_??_1988 by Cytologia, Tokyo Cytologi a 53: 627-634, 1988

Vicia faba-Sister Chromatid Exchanges of the Organophosphorus

Insecticides Methyl parathion, Dimethoate , Oxydemeton methyl, Azinphos methyl and Phoxim

Sandra Gomez-Arroyo, Priscila Castillo-Ruiz1 , Josefina Cortes-Eslava and Rafael Villalobos-Pietrini

Laboratorio de Citogenetica y Mutagenesis Ambientales , Centro de Ciencias de la AtmSsfera, Universidad Nacional Autonoma de Mexico, Coyoacan 04510, Mexico , D. F. Mexico Accepted August 22, 1987

The organophosphorus insecticides are an important group of substances used in agricul tural practice. They can interrupt the nerve impulse of vertebrates by blocking the enzyme cholinesterase which is an essential neurotransmitter (O'Brien 1969, Karczmar et al. 1970, Aldrige 1971, Fukuto 1971.). This blockade is due to the phosphorylation of the hydroxylserine group on the active enzyme site (Fest and Schmidt 1973). Another important aspect of these compounds is their alkylating property (Preussmann 1969, Lofroth 1970, Bedford and Robinson 1972, Wooder and Wright 1981), although it is not known what type of alkylation in nucleic acids is responsible for their various biological effects (Loveless 1969). The damage produced by organophosphorus insecticides to the genetic material is one of the toxicological risks seldom taken into consideration despite their potential effects on human health. It is important to determine the behavior of organophosphorus type compounds on DNA and one way to do so is by carrying out different genotoxic assays with various biological organisms. In general, the direct genotoxic assays in animals are expensive and time con suming. Plants, however, provide a suitable alternative for the detection of the effects of chemical pollutants (de Serres and Shelby 1978, Constantin and Owens 1982). Different characteristics of Viciafaba and its root tip chromosomes make this plant a widely used orga nism in cytogenetics (Ma 1982). A reliable test to evaluate the damage produced by chemicals is the sister chromatid ex change (SCE) assay used to detect the cytogenetic effects of environmental carcinogens and mutagens (Kato and Shimada 1975, Solomon and Brobow 1975, Stetka and Wolff 1976, Latt et al. 1981). It has been shown in Vicia faba that several agents which produce chromosomal aberra tions also increase the number of SCE (Kihiman and Sturelid 1978). Due to the wide distribution and the high toxicity of pesticides as well as their potential genetic risks to the human population, it is important that additional genotoxic studies be carried out. In this paper, the organophosphorus insecticides methyl parathion, dimethoate, oxydemeton methyl, azinphos methyl and phoxim are used to evaluate the induction of SCE's in chromosomes of meristematic root-tip cells of Vicia faba.

Material and methods

Viciafaba seeds were germinated between two cotton layers moistened in tap water. The

1 Present address: Genetics Laboratory, P. O. Box 4000, Department of Plant Science, Macdonald College of McGill University, Ste. Anne de Bellevue, Quebec, Canada H9X ICO. 628 Sandra G6mez-Arroyo, et al. Cytologia 53 roots of the seedlings 2 to 3cm in length were introduced into a solution containing 100ƒÊM 5-bromodeoxyuridine (BrdUrd), 0.1ƒÊM 5-fluorodeoxyuridine (FdUrd) and 5ƒÊM of uridine

(Urd), through one DNA replication of 20 hours. Afterwards, they were treated for two hours with organophosphorus insecticides (Bayer) (Table 1) . Fresh solutions of BrdUrd, FdUrd and Urd were applied in a second replicate cycle of 20 hours. The treatmens were made in the dark at 19•Ž. Controls were exposed to distilled water and were handled in the same manner.

