19

Mutatwn Research, 260 (1991) 19-23 © 1991 ElsevaerSciencePubhshers B.V 0165-1218/91/$03.50 ADONIS 016512189100075F

MUTGEN 01636

Protective effect of vitamin E against chromosomal aberrations and mutation induced by sodium chromate in Chinese hamster V79 cells Masayasu Sugiyama

1, Xinhua

Lin 2 and Max Costa

2

Department of Medwal Btochemtstry, Kurume Umverstty School of Medwme, 67 Asahl-macht, Kurume 830 (Japan) and 2 Department of Enmronmental Medwme, New York Unwerstty Medtcal Center, New York, N Y 10016 (U.S A )

(Received 12 February 1990) (Revlsmn received21 August 1990) (Accepted 28 August 1990)

Keywords Chromate, Vitamin E, Chromosomalaberration, HGPRT mutation assay

Summary The effect of vitamin E on chromosomal aberrations and mutation caused by Na2CrO 4 was investigated m Chinese hamster V79 cells. Pretreatment with 25/xM a-tocopherol succinate (vitamin E) for 24 h prior to chromate exposure (2.5-5 #M) resulted in a decrease of metal-induced chromosomal aberrations. Na2CrO4 (2.5-7.5 btM) induced mutations at the H G P R T locus, but only within a very limited concentration range. This mutagenic response could also be suppressed by pretreatment with vitamin E. These results suggest that vitamin E can protect cells from the clastogenic and mutagenic action of chromate compounds, possibly through its ability to scavenge chromium(V) a n d / o r free radicals.

Chromium(VI) compounds are considered human carcinogens (Enteline, 1974; L6onard and Lauwerys, 1980). They induce chromosome aberrations (Majone and Levis, 1979; Newbold et al., 1979; Tsuda and Kato, 1977; Umeda and Nishimura, 1979) and mutations in cultured mammalian cells (Bianchi et al., 1983; Gainaldi et al., 1982; Paschin et al., 1983). Chromium(VI) compounds have been shown to produce D N A singlestrand breaks and DNA-protein crosslinks (Sugiyama et al., 1986a,b; Cupo and Wetterhahn, 1985),

Correspondence' Dr. M Sug~yama, Department of Medical Blochermstry, Kurume Umverslty School of Medicine, 67 Asaha-machl, Kurume 830 (Japan).

and to inhibit the activity of enzymes such as glutathione reductase in cultured cells (Sugiyama et al., 1989a,b). Hexavalent chromium is more clastogenic and mutagenic than trivalent chromium (Bianchi et al., 1983; Tsuda and Kato, 1977), because the former is readily taken up by cells while the latter is less capable of passing through the cell membrane (Costa et al., 1984; Jennette, 1979). After xt enters the cell, chromium(VI) is reduced to the trivalent form (Connett and Wetterhahn, 1983), through chromium(V) and -(IV) intermediates. This reduction process generates radical species such as active oxygen (Kawanlshi et al., 1986) as well as glutathionyl radicals (Shi and Dalai, 1988) which are thought to be an important underlying mecha-

20 nism for chromate-induced D N A damage. Chromium(V) species formed during the reaction with hydrogen peroxide (Kawanishi et al., 1986) and glutathione (Kortenkamp et al., 1989) have been shown to induce DNA single-strand breaks in vitro. Antioxidants, such as vitamin E, have been shown to protect cells from oxidative damages (Dean and Cheeseman, 1987; Lieber et al., 1986; Summerfield and Tappel, 1984). Vitamin E has also been shown to be effective in protecting against the carcinogemc a n d / o r mutagenic activity of ionizing radiation and chemical agents (Ames, 1983; Borek et al., 1986; Gebhart et al., 1985; Kalinlna et al., 1979; Radner and Kennedy, 1986). Thus, we felt it would be interesting to examine the effect of vitamin E on chromate-induced genotoxlcity as an aid to understanding the mechanism of D N A damage caused by this metal. In previous studies, pretreatment with vitamin E was shown to protect cells from chromate-induced cytotoxacity, enzyme inhibition, and DNA damage as detected by alkaline elution (Suglyama et al., 1987, 1989a). In the present study, these preliminary observations have been extended by showing that vitarnln E suppresses both chromate-induced chromosomal aberrations and mutations at the H G P R T locus. Materials and methods

