess acentrics) and micronuclei at the end of spraying. Compared with the control groups, all results were statistically highly significant (P 4 0.000). A statistically significant (P < 0.003) difference in yield of micronuclei in the first control group compared with the reference control group was observed at the end of the spraying season. With respect to the incidence of micronuclei in the control group in the vine-growing area, a poor but positive correlation (r 4 0.074, P < 0.104) with duration of the spraying season was found. Obviously, the environments of nearby villages and towns were affected by airborne pesticides, which caused an elevated yield of micronuclei in occupationally unexposed citizens. Comparing SCE frequencies and incidence of chromosomal aberrations, as well as yield of micronuclei, it was observed that the values differed. This phenomen had already been described (Madle et al., 1986), and it is explained by different mechanisms in the formation of genetic alterations. There was no correlation between SCE frequency and duration of exposure during the heavy spraying period, but a good correlation between incidence of unstable chromosomal aberrations, especially micronuclei, and duration of exposure was found. Although the results of micronucleus and chromosome aberration assays did correlate, the yield of micronuclei at the end of the season was higher than expected. Since micronuclei can be induced by agents damaging either the chromosomes directly or the spindle apparatus, we suppose that some of the micronuclei are formed by entire chromosomes due to failure of the mitotic spindle. Our present data, particularly the percentage of binucleated cells carrying more than three micronuclei (even up to seven micronuclei per cell) (Table 2) provide some evidence that a portion of the micronuclei were induced by the spindle-inhibiting effects of pesticides. The influence of organic solvents in the applied pesticide preparations also cannot be ruled out. Little further insight into quantifying the portion of micronuclei caused by failure of the mitotic spindle can be gained using in situ hybridization with whole centromere DNA probes in cytochalasin-induced binucleated cells. Improvements in the micronucleus assay in vitro, particularly a method for chromosome painting in micronuclei, developed more recently are expected to elucidate the interrelationship between micronucleus formation and mutagenesis and to evaluate the relative hazards of low dose response to aneugenic chemicals (Parry et al., 1996). This will lead to a better understanding and interpretation of results obtained by examination of in vivo exposed humans.
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Further prospective monitoring of persons with chromosomal and micronuclear findings will make it possible to determine the full significance of exposure to pesticides and to evaluate whether they are prognostically associated with an increased risk of adverse health consequences. CONCLUSION
In summary, from the data presented here, we conclude that environmental exposure to a mixture of pesticides caused genetic hazards in circulating lymphocytes of exposed sprayers. Genetic hazards correlated positively with duration of exposure. In occupationally unexposed subjects, living in an agricultural area, an elevated incidence of spontaneously occurring micronuclei was observed at the end of the spraying season, which is probably due to pollution of the air by airborne pesticides. The positive correlation between the results of the micronucleus and chromosome aberration assays documents that the micronucleus assay, which is a cheaper, simpler, and more sensitive method, can be successfully used for screening purposes. This work illustrates the need to implement more rigid guidelines to minimize or prevent further exposure. ACKNOWLEDGMENTS This study was carried out in cooperation with the Occupational Health Department in Aleksandrovac and the Institute of Occupational and Radiological Health in Belgrade. The authors are grateful for the skillful assistance of the medical staff in both institutions. We are grateful to Dr. Dus˘an Djuric´ for his enthusiastic cooperation and helpful suggestions during this study.
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