Mutation Research, 266 (1992) 85-91 © 1992 Elsevier Science Publishers B.V. All rights reserved 0027-5107/92/$05.00
85
MUT 05056
Effects of L-ascorbic acid on the mutagenicity of ethyl methanesulfonate in cultured mammalian cells H . K o j i m a 1, H . K o n i s h i t a n d Y. K u r o d a 2 I Biochemical Research Institute, Nippon Menard Cosmetic Co. Ltd., Ogaki, Gifu 503 and z Research Institute of Biosciences, Azabu Unicersity, Fuchinobe. Sagamihara, Kanagawa 229 (Japan) (Received 2 April 1991) (Revision received 23 September 1991) (Accepted 26 September 1991)
Keywords: Ethyl methanesulphonate; L-Ascorbic acid; Mutagenicity; Mammalian cells
Summary The effects of L-ascorbic acid (AsA) on the mutations induced by ethyl methanesulfonate (EMS) were examined by means of the 6-thioguanine (6TG)-resistant mutation assay and chromosome aberration assay in cultured Chinese hamster V79 cells. When cells were treated with EMS at various concentrations in the presence of 100 /zg/ml AsA, EMS-induced 6TG-resistant mutations w~re reduced about one third or one fourth. EMS-induced chromosome aberrations were also reduced by AsA. These reductions in the mutagenicity of EMS were also found when cells were treated with mixtures of AsP, and EMS which had previously been incubated at 37.0°C for 2 h. in pre- and post-treatments with AsA, however, the frequencies of EMS-induced mutations were not reduced, but rather increased markedly.
Correspondence: Prof. Dr. Yukiaki Kuroda, Research Institute of Biosciences, Azabu University, 1-17-71, Fuchinobe, Sagamihara, Kanagawa229 (Japan). Tel.: 0427-54-7111(Ext. 430); Fax: 0427-54-7661.
Abbreciations: AsA, L-ascorbic acid; EMS, ethyl methanesulfonate; 6TG, 6-thioguanine, DMN, dimethylnitrosamine; MNNG, N-methyI-N'-nitro-N.nitrosoguanidine; AFB, ariatoxin Bi; TC, tetracycline hydrochloride; CPA, cyclophosphamide; DMBA, 7,12.dimethylbenzoanthracene; SCE, sister-chromatid exchange; N-OH-AAF, N-hydroxy-2-acetylaminofluorene; N-OH-Trp-P2, 3-hydroxyamino-l-methyl-5Hpyrido[4,3-b]indole; BHT, butylhydroperoxide; 8AG, 8azaguanine.
L-Ascorbic acid (AsA) is an antioxidant and a reducing agent. It is abundantly present in green tea, fruits and vegetables. It is effective in preventing scurvy, and its antioxidant activity is useful for the detoxification of some drugs and for the protection against rancidity in foodstuffs. In the present paper, EMS was used as a mutagen with alkylating activity. The effects of AsA on 6TO-resistant mutations and chromosome aberrations induced by EMS were examined in cultured Chinese hamster V79 cells. Some preliminary results have been reported elsewhere (Kuroda, 1986a, b).
86
Materials and methods
Cell culture The cell line used was the strain of Chinese hamster lung V79 cells isolated by Ford and Yerganian (1958). Cells were maintained in Eagle's minimum essential medium (Nissui Seiyaku Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (Gibco Lab., Grand Island, NY) in 60-mm plastic petri dishes (Lux Sci. Corp., Newbury Park, CA, No. 5216, 2-mm grid) under a controlled atmosphere of 5% CO 2 and 95% air at 37.5°C. Tbey were mycoplasma-free and showed a colony-forming ability of more than 80%. The population doubling time was 15.3 h under the above culture conditions. The cells at exponential growth phase in monolayer were dissociated by treatment with 0.25% trypsin (Difco Laboratories, Detroit, MI, 1:250) solution, and centrifuged at 1500 rpm for 5 min. The cells were resuspended in culture medium and used for experiments. Cytotox~city assay The cytotoxic effect of chemicals was examined by determining the colony-forming ability of cells, as described previously (Kuroda, 1984; Kuroda et al., 1985). 2 × 102 cells were incubated each in three 60-ram petri dishes in normal medium for 20 h, washed twice with Hanks' (Nissui Seiyaku Co., Tokyo, Japan) salt solution, and treated with chemicals in Hanks' salt solution at 37,5°C for 3 h. When cells were treated with 2 chemicals, they were treated with the first chemical for 3 h, washed twice with Hanks' salt solution, and treated with the second chemical for another 3 h. The cells were again washed twice with Hanks' salt solution, and incubated in normal medium for 7 days. The colonies formed were fixed in absolute methyl alcohol and stained with May-Griinwaid Giemsa (Merck, Darmstadt) and Giemsa (Merck, Darmstadt), The number of colonies containing more than 50 cells was scored under a binocular microscope. Mutagenicity assays 6TG-resistant mutation assay 2 x 105 cells were incubated each in five 100ram plastic petri dishes (Falcon Labware, Becton