Cell culture Chinese hamster V79 cells were maintained in a-minimal essential medium (MEM) supplemented with 10% fetal bovine serum and a 1% solution of penicillin/streptomycin (Gibco). Cells were pretreated for 24 h with 25/~M a-tocopherol succinate (vitamin E) or with solvent (dimethyl sulfoxide; DMSO) alone in complete growth medium at the time they were plated (Sugiyama et al., 1987, 1989a). The final concentration of DMSO in both treated and untreated cells did not exceed 0.25% (v/v). 24 h after plating, logarithmically growing cells were rinsed 3 times with salts-glucose medium (SGM; 50 mM 4-(2-hydroxyethyl)1-piperazine-ethanesulfonic acid (pH 7.2), 100 mM NaC1, 5 mM KC1, 2 mM CaCI 2, and 5 mM glucose) and then treated for 2 h with Na 2CRO4 at 37 ° C in this maintenance medium.

Chromosome aberratton Following treatment with Na2CrO4, cells were incubated in metal-free complete growth medium for 24 h because Na2CrO 4 induced mitotic delay. Mitotic cells were selected by Colcemid treatment (0.02 /~g/ml for 2 h) and dislodged by gently pipetting the overlying medium. The cells were collected by centrifugation at 1200 rpm, treated with a hypotonic solution (0.56% KC1) for 5 min at room temperature, and fixed in 3 : 1 methanol/glacial acetic acid fixative for 30 min with 2 changes of fixative. The cell suspension was dropped onto clean wet slides and air-dried. Slides were stained with Giemsa and then mounted. From each sample 100 metaphases were analyzed for chromosomal aberrations. The experiments, which were repeated twice, showed essentially similar results. Mutagenests assay Mutagenesis at the H G P R T locus was evaluated as previously described (Chang et al., 1978). To reduce spontaneous mutant frequency, V79 cells were routinely selected in H A T medium ( a - M E M containing 10 # M hypoxanthine, 40 # M aminopterin, and 1.6 mM thymidine) prior to initiating the mutagenesis experiment. The cells were plated at a density of 5 x 105 cells in complete growth medium containing 25 g M vitamin E or DMSO. After 24 h, approximately 1.1 x 10 6 cells were treated for 2 h with NazCrO 4 in SGM and then detached with 0.025% trypsin containing 0.02% EDTA, and seeded into culture dishes to determine cell survival and mutagenesis. Colonies were fixed after 7 days, stained and counted (a surviving colony must have > 50 cells). The cultures for detecting mutagenesis were subcultured twice at a density of 1 x 105 cells per plate during the week and incubated for 7 or 8 days to allow the expression of mutant phenotypes. Following expression, 2 x 105 cells were seeded for mutant selection on each of 6 plates with growth medium containing 5/~g/ml of 6-thioguanine. The plating efficiency of these cells was determined in parallel by seeding 200 cells per plate. Plating efficiency plates were stained after a 7-day incubation, while the plates for the mutation experiment were stained 3 days later. The mutation frequency per 10 6 survivors was calculated by dividing the ob-

21

served mutant colony frequency by the plating efficiency of the cells. N-Methyl-N'-nitro-Nnitrosoguanidine ( M N N G ) was utilized as positive control for the mutagenesis experiment (StoneWolff et al., 1985). Briefly, cells were treated for 15 min with 6.8 g M M N N G in SGM, and plating efficiency and mutation frequency were determined as described above. The experiments were repeated 3 times. Results and discussion

