Microsome-mediated cytotoxicity to CHO cells

Mutation Research, 103 (1982) 359-365

359

Elsevier Biomedical Press

Microsome-mediated cytotoxicity to CHO cells* E . - L . T a n * * , R . L . Schenley a n d A . W . Hsie University of Tennessee-Oak Ridge Graduate School of Biomedical Sciences, and Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 (U. 5.A.)

(Accepted 10 August 1981)

Many short-term assays for the detection o f promutagens require the presence of an exogenous metabolic activation system. The most commonly used system is the 9000 × g supernatant ($9) prepared from the livers of rats which have been pretreated with inducers of the microsomal mixed-function oxidases (Ames et al., 1975). However, most studies on the metabolism of xenobiotics in vitro have utilized purified microsomes, and much information exists on this subject (see, for example, Coon et al., 1980). In investigating mechanisms of mutagenesis, it might be useful to use purified microsomal fractions instead of $9 to better define the enzymatic reactions involved. However, in the present study we found that purified microsomes, unlike $9 (O'Neill et al., 1977b; Machanoff et al., 1981), were highly cytotoxic to Chinese hamster ovary (CHO) cells in culture. The microsomemediated cytotoxic effect was dependent on N A D P , in the presence of an N A D P H regenerating system (G-6-P and G-6-P dehydrogenase), and omission of G-6-P negated this effect. In the absence of microsomes, neither N A D P nor its reduced form was cytotoxic to C H O cells. The microsome-mediated cytotoxicity was inhibited by the addition of either CaC12 or butylated hydroxyanisole (BHA) to the assay system. Cytotoxicity increased with increasing amounts of microsomes, but there was no detectable mutant induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus.

By acceptance of this article the publisher or recipient acknowledgesthe right of the U.S. Government to retain a nonexclusive royalty free license in and to any copyright covering this article. *Research supported jointly by the U.S. Environmental Protection Agency under lnteragency Agreements 40-516-75 and 40-732-78 and by the Office of Health and Environmental Research, U.S. Department of Energy, under contract W-7405-eng-26with the Union Carbide Corporation. **Postdoctoral Investigator supported by subcontract No. 3322 from the BiologyDivision of Oak Ridge National Laboratory to the University of Tennessee. 0165-7992/82/0000-0000/$02.75 © Elsevier Biomedical Press

360 Materials and methods

Cell culture media were purchased from K.C. Biological Co., tissue culture plasticware from Corning Glass Works, and BHA from Sigma Chemical Co. All chemicals were of reagent-grade. $9 was prepared by W. Winton and J.L. Epler according to the procedure of Ames et al. (1975) from liver homogenates of male Sprague-Dawley rats which had been treated or not treated with Aroclor 1254. The $9 was stored in 1-ml aliquots at - 9 0 °C. We prepared microsomal fractions by centrifuging a diluted $9 (1:3 with 0.15 m KCI) at 105000 x g for 1 h. The microsomal pellet was resuspended in 0.15 M KC1 to the original volume of the $9, and the mixture was manually homogenized in a Potter-Elvehjem type tissue homogenizer (A.H. Thomas). The microsomal preparation was kept on ice and used within 1 h. The protein contents of $9 and microsomes, as measured by the method of Lowry et al. (1951), were approx. 26 and 6 mg/ml. The $9 metabolic activation system was made up as follows: 1 ml of the $9 fraction was added to 9 ml of a buffered salt solution; the final 10-ml volume contained 50 mM NaEHPO4 (pH 8.0), 4 mM NADP, 5 mM G-6-P, 30 mM KCI and 10 mM MgC12. This system is referred to as Mg-S9. The system obtained when 10 mM CaCl2 was added to the mixture is denoted Ca,Mg-S9. When microsomes were used instead of $9, 0.5 unit of G-6-P dehydrogenase (Sigma Chemical Co., lyophilized preparation from Leuconostoc mesenteroides) was added, and this system will be appropriately identified. Cells (CHO-KI BH4; Hsie et al., 1975) were grown in monolayer subculture in Ham's F12 medium supplemented with 10%0 fetal bovine serum (F12FCS10) and used throughout the experiments. The procedure for the estimation of cellular cytotoxicity and mutant induction at the H G P R T locus is published elsewhere (O'Neill et al., 1977a). Briefly, 1 ml of the metabolic activation system was added to 25-cm2 flasks containing 4 ml of Ham's F12 medium and approximately 1 x 106 cells which had been grown in Ham's FI2 medium supplemented with 5% dialyzed fetal bovine serum (F12FCM5). The flasks were then incubated for 5 h (37 °C, 5% CO2 in air, 95% humidity) before they were rinsed with Saline G; the medium was replaced with F12FCMS, and the flasks were incubated for 19 h. The next day, 200 cells were plated in triplicate, and cytotoxicity due to the treatment was determined by scoring the colonies after 1 week. The results are expressed relative to the value obtained for untreated control cultures. Mutation induction was determined by plating 1 × 106 cells (5 plates, 2 x 105 cells each) in hypoxanthine-free F12FCM5 containing 10/~M 6-thioguanine after the cells had been subcultured for 8 days for the mutant phenotype to express itself maximally. The results were corrected by taking into account the cloning efficiencies of the cells in non-selective medium determined at the same time as the selection. The results are expressed as mutants/106 clonable cells.

