369

Chem.-Biol. Interactions, 19 (1977) 369--374 © Elsevier/North-Holland Scientific Publishers Ltd.

PESTICIDE INDUCED OUABAIN RESISTANT MUTANTS IN CHINESE HAMSTER V79 CELLS

F A R I D E. A H M E D * , NEIL J. LEWIS and R O N A L D

W. H A R T

Department of Biophysics; Division of Medicinal Chemistry, College of Pharmacy; Department of Radiology, The Ohio State University,Columbus, Ohio 43210 (U.S.A.) (Received June 21st, 1977) (Revision received Sept. 26th, 1977) (Accepted Oct. 3rd, 1977)

SUMMARY

The effect of 3 insecticides (chlordane, dieldrin and carbaryl) and one herbicide (2,4-D-fluid) was studied on the induction of ouabain-resistant mutants in Chinese hamster V79 cells at concentrations approaching the Environmental Protection Agency (EPA) tolerance limits. The kinetics and dose range for cytotoxicity were deterimined by colony formation assay. Results showed that these compounds enhanced the number of ouabainresistant mutants and acted as weak mutagens.

INTRODUCTION

M a n y of the pesticides widely used in agriculture,though not showing an immediate harmful cytotoxic effect in vivo at the concentrations used, m a y have long-term hazardous effects on h u m a n genetic material [1]. Therefore, we have selected some of these pesticides to test their effects on inflicting damage to D N A that will result in mutation induction. Dieldrin was chosen because it was suspected of inducing neoplastic transformation in animals and the E P A had already stopped the marketing of the halogenated pesticide [2]. Recently, a ban was placed on the use of chlordane by the EPA. Carbaryl was selected for testing because it was reported to be too hazardous to m a n and his environment to allow its continued use [3]. The herbicide 2,4-D-fluid was also chosen because there were reports that it had induced mitotic gene conversion in yeast [4]. By employing the ouabain system it was demonstrated that the fungicide captan, either by itself or in its vapour phase, induced resistant variants at low frequencies [5]. W e have chosen ouabain resistance in V 7 9 cells as a * Present address: Department of Biology, Brookhaven National Laboratory, Upton, N e w York 11973, U.S.A. Abbreviation: EPA, Environmental Protection Agency.

370 genetic marker because of the following advantages over other mammalian somatic mutation systems: (i) ouabain-resistant mutants in Chinese hamster are readily induced by ultraviolet radiation and chemical mutagens, (ii) it permits discrimination between weak mutagens because of the low level of spontaneous variants, (iii) it produces data with less intrinsic variability, (iv) there are fewer cell density effects [5] and (v) it has a relatively short m a x i m u m expression time: 40 h [6] and 48 h [7]. MATERIALS

AND METHODS

Cell line and tissue culture An aneuploid cell line (V79), derived from the lung of a male hamster, 2n = 22 [8] was provided by James Trosko, Michigan State University. Cells were grown in modified Eagle's medium [9], supplemented with nonessential amino acids, 1 mM sodium pyruvate and 5% fetal calf serum and kept in humidified CO2 at 37°C. Pesticides 4 pesticides were purchased from Chemical Service Inc., West Chester, Penn. They included 3 insecticides: carbaryl (1-naphthyl methylcarbamate), chlordane (octachloro-4,7-methanotetrahydroindane), dieldrin (hexachloroepoxyoctahydro-endo,exo-dlmethanonaphthalene) and a herbicide 2,4-Dfluid (2,4~lichlorophenoxyacetic acid). The chemical structure and EPA tolerance limits (for toxicity to humans and animals) o f these pesticides are shown in Fig. 1. The insecticides were originally dissolved in acetone, whereas 2,4-D-fluid was water soluble, to 0.1 M and then diluted with the tissue culture medium to the required final concentration. At its highest concentration, acetone did n o t exceed 0.001%.