Table 1. Organophosphorus compounds used for indication of sister chromatid exchanges

Afterwards, the meristems were cut off and treated with colchicine (0.05%) for 3 hours and stained with Feulgen differential technique described by Tempelaar et al. (1982) but modified as follows: root tips were fixed with glacial acetic acid for one hour and then placed in ethanol acetic acid (3:1) for two days at -20•Ž and also in 70% ethanol for 15min at room temper ature. Hydrolysis was carried out in 5N HC1 for 80min at 28•Ž. The root tips were washed three times with distilled water and stained with Schiff reagent (Feulgen procedure) for 12 min in the dark. For maceration, root tips were treated with 2% pectinase dissolved in citrate buf fer (pH 4.7) for 15 min at 28•Ž, then with 45% acetic acid for 10min, and finally transferred to 70% cold ethanol for 30 min. Squashing was done using 45% acetic acid. The slides were made permanent by the dry ice method (Conger and Fairchild 1953), dehydrating with two changes of absolute butanol and mounting in Canada balsam.

Scoring of SCE's was made from 25 metaphase cells for each experiment and its replicate. Statistical analysis was applied by means of a student t test. Slides were coded to avoid knowing to which group they belonged.

Results and discussion

Methyl parathion

The SCE frequencies induced by this insecticide were significantly different from those of the control. There was a dose-effect relationship with a greater effect at a higher concentra tion until a concentration of 1ppm was reached. At 2ppm (Table 2) the tissues were damaged and the mitotic index diminished. These data agree with those of deKergammeaux et al. (1983) who observed a reduction in the mitotic index with increasing concentration of this insec ticide.

Various reports which are not all in agreement on the genetic activity of this compound have been made. It has been reported to induce chromosome clumping and stickiness (de

Kergammeaux et al. 1983) and also chromosomal aberrations in Vicia faba root tips (Gdmez Arroyo et al. 1985). In Hordeum vulgare chlorophyll mutations (Panda and Sharma 1979) 1988 Sister Chromatid Exchanges of the Organophosphorus Insecticides 629

and chromosomal aberrations have been induced in meiotic and mitotic cells (Kaur and Grover 1985a, b). Methyl parathion has been reported to break the plasmid El DNA of E. coli (Griffin and Hill 1978) and induce mitotic gene conversion in Saccharomyces cerevisiae (Fahrig 1974). W aters et al. (1980) also found it mutagenic in Salmonella typhimurium and S. cerevisiae but Si mmon et al. (1976) described it as not mutagenic in S. typhimurium and S. cerevisiae, and in addition, Mohn (1973) and Wild (1975) have reported it as non-mutagenic in E. coli. Likewise, methyl parathion did not induce dominat lethals in mice (Jorgenson et al. 1976). The growth of three human hematopoietic cell lines was inhibited by methyl parathion but it did not induce chromosomal aberrations (Huang 1973) neither in the lymphocyte cultures of plant workers chronically exposed (de Cassia Stocco et al. 1982). This disagrees with Yoder et al. (1973) who found chromosomal aberrations in lymphocytes of agricultural workers during extensive occupational exposure, and with van Bao et al. (1974) who observed a significant in crease of chromosomal aberrations in acutely intoxicated persons. Chen et al. (1981) induced SCE and cell cycle delay in the human lymphoid cell line B35M and in Chinese hamster cell line V79 with methyl parathion . Sobti et al. (1982) also found methyl parathion to increase SCE in another human lymphoid cell line (LAZ-007) and Grover and Malhi (1985) observed the induction of micronuclei in bone marrow cells in rat .

Table 2. SCE frequencies induced by methyl parathion in Vicia fabat

•õ n=50 metaphases in 2 repetitions

•õ•õ SCEs/metaphase * P•á0 .001

Table 3. SCE frequencies induced by dimethoate in Viciafabat

•õ n=50 metaphases in 2 repetitions

•õ•õ SCEs/metaphase N. S. not-significant * P•á0 .001

Dimethoate (Rogor) This insecticide did not increase the number of SCE significantly at 0.5ppm but it did at both 1.0 and 2.0ppm. A dose dependent-effect relationship was not observed. However, the induction of SCE was more than double the control frequency (Table 3). Higher concentra tions damaged the tissues which did not allow us to make an analysis at higher levels. Differences in results have also been described with this compound. In Hordeum vulgare, dimethoate induced chromosomal aberrations in mitotic and meiotic cells (Kaur and Grover 1985a, b). In Vicia faba, chromosomal alterations and a decrease in the mitotic index were 630 Sandra G6mez-Arroyo, et al. Cytologia 53