As shown m Table 1, treatment of V79 cells with Na2CrO 4 resulted in a striking concentration-dependent increase in chromosomal aberrations. The aberrations observed were predominantly breaks and exchanges. These observations were in accord with those of previous studies (Majone and Levis, 1979; Newbold et al., 1979; Tsuda and Kato, 1977). When cells were pretreated with 25 g M vitamin E for 24 h, there was a marked decrease in the frequency of chromosomal aberrations induced by chromate (Table 1). Vitarmn E alone did not induce chromosomal aberrations, since no significant difference in vitamin E vs. spontaneous aberrations was observed. These results demonstrate that vitamin E has a protective effect against the clastogenic activity of chromate. Table 2 shows that treatment with Na2CrO 4 (2.5-7.5 gM) and M N N G resulted in the induction of 6-thioguanine-resistant mutants in V79

TABLE 1 EFFECT OF VITAMIN E ON C H R O M O S O M A L ABERRATIONS I N D U C E D BY Na2CrO 4 Pretreatment

Na 2CrO4 (/~M)

Cells with damages

Types of aberrations a G

B

E

D

F

0 0 2 1 4 4

3 1 37 7 59 19

0 0 13 7 24 11

0 0 4 3 4 4

1 2 4 2 6 2

(%) DMSO Vitamin E DMSO Vitamin E DMSO Vitamin E

0 0 25 25 50 5.0

4 3 24 11 * 29 18 **

V79 cells were pretreated for 24 h with 25 g M vitamin E or with DMSO alone. Following 2-h treatment with NaECrO4 m SGM, the cells were allowed to recover for 24 h prior to collection of matotic cells as described m Materials and methods a G, gaps; B, breaks, E, exchanges, D, dlcentrics, F, fragments. * P < 0.02, ** P < 0.1 compared to unpretreated Na2CrO 4treated values (X 2 test).

cells. The induction of mutants decreases as the chromate dose was raised (5-7.5 gM) and chromate was not mutagenic at the highest concentration tested (10 gM; 5.6 + 1.0 mutants/106 survivors). These results were consistent with previous mutation studies using V79 cells at the H G P R T locus (Bianchi et al., 1983; Newbold et al., 1979). Previous studies showed that pretreatment of V79 cells with vitamin E resulted in a decrease in the cytotoxicity caused by Na2CrO 4 (Sugiyama et

TABLE 2 E F F E C T OF V I T A M I N E ON CYTOTOXICITY A N D M U T A T I O N F R E Q U E N C Y I N D U C E D BY Na2CrO 4 Pretreatment

Treatment

Mutation frequency (mutants/106 surwvors)

Plating efficiency (%)

DMSO Vltanun DMSO Vltarmn DMSO Vitamin DMSO Vitamin DMSO

0 0 2.5/~M 2.5/~M 5 0/zM 5 0/zM 7 5/~M 7 5/~M 6.8 gM

3.1+ 21 1.0_+ 0 6 17.3_+ 4.1 20_+ 1.4 * 12.8_+ 6 4 5.0_+ 4.9 7.4 ± 0.8 6.2_+ 3 4 399.2 _+27.0

95.0+_ 23 93.6_+ 2 5 84.8_+ 6.6 89.5_+ 6.8 61.2+ 9.2 83.5 _+14.3 25 6 _+12 7 68.3_+ 16.5 * 60.3 5= 6.6

E E E E

Na2CrO4 Na2CrO4 Na2CrO 4 NaECrO4 NaECrO4 Na2CrO4 MNNG

V79 cells were pretreated for 24 h with 25 gM vitamin E or with DMSO. Fonowlng 2-h treatment with Na2CrO 4 m SGM, plating efficiency and mutation frequency were deterrmned. The treatment with M N N G for 15 man in SGM was utxhzed as posmve control. Each value is the mean + SD for 3 separate experiments * P < 0.05 compared to unpretreated Na2CrO4-treated values (Student's t-test)