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371 Cell survival

To determine the survival fraction of cells treated with the pesticides, the cells were plated in triplets in 9 cm plates for attachment for 4 h before adding the pesticides. The following concentrations of pesticides were used: 1000, 100, 10, 1 and 0.1 uM. The number of seeded cells varied according to the concentration of the chemicals used (because higher concentrations would result in extensive cell lethality, therefore sufficient cells have to be treated to allow significant percentage to survive to get statistical results): 1000 pM, 2 × 104 cells; 100 ~M, 2 × 103 cells, 10 ~M, 8 X 102 cells; 1 ~M, 2 × 102 cells and 0.1 ~M, 2 × 102 cells. Two controls were used, b u t without chemical addition, with and without acetone at the same concentration as was used to dissolve the pesticides. Control plates were seeded at a density of 2 × 102 cells/plate. After 10 days plates were fixed in 95% ethanol, stained with Giemsa, rinsed in water, dried and colonies were counted. The plating efficiencies were determined from the average percentage of survivors in the 3 plates. In vitro mutation assay

Cells were plated in 9 cm plates for attachment 4 h before adding pesticides. From survival studies a concentration of 0.01 mM was chosen (40--75% survival). 30 plates were used for each chemical and cells were seeded at l 0 s cells/plate. 48 h later [6,7] the selective agent (ouabain, 1 mM; Sigma Chemical Co., St. Louis, Mo.) was added. Replicate plates with and without acetone were assayed for spontaneous mutation frequency in the absence of test compounds. Resistant colonies developed one week later. They were scored and isolated for further tests in ouabain (1 mM)~ontaining medium [5,7]. RESULTS The dose survival curves for the effect of various pesticides on V79 cells are shown in Fig. 2. It is apparent that there is a concentration effect of the chemical on cell survival, with less than 50% survival for concentration above 10 ~M in all cases. Carbaryl and chlordane were very toxic to cells at 100 ~M. Based on these results 10 uM concentration was chosen for mutation studies since cell survival averaged 40--75% at that concentration. Results in Table I show that all 4 compounds stimulated mutation induction. Acetone, the solvent used to dissolve the insecticides, did not have any effect on plating efficiency nor was there any difference in spontaneous mutations from controls without acetone and the rate of spontaneous mutation in its presence was significantly lower than that observed for the chemical induction of ouabain-resistant mutants. In order to show whether the ouabain-resistant phenotypes were due to stable heritable changes, 5 clones were isolated for each compound, allowed to grow and routinely subcultured for 2 months in tissue culture medium without adding ouabain. When subsequently grown in medium containing 1 mM ouabain they all had maintained their resistance over that long period.

372 1.0

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Fig. 2. Dose survival curves o f V 7 9 cells e x p o s e d t o pesticides. C o n c e n t r a t i o n s o f f r o m 0.1 . M to 1 mM: carbaryl, 4 ; c h l o r d a n e , - - - - o - - --; dieldrin, ----- .... ; 2,4-D-fluid, - - - - - . - - - - - w e r e used. Every p o i n t r e p r e s e n t e d an average o f t h r e e 9 c m plates s e e d e d at cell c o n c e n t r a t i o n s o f f r o m 2 × 10 5 t o 2 x 1 0 ' cells/plate.

TABLE I FREQUENCY OF FORWARD MUTATION FROM OUABAIN R E S I S T A N C E O F V 7 9 C E L L S T R E A T E D W I T H PESTICIDES

S E N S I T I V I T Y TO

Pesticide

Concentration a

% Survival

Number of mutants b

Mutation frequency (per 106 survivors)

None Carbaryl Chlordane Dieldrin 2,4-D Fluid

-0.01 0.01 0.01 0.01

93 66 44.4 77.8 41.3

5 42 74 45 70

1.8 15.3 26.9 16.4 25.5

mM mM mM mM

a C o n c e n t r a t i o n o f a c e t o n e e q u i v a l e n t t o t h a t used in dissolving p e s t i c i d e s t o a c o n c e n t r a t i o n o f 0.01 m M did n o t p r o d u c e an increase in m u t a t i o n f r e q u e n c y as c o m p a r e d t o c o n t r o l plates w i t h o u t a c e t o n e t r e a t m e n t . b N u m b e r o f cells a n d plates u s e d : 3 x 1 0 s / 3 0 .