observed (Amer and Farah 1974). In E. coli K12, dimethoate induced 5-methyl tryptophan resistant mutants (Mohn 1973), streptomycin resistant mutants (Wild 1975) and a positive in the reversion assay system in E. coli and in S. typhimuriumTA100 (Moriya et al. 1983). Dimethoate was also positive in S. typhimurium G46 in treated mice using the host mediated assay (Ulna Rani et al. 1980). Shirasu et al. (1976) did not find revertants in the B. subtilis rec-assay. Mitotic gene con version (ade 2 and trp 5 loci) was induced by dimethoate in S. cerevisiae (Fahrig 1974) but in Schizosaccharomyces pombe the mutation frequency was not increased at the ade 6 locus (Gilot-Delhalle et al. 1983). Sobti et al. (1982) observed an increase in SCE in human lymphoid cell lines. Van Bao et al. (1974) found a significant increase in chromosomal aberrations in acutely intoxicated persons. Chen et al. (1981) observed an increase of SCE in the Chinese hamster cell line V79 and Usna Rani et al. (1980) observed an increase in micronuclei in bone marrow cells of mice fed with dimethoate (51.7mg/kg).

Oxydemeton methyl (Metasistox) In all concentrations, the frequencies of SCE were signicantly above the control. A dose response relationship was not observed because the peak frequency was reached at 0.5 ppm which was almost double the control value (Table 4). Higher concentrations damaged the tissues.

Table 4. SCE frequencies induced by oxydemeton methyl in Vicia fabai

•õ n=50 metaphases in 2 repetitions •õ•õ SCEs/metaphase * P•á0 .001

Oxydemeton methyl is reported to be mutagenic in E. coli (Mohn 1973, Wild 1975) and to produce mitotic gene conversion in S. cerevisiae (Fahrig 1973, 1974). It was found non mutagenic or a weak inducer of recessive sex-linked lethal mutations in Drosophila melanogaster depending on the genetic strain used (Vogel 1974). Oxydemeton methyl is considered as to be a weak inducer of SCE since it produced a signi fi cant increase in Chinese hamster V79 cells only at a relatively high dose level (Chen et al. 1982). It also produced micronuclei in mice bone marrow and SCE in human lymphocyte cultures (Pandita 1983).

Azinphos methyl (Guthion or Gusathion) In all concentrations, the induction of SCE was significant although there was not a con centration-response relationship, thus the highest frequency was obtained at 0.5ppm which decreased at 1.0 and 2.0ppm. Higher concentrations inhibited cell division. In this respect, Alam et al. (1974) described drastic chromosomal anomalies that lead to cell death of Chinese hamster cells (line CHO-K). On the other hand, Antunes-Madeira and Madeira (1979) found that this insecticide dramatically increased the permeability of the liposome membrane. This fact is probably the result of the cell death induced in Vicia faba root tip cells which were treated with higher concentrations than 2.0ppm. 1988 Sister Chromatid Exchanges of the Organophosphorus Insecticides 631

Azinphos methyl is not mutagenic to S. cerevisiae (Riccio et al. 1981) nor will it induce dominant lethal mutations in mice (Jorgenson et al. 1974). Likewise, it did not increase the frequency of SCE in Chinese hamster V79 cells (Chen et al. 1982), nor in the central mudmin nows (Umbra limi) (Vigfusson et al. 1983). But it did produce mitotic recombination in S. cerevisiae (Simmon et al. 1976), forward mutations in S. cerevisiae and S. pombe (Gilot-Delhalle et al. 1983), C-mitotic action in plants (Grant 1973) and chromosome anomalies in Chinese hamster line CHO-KI (Alam et al. 1974).