361 TABLE 1 EFFECT OF NADP ON THE CYTOTOX1CITY MEDlATED BY $9 OR MICROSOMAL ACTIVATION SYSTEMS WITH OR WITHOUT CaC12 NADP concentration a

Relative survival (o70) Mg-S9

Mg-microsomes

Ca,Mg-S9

Ca,Mg-microsomes

100 86 97 98 89 87

100 68 44 38 44 39

100 93 88 97 102 96

100(2) c 102 101 98 98 94 (1)

-

100

-

(mM) 0b 0.01 0.10 1.00 2.00 4.00 4.00

d

98

a Concentration of NADP in the metabolic activation system. 4 mM NADP was used in the preparation of a standard mixture. b The absolute cloning efficiency of the untreated control culture was 83°7o. c Figures in parentheses are mutants/l06 clonable cells. d G-6-P was omitted from the activation system.

Results and discussion Table 1 shows that the m i c r o s o m a l activation system plus N A D P was cytotoxic to C H O cells only when Ca 2+ was omitted f r o m the system. $9, with or without the addition o f Ca 2+ in the activation system, was not cytotoxic to C H O cells. The cytotoxicity observed when microsomes were used could be due to toxic products such as C O , malondialdehyde, and hemolytic lipids, generated by N A D P H - c a t a l y z e d m i c r o s o m a l lipid peroxidation (Talcott et al., 1980; H o g b e r g et al., 1973; Schacter et al., 1972; Rodgers et al., 1977; Benedetti et al., 1980). The lack o f effect when $9 was used supports the observation made by others that c o m p o n e n t s in the cytosolic fraction o f rat and m o u s e liver inhibit the peroxidative d a m a g e to microsomal m e m b r a n e s (Talcott et al., 1976, 1980). The presence o f Ca 2+, which forms a calcium p h o s p h a t e precipitate in the p h o s p h a t e - b u f f e r e d metabolic activation system, apparently also protects the cells against the effects o f lipid peroxidation. Omission o f G - 6 - P f r o m the M g - m i c r o s o m e system negates the cytotoxic effect, which indicates an N A D P H - m e d i a t e d mechanism (Talcott et al., 1980). In the absence o f microsomes, N A D P H ( 0 . 1 - 4 . 0 0 mM) had no effect on the C H O cells (results not shown). Table 2 shows that 4 m M CaC12 in the M g - m i c r o s o m e activation system appears to he sufficient to abolish the cytotoxic effects mediated by microsomes. Preliminary experiments show that for a 5-h incubation, M g - m i c r o somes p r o d u c e between 40 and 50 nmoles o f m a l o n d i a l d e h y d e per flask, whereas in the presence o f l0 m M CaC12 in the M g - m i c r o s o m e activation system, malondialdehyde p r o d u c t i o n was not detectable, i.e., < 5 nmoles/flask.

