Chromosomal Aberrations in Cultured Human Lymphocytes Treated with the Mixtures of. Ethyl Methanesulfonate

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1 2000 The Japan Mendel Society Cytologia 65: 83-92, 2000 Chromosomal Aberrations in Cultured Human Lymphocytes Treated with the Mixtures of Carbosulfan, and Ethyl Carbamate Ethyl Methanesulfonate Eyyup Rencuzogullari and Mehmet Topakta cukurova University, Faculty of Arts and Sciences, Department of Biology, Adana, Turkey Accepted December 6, 1999 Summary The aim of this study was to investigate the ability of the mixtures of carbosulfan (C) (the effective ingredient of "marshal", an insecticide/nematocide) and ethyl carbamate (EC)(carcino- gen) and also the mixtures of carbosulfan and ethyl methanesulfonate (EMS)(mutagen) to induce the chromosomal aberrations (CA) in human peripheral lymphocytes and to investigate whether the test substances as a mixture had any synergistic effect. All the mixtures of the test substances increased the formation of chromosomal aberrations at all the concentrations and treatmen times compared to the control groups. The combination of carbosulfan and EC synergistically increased the formation of CA, at the same concentrations of both carbosulfan and EC at 48 h treatmentime. The combination of carbosulfan and EMS also synergistically increased the formation of CA especially at the high concentrations for 48 h treatmentimes. Key words Carbosulfan, Ethyl carbamate, Ethyl methanesulfonate, Human lymphocytes, Chromosomal aberrations, Synergism. We are living in hazardous conditions due to combinations of the environmental pollutants namely pesticides, nuclear chemicals, drugs and airborne pollutants. Humans are exposed to these pollutants as a mixture because of their living conditions. Most of these pollutants are mutagenic or carcinogenic and have synergism on the induction of gene mutations and chromosomal aberrations (CA). For example, Bronzetti et al. (1981) reported that complex mixtures from some factories (especially leather and glasses manufacturing) caused to form gene mutations in D7 strain of Saccharomyces cerevisiae. The formation of CA and frequency of micronuclei were increased in cultured human lymphocytes from rural populations occupationally or environmentally exposed to pesticides (Paldy et al. 1987, Kourakis et al. 1992, Bolognesi et al. 1993, Anwar 1994). CA was also increased in workers exposed to occupational clastogens (Al-Sabti et al. 1992), in workers of rubber factory (Rendon et al. 1994) and in human populations at risk in Egypt occupationally exposed to chemicals and airborne pollutants (Anwar 1994). Dolara et al. (1994) observed that a mixture of 15 pesticides commonly found in foods increased the nonsynchronous centromeric separations in cultured human lymphocytes, however mixture of pesticides did not increase the number of CA and poliploid cells. Degreave et al. (1984) reported that a mixture of 4 insecticides did not induce the formation of CA in mouse bone marrow and did not cause to lethal gene mutations. According to these results, it was observed that, most of the chemicals induced the mutations in prokaryotes and eucaryotes. However Degreave et al. (1985) reported that the mixture of insecticides increased dominant lethal gene mutations in mice. The mixture of methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS) synergistically increased the mutant colonies in mouse lymphoma cells (Tarlo et al. 1988) and increased the 6TG (6-thioguanine) resistant mutants in Chinese hamster V79 cells (Kojima et al. 1992).