Dickinson, U.S.A.) in normal medium for 20 h, washed twice in Hanks' salt solution, and treated with chemicals in Hanks' salt solution at 37.5°C for 3 h. When cells were treated with 2 chemicals, the procedure was the same as described in the cytotoxicity assay. These cells were again washed twice with Hanks' salt solution and incubated in normal medium at 37.5°C for an expression time of 6 days. Then the cells were dissociated by treatment with 0.25% trypsin solution, centrifuged, and inoculated into fresh dishes. 2 x 105 cells were incubated each in five 100-mm petri dishes in culture medium containing 5 /~g/ml 6TG (Wake Pure Chem. Ind., Osaka, Japan) for 10 days. Colonies formed were fixed in methyl alcohol and stained with May-Griiwald Giemsa and Giemsa. The number of colonies containing more than 50 cel!s was scored by the same procedure as described in the cytotoxicity assay. 2 × 102 dissociated cells were incubated each in three 60-mm petri dishes in normal medium for 7 days. The colonies formed were fixed, stained, and scored. The number of mutant colonies was corrected by the colony-forming ability of replated cells cultured in normal medium. The induced mutation frequency was calculated from the number of mutant colonies of treated cells minus the number of mutant colonies of untreated cells in control cultures. The induced mutation frequency was expressed as the number of induced mutant colonies per 105 survivors.
Chromosome aberration assay 2 x l0 s cells were incubated in normal medium for 20 h, washed twice in Hanks' salt solution, and treated with chemicals in Hanks' salt solution at 37.5°C. These cells were again washed twice in Hanks' salt solution and incubated in normal medium at 37.5°C for 18-21 h. Colcemid (Wake Pure Chem. Ind.) at a concentration of 5 ~ g / m l was added to culture medium and incubated for 2 h. The cells were dissociated by treatment with 0.25% trypsin solution, centrifuged and fixed with methanol-acetic acid (3: 1) after hypotonic treatment with 0.075 M KC! solution at 37.5°C for 15 min. Chromosome preparations were made according to the routine air-drying method. After 1 night, they were stained with Giemsa. Scoring of chromosome aberrations was done by the method
87 1.0t
of Cohen and Hirschhorn (1971). A sample of 100 metaphases was examined for chromosome aberrations in cells treated at each concentration of chemicals. Chemicals and treatments
The chemical used for inducing mutations was ethyl methanesulfonate (EMS) (Aldrich Chemical Co., Milwaukee, Wl). AsA (Wako Pure Chem. Ind.) was used to investigate effects on EMS-induced mutations and chromosome aberrations. Cells were treated with EMS alone for 3 h, treated with AsA and EMS simultaneously for 3 h, pre-treated with AsA for 3 h before treatment with EMS for 3 h, post-treated with AsA for 3 h after treatment with EMS for 3 h, and treated with mixtures of AsA and EMS for 3 h, which had previously been incubated at 37.5°C for 2 h. The concentrations used were varied for EMS and fixed at 100 ~,g/ml for AsA.
¢
.o a ..= .>
0.1(
¢R
0.01 !
0
200
I
400
Concentration
Results
Before we examined the effects of AsA on EMS-induced mutations, the cytotoxicity and the mutagenicity (inducing 6TG-resistant mutations and chromosome aberrations) of AsA were tested in Chinese hamster V79 cells. AsA showed strong cytotoxicity at a concentration of 500 p,g/ml in V79 cells treated for 3 h, but did not induce 6TG-resistant mutations and chromosome aberrations. As shown in Table 1, AsA at a concentration of 100 ~ g / m l had no detectable effects on the survival and the indue-
TABLE i CYTOTOXICITY AND MUTAGENICITY OF AsA IN CHINESE HAMSTER V79 CELLS Concentration of AsA (p.g/ml)
Surviving fraclion
Induced mutation frequency (per 105 survivors)
Total number of cells with chromosome aberrations ( ~ ) "
0 100 200 500
1.00 1.13 1.05 0.20
1.35 1.70 0.00 0.80
0.0 0.0 1.0 0.0
a Cells with gaps alone were excluded.