362 TABLE 2 I N H I B I T I O N OF M I C R O S O M E - M E D I A T E D C Y T O T O X I C I T Y BY CaC12 CaCI2 concentrationa

Relative survival (°7o)

(mM) 0b 0

100 41 50 53 98 86 97 103

1

2 4 6 8 10

a Concentration of CaCI2 in the M g - m i c r o s o m e activation system. b Control incubation containing no microsomes. The absolute cloning efficiency o f the cells was 75°7o.

The antioxidant BHA has been shown to inhibit microsomal lipid peroxidation (Talcott et al., 1976; Yang et al., 1974). Table 3 shows that in the absence of N A D P or when no microsomes were used, BHA had no effect on the relative survival of C H O cells. In the presence of N A D P in the M g - m i c r o s o m e activation system, there was 8% relative survival of the cells in the absence of BHA; at 1/zM BHA there was 49O7o survival; and at 2/~M no cytotoxicity was observed. Yang et al. (1974) have shown that 4 #M BHA inhibits about 90% of the NADPH-dependent lipid peroxidation of rat-liver microsomes.

TABLE 3 I N H I B I T I O N OF M I C R O S O M E - M E D I A T E D C Y T O T O X I C I T Y BY B H A BHA

Relative survival (%)

c°ncentrati°na

No

Mg-microsomes

Mg-microsomes b

~M)

microsomes

(without NADP)

(with N A D P )

0c

100

1

-

100 98 102 96 104 93 100

8 (1) d 49 94 (3) 99 90 83 83 (2)

2 4 6 8 l0

108 110 113 ll0 100

a Concentration of B H A in the flasks. b M g - m i c r o s o m e s (without N A D P ) with no added B H A were used as the control in calculation o f the relative survival. c The absolute cloning efficiencies o f the untreated control cultures were 73 and 76O7o. Figures in parentheses are mutants/106 clonable cells.

363 O0 - , - - - ~ •

,'...

50

o

~o

"i

J

.

ztD

o_9

0

80

160

MICROSOMES (F.I/ml activation system)

Fig. 1. Dose-dependent cytotoxicity and mutagenicity of microsomes. The buffered salt solution without CaC12 was made up as described in Materials and Methods. To each ml of this solution was added the indicated amount of microsomes. The mixtures were then used to treat the cells. Cytotoxicity and mutagenicity were determined as described. Cytotoxicity (.) and mutagenicity (L',) of microsomes from Aroclor 1254-treated rats; cytotoxicity (o) of microsomes from untreated rats. The absolute cloning efficiency of the untreated control culture was 85°7o.

Fig. 1 shows that in the presence of a complete metabolic activation system, cytotoxicity increased with increasing amounts of microsomes prepared from rats which were treated or not treated with Aroclor 1254. However, the cytotoxic effects did not result in mutant induction at the H G P R T locus. The results indicate that use of purified microsomes instead of $9 in an in vitro metabolic activation system for the detection o f promutagens were extremely cytotoxic to C H O cells in culture. Thus, this system would be unacceptable for the routine assay of mutagens, since cell survival would be too low to permit any meaningful determinations of cytotoxicity and mutation induction. However, if Ca 2+ or low amounts of BHA were added, this microsome-mediated cytotoxicity could be avoided. At the concentrations used, neither Ca 2+ (Table 1) nor BHA (Table 3) was cytotoxic or mutagenic to C H O cells. Although the exact mechanism whereby BHA exerts its effect is not know, low concentrations of BHA had very

364 slight i n h i b i t o r y effects o n b e n z p y r e n e hydroxylase, a m i n o p y r i n e , b e n z p h e t a m i n e a n d e t h y l m o r p h i n e demethylase activities of rat liver m i c r o s o m e s (Yang et al., 1974). O u r results i m p l y that the cytotoxic effects of a m i c r o s o m e m e t a b o l i c a c t i v a t i o n system are d u e to m i c r o s o m a l lipid p e r o x i d a t i o n . W e expect the m i c r o s o m e m e d i a t e d cytotoxicity to be d e p e n d e n t o n factors such as m e t h o d of m i c r o s o m e p r e p a r a t i o n , presence o f lipid p e r o x i d a t i o n cofactors in the a c t i v a t i o n system, a n d diet of the a n i m a l s f r o m which the m i c r o s o m e s were prepared. It r e m a i n s to be studied whether m i c r o s o m e s o b t a i n e d f r o m the $9 f r a c t i o n p r e p a r e d according to the m e t h o d of A m e s et al. 0 9 7 5 ) , as was d o n e in this study, will similarly affect other cell lines.