22 al., 1989a). As shown in T a b l e 2, a similar p r o t e c tive effect of the v i t a m i n against c h r o m a t e - i n d u c e d cytotoxicity was o b s e r v e d in V79 cells. Table 2 also shows that cellular p r e t r e a t m e n t with v i t a m i n E significantly s u p p r e s s e d the m u t a g e n i c action of N a 2 C r O 4. V i t a m i n E at 25 /~M was neither c y t o t o x i c n o r m u t a g e n i c in V79 cells. F u r thermore, a previous s t u d y showed that p r e t r e a t m e n t with v i t a m i n E dxd n o t affect cellular u p t a k e of this m e t a l ( S u g l y a m a et al., 1989a). Since c h r o m i u m ( V I ) is r e a d i l y r e d u c e d to the trivalent form in cells ( C o s t a et al., 1984; Jennette, 1979), the f o r m a t i o n of c h r o m i u m ( I I I ) a n d / o r o t h e r oxadatlon states such as c h r o m i u m ( V ) a n d -(IV) might b e required for the i n d u c t i o n of D N A d a m a g e . W e have recently shown that cellular levels of c h r o m i u m ( V ) were significantly r e d u c e d b y p r e t r e a t m e n t with v i t a m i n E. P r e t r e a t m e n t with v i t a m i n E also d e c r e a s e d the n u m b e r of D N A b r e a k s caused b y c h r o m a t e ( S u g i y a m a et al., 1987, 1989a). U n d e r similar conditions, the p r e s e n t resuits show that c h r o m a t e - i n d u c e d clastogenicity a n d m u t a g e n i c i t y were r e d u c e d b y this vitamin. I n a n o t h e r study, isolated c h r o m i u m ( V ) i n t e r m e d i ates have b e e n shown to p r o d u c e D N A b r e a k s in vitro a n d to i n d u c e m u t a t i o n in b a c t e r i a l cell systems ( R o d n e y et al., 1989). Collectively, these results suggest that c h r o m i u m ( V ) might be the critical form which is r e s p o n s i b l e for the genotoxic a n d clastogenic as well as the m u t a g e n i c activity of c h r o m a t e c o m p o u n d s . However, c h r o m i u m ( V I ) has been shown to b e m e t a b o l i z e d to c h r o m i u m ( V ) with s i m u l t a n e o u s f o r m a t i o n of active oxygens ( K a w a n i s h i et al., 1986; Shi a n d Dalal, 1989) a n d g l u t a t h l o n y l radicals (Shi a n d Dalai, 1988), which c o u l d be scavenged b y w t a m i n E, so it is difficult to exclude the p o s s i b i l i t y of i n v o l v e m e n t of these r a d i c a l species in c h r o m a t e - i n d u c e d d a m a g e s in cells. N u m e r o u s studies indicate that v i t a m i n E m i g h t b e useful as an a n t i c a n c e r agent in b o t h prevention a n d treatment. T h e present results, along with earlier o b s e r v a t i o n s ( S u g i y a m a et al., 1987, 1989a), in which v i t a m i n E p r o t e c t e d cells f r o m chrom a t e - i n d u c e d e n z y m e inhibition, cytotoxicity, a n d D N A d a m a g e , strongly suggest that v i t a m i n E m i g h t b e an a n U m u t a g e n i c a n d / o r a n t i c a r c i n o genic agent for c h r o m i u m c o m p o u n d s . F u r t h e r studies are necessary to elucidate the p r o t e c t i v e

m e c h a n i s m of v i t a m i n E a g a i n s t c h r o m a t e - i n d u c ed d a m a g e .