I
600
I
800
I 1000
of EMS (/~g/ml)
Fig. I. Effect of AsA on survival of V79 cells treated with EMS. o, treatment with EMS alone: [3, simultaneous treatment with EMS and AsA; ,a, post-treatment with AsA after treatment with EMS. AsA was used at a concentration of 100 p,g/ml.
tion of mutations in V79 cells. In the following experiment, the effects of 100 /J.g/ml AsA on EMS-induced cytotoxicity and mutagenicity were examined. Fig. 1 shows the effect of AsA on the cytotoxicity of EMS. In comparison with the surviving fraction of cells treated with EMS alone, that of cells treated simultaneously with EMS and AsA rose markedly. In post-treatments of AsA, however, no such increase was observed in surviving fractions of EMS-treated cells. Table 2 shows the effect of AsA on EMS-induced 6TG-resistant mutations in V79 cells. EMS strongly induced 6TG-resistant mutations in the absence of AsA. AsA markedly reduced 6TG-resistant mutations induced by EMS in simultaneous treatments. The mutagenic activity of EMS at 1000 p,g/ml was reduced to 28.2% by the addition of 100 p,g/ml AsA. With all concentrations of EMS used, the induced mutation frequencies reduced about one third or one fourth. When mixtures of EMS and AsA which had been prein-
88
cubated for 2 h were used to treat cells for 3 h, 6TG-resistant mutations were also reduced markedly. In pre- and post-treatments of cells with AsA for 3 h, however, no significant reduction of EMS-induced 6TG-resistant mutations was observed. Fig. 2 shows EMS-induced chromosome aberrations in the absence or the presence of AsA in V79 cells. AsA had a marked effect on the reduction of the number of cells with chromosome aberrations induced by EMS in simultaneous treatments. In the post-treatment with AsA, however, EMS-induced chromosome aberrations did not decrease, but were slightly increased. Table 3 shows the frequencies of chromosome aberrations induced by EMS in the absence or the presence of AsA. In the presence of AsA, EMS-induccd chromosome aberrations markedly decreased in all types of aberrations. In the posttreatments with AsA, however, the number of cells with EMS-induccd chromosome aberrations increased markedly (Fig. 2, Table 3). The =lumber of chromosome aberrations increased to twice or more that in the absence of AsA in control cultures.
ao
,= O
20
=D
E O
,.=
10
0~
~
1
o
-i-I--''~
400
I
!
800
13oo
Concentration of EMS (#g/mI) Fig. 2, Effect of AsA on chromosome aberrations of cells treated with EMS. o, treatment with EMS alone; El, simultaneous treatment with EMS and AsA; 4 , post-treatment with AsA after treatment with EMS. AsA was used at a concentration of 100 ~.g/ml.
Discussion
There are a number of reports on carcinogenesis and mutagenesis induced by various chemicals. it was shown that AsA inhibited the gene mutations induced by dimethylnitrosamine (DMN) (Lo and Stich, 1978), N-methyl-N'-nitro.
N-nitrosoguanidin¢ (MNNG) (Outtenplan, 1977; $aruno et al., 1979; Vojtekova and Miertus, 1986), aflatoxin B I (AFB) (Raina and Gurtoo, 1985), malonaldehyde and /3-propiolactone (Sham-
TABLE 2 COMPARISON OF VARIOUS TREATMENTS WITH AsA ON 6TG-RESISTANT MUTATIONS INDUCED BY EMS IN CHINESE HAMSTER V79 CELLS Concentration
Induced mutation frequency (per l0 s survivors)"
of EMS (/~g/ml)
No treatment
Simultaneous treatment ~'
Pre-treatment h
Post-treatment b
Pre-incubated mixtures i~
0 100 200 400 600 800 1000
0.0 3,0 I 1.0 19.5 36.0 43.0 87,5
0.0 2,5 1.2 7.7 12.3 12.2 24.7
0,2 ! 2.0 7,0 19,0 45,0 69.0 6%0
0.4 10.6 18.0 30,9 61,0 77,7 92.3
0.0 2.2 4.9 15.! 20.3 30.6 26.0
;' Treatment procedures arc shown in the legend to Fig. 1. t, AsA was used at a concentration of 100 #g/ml.