Table 5. SCE frequencies induced by azinphos methyl in Viciafabai

•õ n=50 metaphases in 2 repetitions•õ•õ SCEs/metaphase * P•á0 .001

Table 6. SCE frequencies induced by phoxim in Vicia faba•õ

•õ n=50 metaphases in 2 repetitions•õ•õ SCEs/metaphase * P•á0 .001

Phoxim (Bay 77488) The cytotoxicity of this insecticide proved to be higher than the other organophosphorus pesticides. This is the reason that the concentrations used in this work are the lowest for this compound. However, in addition to methyl parathion, phoxim was the best SCE inducer in Vicia faba. All concentrations produced significant differences from the control and with a concentration of 0.5 ppm the increase in SCE frequency was more than double the control (Table 6). This insecticide, which is used mainly in stored grains, is degraded fairly rapidly into non toxic compounds and has a low mammalian toxicity (Mason and Meloan 1976), however, it was highly toxic to Viciafaba root tip cells. There are few studies on the genetic effects of this insecticide. It was negative in response for the production of reverse mutations in bacteria (Moriya et al. 1983).

Summary

Vicia faba root tips were treated for two hours with several concentrations of the orga nophosphorus insecticides methyl parathion, dimethoate, oxydemeton methyl, azinphos methyl and phoxim, after one incroporation cyclewith 5-bromodeoxyuridine (5-BrdUrd). Afterwards, the roots were exposed to a second replication cycle with 5-BrdUrd. The differential stain technique described by Tempelaar et al. (1982) with some modifications was used. The results showed that the two insecticides which are the most effective inducers of SCE are methyl 632 Sandra Gomez-Arroyo, et al. Cytologia 53 parathion and phoxim. In no case was a concentration-response relationship observed. The treatments (except with dimethoate 0.5 ppm) produced SCE frequencies which differed significantly from those of the control.

Acknowledgements

We thank to Dr. William F. Grant for comments and suggestions on the manuscript, Miguel Angel Meneses and Ana Rosa flores-Marquez for technical assistance. This work was supported in part by CONACyT grant PCCBBNA 021988.

References

Alam, M. T., Corbiel, M., Chagnon, A. and Kasatiya, S. A. 1974. Chromosomal anomalies induced by the orga nic phosphate pesticide guthion in Chinese hamster cells. Chromosoma (Berl.) 49: 77-86. Aldrige, W. N. 1971. The nature of the reaction of organophosphorus compounds and carbamates with esterases. Bull. Wld. Hlth. Org. 44: 25-30. Amer, S. M. and Farah, O. R. 1974. Cytological effects of pesticides. VI. Effects of the insecticide "Rogor" on the mitosis of Vicia faba and Gossypium barbadense. Cytologia 39: 507-514. Antunes-Madeira, M. C. and Madeira, V. M. 1979. Interaction of insecticides with lipid membranes. Biochem. Biophys. Acta 550: 384-392. Bedford, C. T. and Robinson, J. 1972. The alkylating properties of organophosphates. Xenobiotica 2: 307 -337. Chen, H. H., Hsueh, J. L., Sirianni, S. R. and Huang, C. C. 1981. Induction of sister chromatid exchanges and cell cycle delay in cultured mammalian cells treated with eight organophosphorus pesticides. Mutat. Res. 88: 307-316. -, Sirianni , S. R. and Huang, C. C. 1982. Sister-chromatid exchanges and cell-cycle delay in Chinese hamster V79 cells treated with 9 organophosphorus compounds (8 pesticides and 1 defoliant). Mutat. Res. 103: 307-313. Conger, A. D. and Fairchild, L. M. 1953. A quick-freeze method for making smear slides permanent. Stain Technol. 28: 281-283. Constantin, J. M. and Owens, E. T. 1982. Introduction and perspectives of plant genetic and cytogenetic as says. A report of the U. S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. 99: 1-11. de Cassia Stocco, R., Besak, W., Gaeta, R. and Rabello-Gay, N. 1982. Cytogenetic study of workers exposed to methyl parathion. Mutat. Res. 103: 71-76. de Kergommeaux, D. J., Grant, W. F. and Sandhu, S. S. 1983. Clastogenic and physiological response of chro mosomes to nine pesticides in the Vicia faba in vivo root tip assay system. Mutat. Res. 124: 69-84. de Serres, F. J. and Shelby, M. D. 1978. Higher plant systems as monitors of environmental mutagens. En viron. Health Perspect. 27: 1-206. Fahrig, R. 1973. Nachweis einer genetischen W irkung von Organophosphorinsektiziden. Naturwissenschaften 60: 50-51.- 1974. Comparative mutagenicity studies with pesticides. IARC Sci. Pub. 10: 161-181. Fest, C. and Schmidt, K. J. 1973. The Chemistry of Organophosphorus Pesticides. Springer Verlag, Berlin. Fukuto, T. R. 1971. Relationships between the structure of organophosphorus compounds and their activity as acetylcholinesterase inhibitors. Bull. Wld. Hlth. Org. 44: 31-42. Gilot-Delhalle, J., Colizzi, A., Moutschen, J. and Moutschen-Dahmen, J. 1983. Mutagenicity of some organo phosphorus compounds at the ade 6 locus of Schizosacchromyces pombe. Mutat. Res. 117: 139-148. Gomez-Arroyo, S., Baiza, A. M., Lopez, G. and Villalobos-Pietrini, R. 1985. A comparative study of the cyto genetic effects of the insecticides heptachlor, malathion and methyl parathion in Vicia faba. Contam. Ambient. 1: 7-16. Grant, W. F. 1973. Cytological effects of environmental mutagens-pesticides. Mutat. Res. 21: 221-222. Griffin III, D. E. and Hill, W. E. 1978. In vitro breakage of plasmid DNA by mutagens and pesticides. Mutat. Res. 52: 161-169. Grover, I. S. and Malhi, P. K. 1985. Genotoxic effects of some organophosphorus pesticides. Mutat. Res. 155: 131-134. 1988 Sister Chromatid Exchanges of the Organophosphorus Insecticides 633