Acknowledgements This w o r k was s u p p o r t e d b y the F u k u o k a C a n c e r Society, G r a n t s - i n - A i d for Scientific Research f r o m the M i n i s t r y o f E d u c a t i o n , Science a n d Culture of J a p a n , a n d b y N I E H S G r a n t s ES04895 a n d ES04715. W e t a n k Dr. T o b y Rossm a n a n d co-workers at the I n s t i t u t e of E n v i r o n mental Medicme, New York University Medical Center, for advice a b o u t the m u t a t i o n assay.

References Ames, B N (1983) Daetary carcinogens and antxcarcmogens, oxygen radacals and degeneratave diseases, Soence, 221, 1256-1264. Blanchi, V., L Celom, G Lanfranchi, F. Majone, G Mann, A Montaldl, G Sponza, G. Tamlno, P. Venler, A Zantedesclu and A G. Lews (1983) Genetac effects of chromium compounds, Mutatxon Res, 117, 279-300 Borek, C., A Ong, H Mason, L Onahue and J E Bmglow (1986) Selemum and vltanun E mlublt radiogenic and chemically reduced transformaUon an vatro vm different mechamsms, Proc Natl Acad SeE (U.S.A.), 82, 6755-6759 Chang, C.C, M. Caztellazzl, T.W Glover and J.E. Trosko (1978) Effects of harmon and nor-harmon on spontaneous and ultravaolet hght mduced mutagenesls in cultured Chinese hamster cells, Cancer Res, 38, 4527-4533 Connett, P.H., and K.E. Wetterhahn (1983) Metabohsm of the carcinogen chromate by cellular constituents, Struct. Bond, 54, 93-124. Costa, M., A.J. Kraker and S R PaUerno (1984) Toxaoty and carcmogemclty of essentml and non-essential metals, Prog Chn Blochem., 1, 1-49. Cupo, D Y., and K.E Wetterhahn (1985) Modification of chromium (VI)-lnduced DNA damage by glutathione and cytochrome P-450 an chicken embryo hepatocytes, Proc Natl Acad. So (U S.A ), 82, 6755-6759. Dean, R.T., and K.H. Cheeseman (1987) Vitamin E protects proteins agamst free radical damage an hpld envaronment, Blochem. Blophys. Res. Commun., 148, 1277-1282 Enterhne, P E. (1974) Resparatory cancer among chromate workers, J. Occup. Med., 16, 523-526 Gaanaldl, G , C M Colella, A Paras and T. Manam (1982) Thioguamne resistance, ouabain resistance and sister chromaud exchanges in V79/AP4 Chinese hamster cells treated with potassium dichromate, Chem-Bxol. Interact., 42, 4551. Gebhart, E, H Wagner, K Grzawok and H. Behnsen (1985) The actaon of antlclastogens m human lymphocyte cultures and their modlficataon by rat-hver $9 mix. Studies wxth vitamins C and E, Mutation Res., 149, 83-94.