89 berger et al., 1979) in microbial assay systems and tetracycline hydrochloride (TC) (Bhattacharjee and Pal, 1982) in mammalian assay systems. A s A reduces the c h r o m o s o m e aberrations ind u c e d by trenimon, cyclophosphamide ( C P A ) ( G e b h a r t , 1984), 7,12-dimethylhenzoanthracene ( D M B A ) (Shamberger et al., 1973) and H202 (Dailapiccola et al., 1985), and SCEs (sister-chromatid exchanges) induced by M N N G (Galloway and Painter, 1979), CPA, mitomycin C (Krishna et al., 1986), thiotepa, L-ethionine (Lialiaris et al., 1987) and oxygen radicals (Weitberg and Weitzman, 1985), and micronueleus formation induced by d i i o d o h y d r o x y q u i n o l i n e ( G h a s k a d b i a n d Vaidya, 1989) and aminopyrine plus nitrite (Pienkowska et al., 1985). O n the o t h e r hand, t h e r e is evidence to show that AsA enhances the gene mutations induced by M N N G (Galloway and Painter, 1979), N-
hydroxy-2-acetylaminofluorene (N-OH-AAF) (Andrews et al., 1978; Wirth and Thorgeirsson, 1980), 3-hydroxyamino- 1-methyl-5 H-pyrido[4,3b]indole (N-OH-Trp-P-2) (Mita et al., 1982) and benzo[a]pyrene (Alzieu et al., 1987), and SCEs induced by thiotepa and L-ethionine (Lialiaris et ai., 1987). In the present study, it was shown that the cytotoxicity, 6TG-resistant mutations and chromosome aberrations induced by E M S in Chinese hamster V79 cells decreased markedly by simultaneous t r e a t m e n t with AsA and t r e a t m e n t with preincubated mixtures of EMS and AsA. Several compounds with antioxidant properties, such as vitamin E (Saruno et al., 1979; S h a m b e r g e r et al., 1979; G e b h a r t , 1984), cysteine (Saruno et al., 1979; Osawa et al., 1980), butylhydroperoxide ( B H T ) and selenium (Shamberger et al., 1973; 1979), also have similar antimutagenic activity to
TABLE 3 COMPARISON OF VARIOUS TREATMENTS WITH AsA ON CHROMOSOME ABERRATIONS INDUCED BY EMS IN CHINESE HAMSTER V79 CELLS Concentration of EMS (p,g/ml)
Concentration of AsA (#g/ml)
Number of cells examined
Cells with aberration,~" (~)
Number of aberrations per cell cdg cdb icdg icdb b
cdx
Totals
I) 401}
0 0
I00 IIH}
ILl} 2.0
2 3
0 I
0 II
0 I
0 0
0 5
~lll)
0 I)
I IHI I(H)
4,1} 9.I)
7 9
3 8
0 I)
0 2
2 I}
12
0
I(H)
16.0
12
16
0
5
2
19 35
100 I00 11}0 IO0 I00 I00
II.0 1.0 0.0 1.0 2.0 5.0
2 0 2 2
0 I 0 0
0 I 0 0
0 0 0 I
0 0 4) 0
2 2 2 3
3
2
0
0
0
5
3
4
0
I
0
8
I) I()) I O0
I O0 I00 I O0
I ,I) 0.0 5.0
2 4 7
I 4) 5
{I 0 I
0 0 0
(l 0 4
3 4 17
IO0 I00 Ill0
I O0 I{}0 100
7.0 14,(I 28.0
13 16 25
8 17 43
2
I
0
I
0
3
3 13 14
27 47 85
8011 1200
Sinlldtallt, olis tredtnle#ll with A+,4 c
0 0 400 600 800 1200
0 lifo I00 II}O I0{} I00
Post-treatment with AxA c
(I II
400 600 801} 1 200
cdg, chromatid gap; cdb, chromatid break: icdg, isochromatid gap: icdb. isochromatid break: cdx+chromatid exchange. " Cells with gaps alone were excluded. I, Acentric fragments not associated with dicentrics and rings were scored as isochromatid breaks. c AsA was used at a concentration of 100 ktg/ml.