373 DISCUSSION

The use of cell cultures for the study of carcinogenesis and mutagenesis in mammalian systems will have a significant value in understanding and detection of the effects of environmental agents. For example, a frequency of mutation of 1 in 106 , which can be detected in cell cultures is outside the scope of tests in whole animals such as mice, rats, etc. Mutation leading to neoplasia m a y well be of this frequency [10]. A dose-response curve describing cell survival as a function of dose of the potential mutagen must be obtained in order to determine the kinetics and dose range for cytotoxicity [11]. This is an important step before proceeding to determine mutation frequencies. The assay for mutation induction is done with a mutagen dose that results in 40--90% survival so as to ensure the effective exposure to the mutagen and at the same time minimize the possibility of selective killingof the mutant or wild type cells.Comparison of the dose response kinetics for cell killing and mutation induced m a y indicate whether there is a relation between lethal and mutagenic lesions [12]. Carbaryl and chlordane were the most toxic agents at 100 ~M concentrations (Fig. 2). Metabolic studies on carbaryl, chlordane and dieldrin in mammalian systems have provided data indicating that all 3 of these chemicals are converted to epoxide intermediates or hydroxylated metabolites derived from epoxides [13--15]. These metabolic conversions modify the structures of chlordane and carbaryl from relatively non-reactive chemical intermediates to labile moieties similar in character to those proposed for carcinogenic aromatic hydrocarbons [16]. Dieldrin which already contains an epoxide function does not have to be further metabolized, but will exert its effect directly on macromolecules. Interestingly, the herbicide 2,4-D-fluid was the most toxic chemical at lower doses and least toxic at higher doses. The metabolism of phenoxyacetic acid herbicides such as 2,4-D-fluid proceed via pathways substantially different than those reported for chlordane, carbaryl or dieldrin [17]. These pathways may be alterable with varying concentrations of 2,4-D-fluid as a metabolic substrate. We have previously reported that these 4 pesticides were capable of producing DNA damage in human fibroblasts. The herbicide 2,4-D-fluid demonstrated X-ray type damage while chlordane, carbaryl and dieldrin produced UV-type damage [1]. Several other studies have shown that somatic cell genetic markers can be successfully applied for evaluating the mutagenicity of various carcinogens [ 18,19]. The assay for mutation induction, if coupled with other assays (unscheduled DNA synthesis, bromodeoxyuridine photolysis and the endonuclease sensitive site assay in mammalian cell cultures) may be used as a prescreen for environmental carcinogens and mutagens [20--22]. We are currently investigating the mutability of several repair deficient human lines to determine their utilization in this assay system. ACKNOWLEDGEMENTS

This work was supported by NCI Grant No. CA17917 and EPA Grant No. R804201.