23 Jennette, K.W. (1979) Chromate metabohsm m hver nucrosomes, Biol. Trace Elem Res., 1, 55-62. Kahnma, L.M., G.M Sardarly and U.K Alekperov (1979) Antimutagemc effect of ~t-tocopheroi on the frequency of gene mutations in Salmonella, Genettka, XV, 1880-1888. Kawanlshl, S., S Inoue and S. Sano (1986) Mechamsm of DNA cleavage reduced by sodium chromate (VI) m the presence of hydrogen peroxade, J Blol Chem., 261, 59525958. Kortenkamp, A., Z Ozohns, D Beyersmann and P. O'Brlen (1989) Generation of PM2 DNA breaks in the course of reduction of chrormum(VI) by glutatinone, Mutauon Res, 216, 19-26 L6onard, A., and R.R. Lauwerys (1980) Carcmogemclty and mutagenlcity of chromium, Mutation Res, 76, 227-239 Lieber, D.C., D.S. Khngs and D.J. Read (1986) Antloxldant protection of phosphohpid bilayers by a-tocopherol, control of a-tocopherol status and hpld peroxldatlon by ascorbic aod and glutatinone, J Biol. Chem., 261, 1211412119 Malone, F., and A.G Lexas (1979) Chromosomal aberrations and sister chromatld exchanges m Cinnese hamster cells treated in vitro with hexavalent chronuum compounds, Mutation Res., 67, 231-238 Newbold, R.F., J. Amos and J.R. Connell (1979) The cytotoxic, mutagemc and clastogenlc effect of chromaum-contmmng compounds on mammalian cells m culture, Mutation Res., 67, 55-63. Pascinn, Y.V, V.I. Kozachenko and L.E. Sal'nlkova (1983) Differential mutagenlc response at HGPRT locus in V79 and CHO Chinese hamster cells after treatment with chromate, Mutation Res, 122, 361-365. Radner, B.S., and A.R. Kennedy (1986) Suppression of X-ray reduced transformation by vitamin E in mouse C3H/10T1/2 cells, Cancer Lett., 32, 25-32 Rodney, P.F, J.J. Robert, P A Lay, N E Dixon, R.S U. Raker and A.M. Boron (1989) Chronuum(V)-mduced cleavage of DNA. are chrormum(V) complexes the active carcinogens m chrormum(VI)-lnduced cancerg, Chem. Res. Toxacol, 2, 227-229. Sin, X., and N.S. Dalal (1988) The mechamsm of the chromate reduction by glutatinone: ESR exadence for the glutathionyl

radical and an isolable Cr(V) intermediate, Biochem. Biophys. Res. Commun., 156, 137-142 Sin, X., and N.S. Dalai (1989) Chrormum(V) and hydroxyl ra&cal formation dunng the glutatinone reductase-catalyzed reduction of chrormum(VI), Biochem Blophys Res. Commun., 163, 627-634. Stone-Wolff, D S, C.B. Klein and T G Rossman (1985) HGPRT- mutants of V79 cells that revert specifically by base pair substitution and framesinft mutations, Environ. Mutagen, 7, 281-291 Suglyama, M., X -W. Wang and M Costa (1986a) Comparison of DNA lesions and cytotoxlclty reduced by calcium chromate in human, mouse and hamster cell hnes, Cancer Res., 46, 4547-4551 Suglyama, M., S.R Patierno, O. Cantom and M Costa (1986b) Characterization of DNA lesions reduced by CaCrO4 m synchronous and asynchronous cultured mammahan cells, Mol Pharmacol., 29, 606-613. Sugtyama, M., A Ando, H. Furuno, N B Furlong, T. Hldaka and R. Ogura (1987) Effects of vltanun E, vitamin B2 and selemte on DNA single strand breaks induced by sodmm chromate (VI), Cancer Lett, 38, 1-7. Suglyama, M., A. Ando and R Ogura (1989a) Effect of vitanun E on survival, glutatinone reductase and formation of chromium (V) m Chmese hamster V-79 cells treated with sodmm chromate (VI), Carcinogenesis, 10, 737-741 Suglyama, M., A. Ando, K. Nakao, H Ueta, T. HIdaka and R. Ogura (1989b) Influence of vltarmn B2 on formaUon of chromium (V), alkah-labde sites, and lethahty of sodium chromate (VI) in Chinese hamster V-79 cells, Cancer Res., 49, 6180-6184. Summerfleld, F.W, and A L Tappel (1984) Vitarmn E protects agmnst methyl ethyl ketone peroxade-mduced peroxadatlve damage to rat brain DNA, Mutation Res., 126, 113-120. Tsuda, H , and K. Kato (1977) Chromosomal aberraUons and morphological transformation in hamster embryomc cells treated w~th potassium dichromate in vitro, Mutation Res, 46, 87-94 Umeda, M , and M Nlshimura (1979) Induclbihty of chromosomal aberrations by metal compounds In cultured mammahan cells, Mutation Res., 67, 221-229