90 AsA. When mutagens induce damage in DNA and protein, antioxidants may protect against or reduce the production of the mutational endpoint. As an action mechanism of AsA, AsA is composed of indole-3-methanol anion at physiological pH, which causes alkylation at nucleophilic sites of DNA and protein (Edger, 1974). Competition for alkylation of AsA and ethylation of EMS (Dion et al., 1982) may result in a decrease of the cytotoxicity and mutagenicity of EMS. Treatment with preincubated mixtures of AsA and EMS also reduced the 6GT-resistant mutations induced by EMS. These results suggest that the action of AsA on EMS may be desmutagenic. There were some differences in the reduction of mutations between the simultaneous treatments and treatments with preincubated mixtures of AsA and EMS. These differences may be due to some bio-antimutagenic activity of AsA incorporated into cells during the simultaneous treatment with AsA and EMS. On the other hand, AsA is an important vitamin to support redox reactions in vivo. The possibility of its connection with repair enzymes of DNA cannot be excluded. The effects of AsA on EMS-induced mutations were examined in proand post.treatments with AsA. The results (Table 2) of both treatments with AsA indicated that the frequencies of EMS.induced mutations were not reduced but increased slightly. This suggests that AsA may not protect DNA against mutagenie agents, and it may not be concerned with DNArepair mechanisms. The possibility that AsA enhanced the mutagenicity due to damaged DNA and protein may be considered, These results support the reports of Galloway and Painter (1979) and Mira et al. (1982). Bhattacharjce and Pal (1982)suggested, however, that AsA slightly decreased the frequency of 8azaguanine (8AG)-resistant mutations in Chinese hamster cells induced by TC in simultaneous treatments with AsA, and also significantly reduced the frequency in pre- and post-treatments with AsA. They considered that AsA shielded the genetic material from mutagens. These results are contrary to those in the present study, The mechanism of induction of mutations by EMS may be different from that by TC. It is known
that TC inhibits protein synthesis (Bhattacharjee and Pal, 1982). A concentration of AsA used in the present experiments was 100 /.~g/ml. At this concentration AsA has little cell to,~icity. High concentrations of AsA were not tested in simultaneous treatment of cells with EMS. Many results using various mutagens and methods are different from each other. Comparison of the results of antimutagenic effects of AsA indicates that the action of AsA varies significantly among mutagens used, mutation endpoints, kinds of DNA damages, and assay systems with different organisms. More investigations are needed to understand the mechanism of activity of AsA on chemical-induced mutagenicity. Acknowledgements
We acknowledge the helpful advice of K. Shinkawa (Tokyo University), H. Tezuka, A. Yokoiyama and Dr. T. Kada (National Institute of Genetics). We also wish to thank Miss Y. Takada for her technical assistance during this work. References
Alzieu, P,, P. Cassand,C. Colin,P. Grolier and J.F. Narbonne (1987) Effect of vitamin A, C and glutathione on the mutagenicityof benzo[a]pyrenemediatedby $9 fromvitamin A-deficientrats, Mutation Res., 192,227-231. Andrews, L.S., J.A. Hinson and J.R. Gillette (1978) Studies on the mutagenieityof N-hydro~.2-acetylaminofluorene in the Ames-Salmonellamutagenesistest system,Biochem. Pharmacol.,27, 2399-2408. Bhattacharjee, S.B., and B. Pal (1982) Tetracycline induced mutationin cultured Chinesehamstercells, MutationRes., 101,329-338. Cohen, M.M., and K. l-!irschhorn(1971) Cytogeneticstudies in animals, in: A. |-|o!laender(Ed.), Chemic,~lMutagens, Principles and Methods for Their Detection, Vol. 2, Plenum, New York, pp. 515-534. Dallapiccola, B,, B, Porfiro, V, Mokini, G. Alimena, G. lsacchi and E, Gandini (1985) Effect of oxidants and antioxidants on chromosomalbreakage in Fanconi anemia iymphocytes,Hum. Genet., 69, 62-65. Dion, P.W., E.B. Bryght-See,C.C. Smith and W.R. Bruce (1982)The effect of dietaryascorbicacid and a-tocopherol on fecal mutagenicity,Mutation Res., 102, 27-37. Edger, J.&. (1974) Ascorbie acid and biological aikylating agents, Nature (London),248, 136.