Huang, C. C. 1973. Effect on growth but not on chromsomes of the mammalian cells after treatment with three organophosphorus insecticides. Proc. Soc. Exp . Biol. Med. 142: 36-40. Jorgenson, T. A., Rushbrook, C. J. and Newel , G. W. 1976. In vivo mutagenesis investigations of ten com mercial pesticides. Toxicol. Appl . Pharmacol. 37: 109 (abstr.). Karczmar, A. G., Nishi, S. and Blaber , L. C. 1970. Investigations, particularly by means of anticholinesterase agents, of the multiple peripheral and central cholinergic mechanisms and of their behavioral implica tions. Acta Vitaminol. Enzimol. 24: 131-189 . Kato, H. and Shimada, H. 1975. Sister chromatid exchanges induced by mitomycin C, a new method of detecting DNA damage at chromosomal level. Mutat . Res. 28: 459-464. Kaur, P. and Grover, I. S. 1985a. Cytological effects of some organophosphorus pesticides I . Mitotic effects. Cytologia 50: 187-197.- and -1985b. Cytological effects of some organophosphorus pesticides II. Meiotic effects. Cytologia 50: 199-211. Kihlman, B. A. and Sturelid, S. 1978. Effects of caffeine on the frequencies of chromosomal exchanges induced by chemical mutagens in root tips cells of Viciafaba. Hereditas 88: 35-41. Latt, S. A., Allen, J., Bloom, S. E., Carrano, A., Falke, E., Kram, D., Scheider, E., Schreck, R., Tice, R., Wit fi eld, B. and Wolff, S. 1981. Sister chromatid exchange: a report of the gene-tox program. Mutat. Res. 87: 17-62. Lofroth, G. 1970. Alkylation of DNA by dichlorvos. Naturwissenschaften 8: 393-394. Loveless, A. 1969. Possible relevance of 0-6 alkylation of deoxyguanosine to the mutagenicity and carcinogenici ty of nitosamines and nitrosamides. Nature 223: 206-207. Ma, T. H. 1982. Vicia cytogenetic test for environmental mutagens: A report of the U. S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. 99: 257-271. Mason, W. A. and Meloan, C. E. 1976. Degradation products of phoxim (Bay 77488) on stored wheat. J. Agric. Food Chem. 24: 299-304. Mohn, G. 1973. 5-Methyl tryptophan resistance mutation in Escherichia coli K12; mutagenic activity of mono functional alkylating agents including organophosphorus insecticides. Mutat. Res. 20: 7-15. Moriya, M., Ohta, T., Watanabe, K., Miyazawa, T., Kato, T. and Shirasu, Y. 1983. Further mutagenicity studies on pesticides in bacterial reversion assay systems. Mutat. Res. 116: 185-216. O'Brien, R. B. 1969. Phosphorylation and carbamylation of cholinesterase. Ann. N. Y. Acad. Sci. 160: 204 -214. Panda, B. B. and Sharma, C. B. 1979. Organophosphate induced chlorophyll mutations in Hordeum vulgare. Theor. Appl. Genet. 