374 REFERENCES 1 F.E. Ahmed, R.W. Hart and N.J. Lewis, Pesticide induced DNA damage and its repair in cultured human cells, Mutat. Res., 42 (1977) 161. 2 Report of Aldrin/Dieldrin Advisory Committee to William D. Ruckelshause. Environmental Protection Agency, March 28, 1972. 3 EPA lists pesticide that may be too dangerous to use, Chem. Eng. News, 14 (1976) 18. 4 D. Siebert and E. Lemperle, Genetic effects of herbicides: induction of mitotic gene conversion in Saccharomyces cerevisiae, Mutat. Res., 22 (1974) 111. 5 C.F. Arlett, D. Turnbull, S.A. Harcourt, A.R. Lehmann and C.M. Colella, A comparison of the 8-azaguanine and ouabaln-resistance systems for the selection of induced mutant Chinese hamster cells, Mutat. Res., 33 (1975) 261. 6 P.J. Davies and J. Parry, The induction of ouabain-resistant mutants by N-methylN-nitro-N-nitrosoguanidinein Chinese hamster cells, Genet. Res., 24 (1974) 311. 7 J.E. Trosko, C.C. Chang, L.P. Yotti and E.H.Y. Chu, Effect of phorbol myristate acetate on the recovery of spontaneous and ultraviolet-light induced 6-thioguanine and ouabain-resistant Chinese hamster cells, Cancer Res., 37 (1977) 188. 8 D.K. Ford and G. Yerganian, Observation on the chromosomes of Chinese hamster cells in tissue culture, J. Natl. Cancer Inst., 21 (1958) 393. 9 H. Eagle, Amino acid metabolism in mammalian cell cultures, Science, 130 (1959) 432. 10 P. Brookers, Quantitative aspects of the reaction of some carcinogens with nucleic acids and the possible significance reaction in the process of carcinogenesis, Cancer Res., 26 (1966) 1994. 11 F.T. Kao and T.T. Puck, Genetics of somatic mammalian cells, IX. Quantitation of mutagenesis by physical and chemical agents, J. Cell Physiol., 74 (1969) 245. 12 H.R. Haynes, R.M. Baker and E.G. Jones, in: G.O. Philips (Ed.), Energetics and Mechanisms in Radiation Biology, Academic Press, New York, 1968, p. 425. 13 T.R. Fukuts and J.J. Sims, in: R. Whitestevens (Ed.), Pesticides in the Environment, Vol. 1, Marcel Dekker, New York, 1971, p.1. 14 W.J. Hayes, Jr., Review of the metabolism of chlorinated hydrocarbon insecticides especially in mammals, Ann. Rev. Pharmacol, 5 (1965) 27. 15 F. Matsumura, Toxicology of Insecticides, Plenum, New York, 1975, p.1. 16 D.M. Jerina, J.W. Daly, B. Witkop, P. Zaltzman-Nirenberg and S. Udenfriend, 1,2Naphthalene oxide as an intermediate in the microsomal hydroxylation of naphthalene, Biochemistry, 9 (1970) 147. 17 F. Matsumura, G.M. Bousch and T. Misato (Eds.), Environmental Toxicology of Pesticides, Academic Press, New York, 1972, p. 193. 18 E. Huberman, L. Aspiras, C. Heidelberger, P.L. Grover and P. Sims, Mutagenicity to mammalian cells of epoxides and other derivatives of polycyclic hydrocarbons, Proc. Natl. Acad. Sci. USA, 68 (1971) 3195. 19 F.T. Kao and T.T. Puck, Genetics of somatic mammalian cells XII: mutagenesis by carcinogenic nitroso compounds, J. Cell. Physiol., 78 (1971) 139. 20 F.E. Ahmed, Environmental genetics: a model to investigate pollutants producing genetic damage, Ph.D. Dissertation, The Ohio State University, 1975. 21 F.E. Ahmed and R.B. Setlow, Different rate limiting steps in excision repair of ultraviolet- and N-acetoxy-2-acetylaminofluorene damaged DNA in normal human fibroblasts, Proc. Natl. Acad. Sci. USA, 74 (1977) 1548. 22 F.E. Ahmed and R.B. Setlow, DNA repair in V-79 cells treated with combinations of ultraviolet radiation and N-acetoxy-2-acetylaminofluorens, Cancer Res, 37 (1977) 3414.

Pesticide induced ouabain resistant mutants in Chinese hamster V79 cells.

369 Chem.-Biol. Interactions, 19 (1977) 369--374 © Elsevier/North-Holland Scientific Publishers Ltd. PESTICIDE INDUCED OUABAIN RESISTANT MUTANTS IN...
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