91 Ford, D.K., and G. Yerganian (1958) Observations on the chromosomes of Chinese hamster cells in tissue culture, J. Natl. Cancer Inst., 21, 393-425. Galloway, S.M., and R.B. Painter (1979) Vitamin C is positive in the DNA synthesis inhibition and sister-chromatid exchange tests, Mutation Res., 60, 321-327. Gebhart, E. (1984) The action of anticlastogens on chemically induced SCE, Basic Life Sci., 29A, 319-332. Ghaskadbi, S., and V.G. Vaidya (1989) In vivo antimutagenic effect of ascorbic acid against mutagenicity of the common antiamebic drug diiodohydroxyquinoline, Mutation Res., 222, 219-222. Guttenplan, J.B. (1977) Inhibition by L-ascorbat,~, of bacterial mutagenesis induced by two N-nitroso compounds, Nature (London), 268, 368-370. Krishna, G., J. Nath and T. Ong (1986) Inhibition of cyclophosphamide and mitomycin C-induced sister chromatid exchanges in mice by vitamin C, Cancer Res., 46, 2670-2674. Kuroda, Y. (1984) Dose-rate effects of chemicals on mutation induction in mammalian cells in culture, in: Y. Tazima, S. Kondo and Y. Kuroda (Eds.), Problems of Threshold in Chemical Mutagenesis, Environmental Mntagen Society of Japan, Mishima, Shizuoka, pp. 99-108. Kuroda, Y, (1986a) Genetic and chemical factors affecting chemical mutagenesis in cultured mammalian cells, in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis: Mechanisms, Plenum, New York, pp. 359-375. Kuroda, Y, (1986b) Antimutagenic activity of vitamin C in cultured mammalian cells, Mutation Res., 164, 273. Kuroda, Y., A. Yokoiyama and T. Kada (1985) Assay for the induction of mutations to 6-thioguanine resistance in Chinese hamster V79 cells in culture, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B,H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, World Health Organization, Elsevier, Amsterdam, pp. 537-545. Lialiaris. T., D. Mourelatos and J.D. Vassiliades (1987) Enhancement and attenuation of ¢ytogenetic damage by vitamin C in cultured human lymphocytes exposed to Thiotepa or L-ethionine, Cytogenet. Cell Genet., 44, 209-214. Lo, L.W., and H.F. Stich (1978) The use of short-term test to
measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds, Mutation Res., 57, 57-67. Mita, S., Y. Yamazoe, T. Kamataki and R. Kato (1982) Effects of ascorbic acid on the nonenzymatic binding to DNA and the mutagenicity of N-hydroxylated metabolite of a tryptophan-pyrolysis product, Biochem. Biophys. Res. Commun., 105, 1396-1401. Osawa, T., H. Ishibashi, M. Namiki and T. Kada (1980) Desmutagenic actions of ascorbie acid and cystein on a new pyrole mutagen formed by the reaction between food additives: sorbic acid and sodium nitrite, Biochem. Biophys. Res. Commun., 95, 835-841. Pienkowska, K., H. Gajcy and J. Koziorowka (1985) Protective action of ascorbic acid against mutagenicity of aminopyrine plus nitrite, Pol. J. Pharmacol. Pharm., 37, 601-607. Raina, V., and H.L. Gurtoo (1985) Effects of vitamins A, C and E on aflatoxin Bi-induced mutagenesis in Salmonella typhimurium TA98 and TA100, Teratogen. Carcinogen. Mutagen., 5, 29-40. Saruno, R., M. Yoshida, F. Kato and A. Murata (1979) Screening of desmutagenic substances on mutagen NmethyI-N'-nitro-N-nitrosoguanidine, Hakkoogaku, 57, 8691. Shamberger, R.J., F.F. Banghman, S,L. Kalchert, C.E. Willis and G.C. Hoffman (1973)Carcinogen induced chromosomal breakage decreased by antioxidanls, Proc. Natl. Acad. Sci. (U.S.A.), 70, 1461-1463. Shamberger, R.J., C.L. Corlett, K.D. Beamam and B.L. Kasten (1979) Antioxidants reduce the mutagenic effect of malonaldehyde and g-pirolactone, Part IX, Antioxidants and cancer, Mutation Res., 66, 349-355. Vojtekova, H., and S. Miertus (1986) Influence of ascorbic acid on the mutagenicity of N-methyI-N=nitrosoguanidine and nitrofurans studied by SOS chromotest, Neoplasma, 33, 691-698. Weitherg, A,B., and S,A. Weitzman (1985) The effect of vitamin C on oxTgen radical-induced sister chromatid exchanges, Mutation Res,, 144, 23-26. Wirth, P,J., and S.S. Thorgeirsson (1980) Mechanism of N-hydroxv.2-acetyl-aminofluorene mutagenicity in the Salmonella test system, Mol. Pharmacol., 19, 337-344.