Acknowledgements W e t h a n k W . W i n t o n a n d J.L. Epler for their gift o f $9 a n d J.K. Selkirk, K.R. T i n d a l l , L.C. W a t e r s a n d H . R . Witschi for reviewing the m a n u s c r i p t .

References Ames, B.M., J. McCann and E. Yamasaki (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test, Mutation Res., 3 l, 347-369. Benedetti, A., M. Comporti and H. Esterbauer (1980) Identification of 4-hydroxynonenal as a cytotoxic product originating from the peroxidation of liver microsomal lipids, Biochim. Biophys. Acta, 620, 281-296. Coon, M.J., A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O'Brien (Eds.) (1980) Microsomes, Drug Oxidations, and Chemical Carcinogenesis, Vols. I and II, Academic Press, New York. Hogberg, J., A. Bergstrand and S.V. Jakobsson (1973) Lipid peroxidation of rat liver microsomes, Its effect on the microsomal membrane and some membrane bound microsomal enzymes, Eur. J. Biochem., 37, 51-59. Hsie, A.W., P.A. Brimer, T.J. Mitchell and D.G. Gosslee (1975) The dose-response relationship of ethyl methanesulfonate-induced mutations at the hypoxanthine-guaninephosphoribosyl transferase locus in Chinese hamster ovary cells, Somat. Cell Genet., l, 247-261. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall (1951) Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193,265-275. Machanoff, R., J.P. O'Neill and A.W. Hsie (1981) Quantitative analysis of cytotoxicity and mutagenicity of benzo[a]pyrene in mammalian cells (CHO/HGPRT system), Chem.-Biol. Interact., 34, 1-10. O'Neill, J.P., P.A. Brimer, R. Machanoff, G.P. Hirsch and A.W. Hsie (1977a) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Development and definition of the system, Mutation Res., 45, 91-101. O'Neill, J.P., D.B. Couch, R. Machanoff, J.R. San Sebastian, P.A. Brimer and A.W. Hsie (1977b) A quantitative assay of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in Chinese hamster ovary cells (CHO/HGPRT system): Utilization with a variety of mutagenic agents, Mutation Res., 45, 103-109.

365 Rodgers, M.K., E.A. Glende Jr. and R.D. Recknagel (1977) Prelyptic damage of red cells in filtrates from peroxidizing microsomes, Science, 196, 1221-1222. Schacter, B.A., H.S. Marver and U.A. Meyer (1972) Hemoprotein catabolism during stimulation of microsomal lipid peroxidation, Biochim. Biophys. Acta, 279, 221-227. Talcott, R.E., H. Denk, R. Eckerstorfer and J.B. Schenkman (1976) Inhibition of NADPH-driven microsomal lipid peroxidation by cytosol factor(s) Effect of a fat-free, high carbohydrate diet, Chem.Biol. Interact., 12, 355-361. Talcott, R., A. Ketterman, W. Harger, H. Denk, D. Kerjaschki, I. Zeiler and R. Eckerstorfer (1980) Microsomal lipid peroxidation: Catalysis, effect, and inhibition by cytosolic protein, in: M.J. Coon, A.H. Conney, R.W. Estabrook, H.V. Gelboin, J.R. Gillette and P.J. O'Brien (Eds.), Microsomes, Drug Oxidations, and Chemical Carcinogenesis, Vol. II, Academic Press, New York, pp. 753 759. Yang, C.S., F.S. Strickhart and G.K. Woo (1974) Inhibition of the mono-oxygenase system by butylated hydroxyanisole and butylated hydroxytoluene, Life Sci., 15, 1497 1505.