2 84 Eyyup Rencuzogullari and Mehmet Topaktas Cytologia 65 As shown in these results, the chemicals exhibited synergism on the induction of chromosomal aberrations (CA) when the populations exposed to a mixture of these chemicals. Carbamate pesticides have been used extensively in the cukurova region which is one of the most important agricultural regions in Turkey. An estimated kg of carbamate pesticides were used in province of Adana in this region in In the same year kg of Carbosulfan (trade name "marshal") was used in province of Adana of cukurova. As understood, the aim of this study was to investigate the effects of the mixtures of carbosulfan and ethyl carbamate (EC) (carcinogen), and also the mixtures of carbosulfan and EMS (mutagen) on the chromosomal aberrations (CA) in human lymphocytes and also to investigate whether the test substances as a mixture had any synergistic effect. Materials and methods The chromosomal aberration test in human peripheral blood was used as the test system and carbosulfan (C) (a carbamate pesticide, extensively used in the cukurova region as insecticide/nematocide), ethyl carbamate (EC) (carcinogen) and ethyl methanesulfonate (EMS) (mutagen) were used as the test substance. The chemical structure and formula of test substances are shown in Fig. 1. Whole blood (0.2 ml) from two healthy donors (one male, one female, non smokers, age 30) was added to 2.5 ml chromosome medium B (Biochrom cat. no: F5023). The cultures were incubated at 37 C for 72 h. Firstly, the effects of the test substances separately on the chromosomal aberrations were investigated and then the cells were treated with the mixtures of different concentrations of the test substances as below: Carbosulfan (C)+2 ~ 10-3 M EC 2. tube: 80% ethanol 10ƒÊl+d.water 10ƒÊl 3. tube: 2.5 ~10-5 M C+2 ~ 10-3 M EC 4. tube: 5 ~10-5 M C+2 ~10-3 M EC 5. tube: 10-4 M C+2 ~ 10-3 M EC Carbosulfan (C)+4 ~ 10-3 M EC 2. tube: 80% ethanol 10 ƒêl+d.water 10ƒÊl 3. tube: 2.5 ~10-5 M C+4 ~ 10-3 M EC 4. tube: 5 ~10-5 M C +4 ~ 10-3 M EC 5. tube: 10-4M C+4 ~10-3 M EC Carbosulfan (C)+8 ~ 10-3 M EC 2. tube: 80% ethanol 10ƒÊl+ d.water 10ƒÊl 3. tube: 2.5 ~1 ~-5 M C+8 ~10-3 M EC 4. tube: 5 ~10-5 M C+8 ~10-3 M EC 5. tube: 10-4 M C+ 8 ~ 10-3 M EC Carbosulfan (C)+5 ~ 10-4 M EMS 2. tube: 80% ethanol % ethanol 10ƒÊl 3. tube: 2.5 ~10-5 M C+5 ~ 10-4 M EMS 4. tube: 5 ~10-5 M C+ 5 ~ 10-4 M EMS 5. tube: 10-4 M C+ 5 ~ 10-4 M EMS Carbosulfan (C)+10-3 M EMS 2. tube: 80% ethanol 10ƒÊl+50% ethanol 10ƒÊl 3. tube: 2.5 ~10-5 M C+10-3 M EMS 4. tube: 5 ~10-5 M C+10-3 M EMS 5. tube: 10-4 M C+10-3 M EMS Carbosulfan (C)+2 ~ 10-3 M EMS 2. tube: 80% ethanol 10ƒÊl+50% ethanol 10ƒÊl 3. tube: 2.5 ~10-5 M C+2 ~10-3 M EMS 4. tube: 5 ~10-5 M C+2 ~ 10-3 M EMS 5. tube: 10-4 M C+2 ~ 10-3 M EMS The human lymphocytes were treated with the mixtures of the test substances for 24 h (the test substances were added 48 h after initiating the culture) and 48 h (the test substances were added 24 h after initiating the culture). A normal control and solvent control (for carbosulfan 80% ethanol, for EC distilled water and for EMS 50% ethanol) were present at each treatment time. Colchicine (0.06ƒÊg/ml) was present for the last 2 h of the culture. To collect the cells, the cultures were centrifuged (1,200 rpm, 15 min), treated with hypotonic solution (0.4% KCl) for 30 min at 37 Ž, and then fixed in fixative (methanol : glacial acetic acid 3 : 1) for 20 min at room temperature. The treatment with fixative was repeated 3 times. The cells were spread on glass slides and air dried. One

3 Chromosomal Aberrations in Cultured Cells Treated with Chemicals 85 Fig. 1. a) Carbosulfan (3,3-dihydro-2,2-dimethyl-7-benzofuranyl((dibutylamino)thio)methylcarbamate) (CAS registry number: ). b) Ethyl carbamate (urethane) (CAS registry number: ). c) Ethyl methanesulfonate (methanesulfonic acid ethylester) (CAS registry number: ). day old slides were stained with 5% Giemsa stain (ph 6.72) prepared in Sorensen buffer solution, for 15 min, washed in distilled water, dried and mounted with entellan. From each donor 100 and a total 200 well spread metaphases per dose were examined at 1,000 ~ magnification for occurrence of different types of CA. Numerical and structural chromosomal abnormalities within each metaphase were recorded. These data were used to determine the percentage of the cells with abnormalities (abnormal cells) and the chromosomal aberrations per cell (CA/cell). In addition, for the occurrence of the mitotic index (MI) a total of 6,000 cells were scored and the percentage of the cells in mitotic division was determined. The significance between the mean percentage of abnormal cells and the CA/cell of the treated cultures and their controls were determined using the Chi-square test and also between the MI of the treated cultures and their controls were determined using the t-test. Therefore, for determined the synergism between the mixtures of carbosulfan and EC, and also between carbosulfan and EMS, the results obtained from mixtures of the test substances were compared with the results obtained from the test substances separately. For example, the result obtained from 2.5 ~ 10-5 M carbosulfan+2 ~ 10-3 M EC was compared with the results obtained from both 2.5 ~ 10-5 M carbosulfan and 2 ~ 10-3 M EC separately. Results The mixtures of carbosulfan and 2 ~ 10-3 M EC rarely increased the percentage of abnormal cells and the CA/cell at 24 h compared to the control groups. However, the mixtures of carbosulfan and 4 ~ 10-3 M EC or carbosulfan and 8 ~ 10-3 M EC increased both the percentage of abnormal cells and the CA/cell at all concentrations for 24 h treatment time when compared with the control groups. The mixtures of carbosulfan and the different concentrations of EC decreased the MI when compared with both the normal and solvent control group for 24 h treatment time (Table 1). There was not any synergism between the mixtures of carbosulfan and different concentrations of EC by increasing of the percentage of the abnormal cells and the CA/cell at 24 h, excluding the mixture of 10-4M carbosulfan and 8 X 10-3 M EC which synergistically increased the CA/cell. The mixtures of 2.5 ~ 10-5 and 5 ~ 10-5 M carbosulfan and 2 ~ 10-3 M EC synergistically increased the MI, while the mixture of 2.5 ~10-5 and 10-4 M carbosulfan and 4 ~ 10-3 M EC synergistically decreased the MI at 24 h. Any effects of mixture of carbosulfan and 8 ~ 10-3 M EC on the MI were not observed (Table 1). The mixtures of carbosulfan and EC increased both the percentage of abnormal cells and the CA/cell at all concentrations for 48 h treatment time when compared with the control groups. The mixtures of carbosulfan and the different concentrations of EC decreased the MI when compared with both the normal and solvent control group for 48 h treatment time (Table 2).

4 86 Eyyup Rencuzogullarl and Mehmet Topaktas Cytologia 65 The combinations of 2.5 ~ 10-5 M carbosulfan+2 ~ 10-3 M EC and 2.5 ~ 10-5 M carbosulfan+ 4 ~ 10-3 M EC synergistically increased the CA/cell at 48 h treatment time. And also the mixtures of 2.5 ~ 10-5 M carbosulfan+ 8 ~ 10-3 M EC synergistically increased the percentage of the abnormal cells. There was not any synergism when the MI was decreased except 2.5 ~ 10-5 M carbosulfan+ 8 ~ 10-3 M EC (Table 2). The mixtures of carbosulfan and different concentrations of EMS increased both the percentage of the abnormal cells and the CA/cell at all concentrations of EMS for 24 h treatment time compared to the control groups. The mixtures of carbosulfan and the different concentrations of EMS rarely decreased the MI at 24 h (Table 3). Generally, it was observed that there was a synergism between carbosulfan and 5 ~ 10-4 M EMS, and between 5 ~10-5 M carbosulfan and 10-3 M EMS when increased of both the percentage of the abnormal cells and the CA/cell at 24 h. On the MI, there was not any synergism between carbosulfan and EMS, except the mixture of 5 ~ 10-5M carbosulfan and 5 ~ 10-4 M EMS at 24 h treatment time (Table 3). The mixtures of carbosulfan and different concentrations of EMS increased both the percentage of the abnormal cells and the CA/cell at all concentrations of EMS for 48 h treatment time compared to the control groups. They also decreased the MI at 48 h treatment time (Table 4). A synergism was observed between carbosulfan and EMS in percentage of the abnormal cells and the CA/cell at 10-3 M and 2 ~ 10-3 M concentrations of EMS when mixtured with carbosulfan at 48 h. The mixtures of carbosulfan and EMS synergistically decreased the MI especially at the two highest concentrations of EMS when mixtured with carbosulfan at 48 h treatment time (Table 4). Chromatid break, isochromatid break and chromatid exchange which