85
MUT 05056
Effects of L-ascorbic acid on the mutagenicity of ethyl methanesulfonate in cultured mammalian cells H . K o j i m a 1, H . K o n i s h i t a n d Y. K u r o d a 2 I Biochemical Research Institute, Nippon Menard Cosmetic Co. Ltd., Ogaki, Gifu 503 and z Research Institute of Biosciences, Azabu Unicersity, Fuchinobe. Sagamihara, Kanagawa 229 (Japan) (Received 2 April 1991) (Revision received 23 September 1991) (Accepted 26 September 1991)
Keywords: Ethyl methanesulphonate; L-Ascorbic acid; Mutagenicity; Mammalian cells
Summary The effects of L-ascorbic acid (AsA) on the mutations induced by ethyl methanesulfonate (EMS) were examined by means of the 6-thioguanine (6TG)-resistant mutation assay and chromosome aberration assay in cultured Chinese hamster V79 cells. When cells were treated with EMS at various concentrations in the presence of 100 /zg/ml AsA, EMS-induced 6TG-resistant mutations w~re reduced about one third or one fourth. EMS-induced chromosome aberrations were also reduced by AsA. These reductions in the mutagenicity of EMS were also found when cells were treated with mixtures of AsP, and EMS which had previously been incubated at 37.0°C for 2 h. in pre- and post-treatments with AsA, however, the frequencies of EMS-induced mutations were not reduced, but rather increased markedly.
Correspondence: Prof. Dr. Yukiaki Kuroda, Research Institute of Biosciences, Azabu University, 1-17-71, Fuchinobe, Sagamihara, Kanagawa229 (Japan). Tel.: 0427-54-7111(Ext. 430); Fax: 0427-54-7661.
Abbreciations: AsA, L-ascorbic acid; EMS, ethyl methanesulfonate; 6TG, 6-thioguanine, DMN, dimethylnitrosamine; MNNG, N-methyI-N'-nitro-N.nitrosoguanidine; AFB, ariatoxin Bi; TC, tetracycline hydrochloride; CPA, cyclophosphamide; DMBA, 7,12.dimethylbenzoanthracene; SCE, sister-chromatid exchange; N-OH-AAF, N-hydroxy-2-acetylaminofluorene; N-OH-Trp-P2, 3-hydroxyamino-l-methyl-5Hpyrido[4,3-b]indole; BHT, butylhydroperoxide; 8AG, 8azaguanine.
L-Ascorbic acid (AsA) is an antioxidant and a reducing agent. It is abundantly present in green tea, fruits and vegetables. It is effective in preventing scurvy, and its antioxidant activity is useful for the detoxification of some drugs and for the protection against rancidity in foodstuffs. In the present paper, EMS was used as a mutagen with alkylating activity. The effects of AsA on 6TO-resistant mutations and chromosome aberrations induced by EMS were examined in cultured Chinese hamster V79 cells. Some preliminary results have been reported elsewhere (Kuroda, 1986a, b).
86
Materials and methods
Cell culture The cell line used was the strain of Chinese hamster lung V79 cells isolated by Ford and Yerganian (1958). Cells were maintained in Eagle's minimum essential medium (Nissui Seiyaku Co., Tokyo, Japan) supplemented with 10% fetal bovine serum (Gibco Lab., Grand Island, NY) in 60-mm plastic petri dishes (Lux Sci. Corp., Newbury Park, CA, No. 5216, 2-mm grid) under a controlled atmosphere of 5% CO 2 and 95% air at 37.5°C. Tbey were mycoplasma-free and showed a colony-forming ability of more than 80%. The population doubling time was 15.3 h under the above culture conditions. The cells at exponential growth phase in monolayer were dissociated by treatment with 0.25% trypsin (Difco Laboratories, Detroit, MI, 1:250) solution, and centrifuged at 1500 rpm for 5 min. The cells were resuspended in culture medium and used for experiments. Cytotox~city assay The cytotoxic effect of chemicals was examined by determining the colony-forming ability of cells, as described previously (Kuroda, 1984; Kuroda et al., 1985). 2 × 102 cells were incubated each in three 60-ram petri dishes in normal medium for 20 h, washed twice with Hanks' (Nissui Seiyaku Co., Tokyo, Japan) salt solution, and treated with chemicals in Hanks' salt solution at 37,5°C for 3 h. When cells were treated with 2 chemicals, they were treated with the first chemical for 3 h, washed twice with Hanks' salt solution, and treated with the second chemical for another 3 h. The cells were again washed twice with Hanks' salt solution, and incubated in normal medium for 7 days. The colonies formed were fixed in absolute methyl alcohol and stained with May-Griinwaid Giemsa (Merck, Darmstadt) and Giemsa (Merck, Darmstadt), The number of colonies containing more than 50 cells was scored under a binocular microscope. Mutagenicity assays 6TG-resistant mutation assay 2 x 105 cells were incubated each in five 100ram plastic petri dishes (Falcon Labware, Becton
Dickinson, U.S.A.) in normal medium for 20 h, washed twice in Hanks' salt solution, and treated with chemicals in Hanks' salt solution at 37.5°C for 3 h. When cells were treated with 2 chemicals, the procedure was the same as described in the cytotoxicity assay. These cells were again washed twice with Hanks' salt solution and incubated in normal medium at 37.5°C for an expression time of 6 days. Then the cells were dissociated by treatment with 0.25% trypsin solution, centrifuged, and inoculated into fresh dishes. 2 x 105 cells were incubated each in five 100-mm petri dishes in culture medium containing 5 /~g/ml 6TG (Wake Pure Chem. Ind., Osaka, Japan) for 10 days. Colonies formed were fixed in methyl alcohol and stained with May-Griiwald Giemsa and Giemsa. The number of colonies containing more than 50 cel!s was scored by the same procedure as described in the cytotoxicity assay. 2 × 102 dissociated cells were incubated each in three 60-mm petri dishes in normal medium for 7 days. The colonies formed were fixed, stained, and scored. The number of mutant colonies was corrected by the colony-forming ability of replated cells cultured in normal medium. The induced mutation frequency was calculated from the number of mutant colonies of treated cells minus the number of mutant colonies of untreated cells in control cultures. The induced mutation frequency was expressed as the number of induced mutant colonies per 105 survivors.