55: 253-255. Pandita, T. K. 1983. Mutagenic studies on the insecticide metasystox-R with different genetic systems. Mutat. Res. 124: 97-102. Preussmann, R. 1969. Untersuchungen zum Nachweis alkylierender Agentien. Arzneim. Forsch. 19: 1059 -1073. Riccio, E., Shepherd, G., Pomeroy, A., Mortelmans, K. and Waters, M. D. 1981. Comparative studies between the S. cerevisiae D3 and D7 assay of eleven pesticides. 12th Ann. Meeting Environ. Mutagen Soc. p. 82. Shirasu, Y., Moriya, M., Kato, K., Furuhashi, A. and Kada, T. 1976. Mutagenicity screening of pesticides in the microbial system. Mutat. Res. 40: 19-30. Simmon, V. F., Poole, D. C. and Newell, G. W. 1976. In vitro mutagenic studies of twenty pesticides. Toxicol. Appi. Pharmacol. 37: 109 (abstr.). Sobti, R. C., Krishan, A. and Pfaffenberger, C. D. 1982. Cytokinetic and cytogenetic effects of some agricul tural chemicals on human lymphoid cells in vitro: organophosphates. Mutat. Res. 102: 89-102. Solomon, E. and Bobrow, M. 1975. Sister chromatid exchanges-a sensitive assay of agents damaging human chromosomes. Mutat. Res. 30: 273-278. Stetka, D. G. and Wolff, S. 1976. Sister chromatid exchange as an assay for genetic damage induced by mutagen carcinogens. II. In vitro test for compounds requiring metabolic activation. Mutat. Res. 41: 333-342. Tempelaar, M. J., de Both, M. T. J. and Versteegh, J. E. G. 1982. Measurement of SCE frequencies in plants: a simple Feulgen-staining procedure for Vicia faba. Mutat. Res. 103: 321-326. Usna Rani, M. V., Reddy, O. S. and Reddy, P. P. 1980. Mutagenicity studies involving aldrin, endosulfan, dimethoate, phosphamidon, carbaryl and ceresan. Bull. Environ. Contam. Toxicol. 25: 277-282. van Bao, T., Szab6, I., Ruzicska, P. and Czeizel, A. 1974. Chromosome aberrations in patients suffering acute organic phosphate insecticide intoxication. Humangenetik 24: 33-57. Vigfusson, N. V., Vyse, E. R., Pernsteiner, C. A. and Dawson, R. J. 1983. In vivo induction of sister-chro matid exchange in Umbra limi by the insecticides endrin, chlordane, diazinon and guthion. Mutat. 634 Sandra Gomez-Arroyo, et al.

Res. 118: 61-68. Vogel, E. 1974. Mutagenic activity of the insecticide oxydemetonmethyl in a resistant strain of Drosophila melanogaster. Experientia 30: 396-397. Waters, M. D., Simmon, V. F., Mitchel, A. D., Jorgenson, T. A. and Valencia, R. 1980. An overview of short term test for the mutagenic and carcinogenic potential of pesticides. J. Environ. Sci. Health 15: 867 -906. Wild, D. 1975. Mutagenicity studies on organophosphorus insecticides. Mutat. Res. 32: 133-150. Wooder, M. F. and Wright, A. S. 1981. Alkylation of DNA by organophosphorus pesticides. Acta Pharmacol. Toxicol. 48: (Suppl.) 51-55. 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.