were the most common abnormalities were shown in Fig. 2. Discussion In this study, the mixtures of carbosulfan and EC and also the mixtures of carbosulfan and EMS increased the CA and decreased the MI when compared with control groups. The mixtures of carbosulfan and EC synergistically increased the CA at 48 h treatment time and also a synergism was observed between carbosulfan and EMS. However, Degreave et al. (1984) reported that a mixture of 4 insecticides did not induce the CA in mouse bone marrow cells and did not cause to form lethal gene mutations and Dolara et al. (1994) also reported that the mixtures of 15 pesticides commonly found in foods did not induce significant variations in the number of poliploid cells, in the number of CA, however, the mixtures of the pesticides increased dose-dependently the number of nonsynchronous centromeric separations in human lymphocytes. However, it was reported that the mixture of pesticides (Degreave et al. 1985) and the complex mixtures of some factories (Bronzetti et al. 1981) caused dominant lethal mutation in mice and gene mutation in S. cerevisiae, respectively. It was also reported that, the chromosomal aberration, SCE and micronuclei were increased in cultured human lymphocytes from rural population and from workers exposed to occupational clastogenes, pesticides and airborne pollutants (Paldy et al. 1987, Kourakis et al. 1992, Al-Sabti et al. 1992, Bolognesi et al. 1993, Sasiadek 1993, Anwar 1994, Rendon et al. 1994). As shown, the chemicals caused, the greatest genetic damage when used as a mixture. It was previously reported that carbosulfan and ethyl carbamate have been a higher genotoxic risk following in vivo exposures more than the in vitro treatment. Because carbamates are able to convert into the nitrosated derivatives and metabolites following in vivo exposures (Boyland and Williams 1969, according to Nerry 1969 from Bateman 1976, Topaktas and Rencuzogullarl 1996, Topaktas et al. 1996). Carbosulfan was metabolised to 26 different forms in mature and immature fruit of Valencia oranges (Clay and Fucuto 1984). Ethyl carbamate was also metabolised to ethyl ester of cytosine-5-carboxylic acid which plays a part in carcinogenesis (Boyland and Williams

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7 Chromosomal Aberrations in Cultured Cells Treated with Chemicals 89

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9 Chromosomal Aberrations in Cultured Cells Treated with Chemicals ). Therefore, EMS is known as alkylating agent causing alkylation of cellular substances. Kojima et al. (1992) reported that EMS may react by mixed types of SN1-type (substitution, nucleophilic unimolecular) and SN2-type (substitution, nucleophilic bimolecular) mechanisms. According to this result, EMS is able to alter coupling of the nucleotides by alkylating and causing to gene mutations. As shown, the mixture of carbosulfan and ethyl carbamate and also the mixture of carbosulfan and EMS synergistically increased the abnormalities. MMS and EMS are known as alkylating agents. These two alkylating agents differ only in having a methyl and ethyl group, respectively, in their chemical structure and also showed a synergism on the induction of mutations. Carbosulfan also has three methyl groups in its chemical structure. Therefore, it can be concluded that the synergism between carbosulfan and EMS may react by their methyl and ethyl groups like the synergism showed between MMS and EMS. The synergistic effect of the mixture of carbosulfan and ethyl carbamate were less than the effect of the mixture of carbosulfan and EMS. This difference might be caused from the "S" group which localised in chemical structure of EMS. In the same way, the thiocarbamates have a higher genotoxic effect than the carbamates because of their "S" groups (Topaktas et al. 1999). So that, the chemicals can also acquire mutagenic properties when "S" group is introduced to the their chemical structure. In this study, the mixtures of carbosulfan and EMS synergistically decreased the MI especially at 48 h treatment time. The decreases of the MI might be caused by the decrease of the ATP level and the pressure from the functioning of the energy production centre (Epel 1963, Jain and Andsorbhoy 1988). Alkylating agent like EMS, caused to form mutations at the concentrations which are not cytotoxic. Although the EMS separately could not decrease Fig. 2. Typical chromosomal abnormalities. a) chromatid isochromatid break (5 ~ 10-5 M carbosulfan + 2 ~ 10-3M the MI (except the highest concentrations for EMS, 48 h), c) chromatid exchange (10-4 M carbosulfan+ 24 and 48 h treatment times), while the mixtures of the carbosulfan and EMS showed syn- 2 ~10-3 M EMS 48 h). ~1000. ergism on the decreasing of the MI. As a result, it can be concluded that the combined treatment of carbosulfan with a carcinogen break (10-4 M carbosulfan+2 ~10-3 M EMS, 48 h), b)

10 92 Eyyup Rencuzogullari and Mehmet Topaktas Cytologia 65 (EC) or with a mutagen (EMS) might cause the highest genotoxic risk for humans. According to the result obtained from this study, it was clearly shown that the mutagens and the carcinogens caused the greatest genetic damage in the regions in which pesticides were extensively used. For this reason, it is necessary to inform the populations exposed to the environmental pollutants and rural populations exposed to the pesticides. And it is also necessary to be careful when exposed to these pollutants for the health of future generations. Acknowledgements This study was supported by Q. U. Research Fund, FBE References Al-Sabti, K., Lloyd, D. C., Edwards, A. A. and Stegnar, P A survey of lymphocyte chromosomal damage in Slovenian workers exposed to occupational clastogens. Mut. Res. 280: Anwar, W. A Assessment of cytogenetic changes in human populations at risk in Egypt. Mut. Res. 313: Bateman, A. J The mutagenic action of urethane. Mut. Res. 39: Bolognesi, C., Parrini, M., Bonassi, S., Ianello, G. and Salanitto, A Cytogenetic analysis of a human population occupationally exposed to pesticides. Mut. Res. 285: Boyland, By E. and Williams, K Reaction of urethane with nucleic acids in vivo. Biochem. J. 111: Bronzetti, G., Leporini, C., Bauer, C., Corsi, C., Nieri, R. and Del Carratore, R Use of short-term tests to detect genetic effects of complex mixtures of environmental interest. Mut. Res. 85: Clay, V. E. and Fucuto, T. R Metabolism of carbosulfan in Valencia orange tree leaves and fruit. Arch. Environ. Contam. Toxicol. 13: Degreave, N., Chollet, M. C. and Moutschen, J Evaluation of the mutagenic potential of four commerical mixtures of insecticides. Food. Chem. Toxicol. 22(8): and \ Mutagenic efficiency of organophosphorus insecticides used in combined treatments. Environmental Health Perspectives 60: Dolara, P., Torricelli, E and Antonelli, N Cytogenetic effects of human lymphocytes of a mixture of fifteen pesticides commonly used in Italy. Mut. Res. 325: Epel, D The effects of carbonmonoxide inhibition of ATP level and the date of mitosis in Sea Urchin Egg. J. Cell. Biol. 17: Jain, A. K. and Andsorbhoy, R. K Cytogenetical studies on the effects of some chlorinated pesticides III. concluding remarks. Cytologia 53: Kojima, H., Konishi, H. and Kuroda, Y Combined mutagenicity of methyl methanesulfonate and ethyl methanesulfonate in chinese hamster V79 cells. Mut. Res. 266: Kourakis, A., Mouratidou, M., Kokkinos, G., Barbouti, A., Kotsis, A., Mourelatos, D. and Dozi-Vassiliades, J Frequencies of chromosomal aberrations in pesticide sprayers working in plastic green houses. Mut. Res. 279: Paldy, A., Puskas, N., Vincze, K. and Hadhazi, M Cytogenetic studies on rural populations exposed to pesticides. Mut. Res. 187: Rendon, A., Rojas, A., Fernandez, S. I. and Pineda, I Increases in chromosome aberrations and in abnormal sperm morphology in rubber factory workers. Mut. Res. 323: Sasiadek, M Sister-chromatid exchanges and cell-cycle kinetics in the lymphocytes of workers occupationally exposed to a chemical mixture in the tyre industry. Mut. Res. 302: Tarlo, K. S., Boehnke, M. and Chin, B Synergism of mutant frequencies in the mouse lymphoma cell mutagenicity assay by binary mixtures of methyl methanesulfonate and ethyl methanesulfonate. Mut. Res. 206: Topakta, M. and Rencuzogullari, E Genotoxic effects of Marshal in Allium cepa L. Tr. J. of Botany 20: and Ila, H. B In vivo chromosomal aberrations in bone marrow cells of rats treated with marshal. Mut. Res. 371: and Logoglu, G Mutagenic, carcinogenic and cytogenetic effects of carbamate, thiocarbamate and dithiocarbamate chemicals. In: Sobti, R. C., Obe, G. and Quillardet, P. (eds.) Trends in Environmental Mutagenesis, Tausco Book Distributors, New Delhi, India.