Chromosome aberration assay 2 x l0 s cells were incubated in normal medium for 20 h, washed twice in Hanks' salt solution, and treated with chemicals in Hanks' salt solution at 37.5°C. These cells were again washed twice in Hanks' salt solution and incubated in normal medium at 37.5°C for 18-21 h. Colcemid (Wake Pure Chem. Ind.) at a concentration of 5 ~ g / m l was added to culture medium and incubated for 2 h. The cells were dissociated by treatment with 0.25% trypsin solution, centrifuged and fixed with methanol-acetic acid (3: 1) after hypotonic treatment with 0.075 M KC! solution at 37.5°C for 15 min. Chromosome preparations were made according to the routine air-drying method. After 1 night, they were stained with Giemsa. Scoring of chromosome aberrations was done by the method
87 1.0t
of Cohen and Hirschhorn (1971). A sample of 100 metaphases was examined for chromosome aberrations in cells treated at each concentration of chemicals. Chemicals and treatments
The chemical used for inducing mutations was ethyl methanesulfonate (EMS) (Aldrich Chemical Co., Milwaukee, Wl). AsA (Wako Pure Chem. Ind.) was used to investigate effects on EMS-induced mutations and chromosome aberrations. Cells were treated with EMS alone for 3 h, treated with AsA and EMS simultaneously for 3 h, pre-treated with AsA for 3 h before treatment with EMS for 3 h, post-treated with AsA for 3 h after treatment with EMS for 3 h, and treated with mixtures of AsA and EMS for 3 h, which had previously been incubated at 37.5°C for 2 h. The concentrations used were varied for EMS and fixed at 100 ~,g/ml for AsA.
¢
.o a ..= .>
0.1(
¢R
0.01 !
0
200
I
400
Concentration
Results
Before we examined the effects of AsA on EMS-induced mutations, the cytotoxicity and the mutagenicity (inducing 6TG-resistant mutations and chromosome aberrations) of AsA were tested in Chinese hamster V79 cells. AsA showed strong cytotoxicity at a concentration of 500 p,g/ml in V79 cells treated for 3 h, but did not induce 6TG-resistant mutations and chromosome aberrations. As shown in Table 1, AsA at a concentration of 100 ~ g / m l had no detectable effects on the survival and the indue-
TABLE i CYTOTOXICITY AND MUTAGENICITY OF AsA IN CHINESE HAMSTER V79 CELLS Concentration of AsA (p.g/ml)
Surviving fraclion
Induced mutation frequency (per 105 survivors)
Total number of cells with chromosome aberrations ( ~ ) "
0 100 200 500
1.00 1.13 1.05 0.20
1.35 1.70 0.00 0.80
0.0 0.0 1.0 0.0
a Cells with gaps alone were excluded.
I
600
I
800
I 1000
of EMS (/~g/ml)
Fig. I. Effect of AsA on survival of V79 cells treated with EMS. o, treatment with EMS alone: [3, simultaneous treatment with EMS and AsA; ,a, post-treatment with AsA after treatment with EMS. AsA was used at a concentration of 100 p,g/ml.
tion of mutations in V79 cells. In the following experiment, the effects of 100 /J.g/ml AsA on EMS-induced cytotoxicity and mutagenicity were examined. Fig. 1 shows the effect of AsA on the cytotoxicity of EMS. In comparison with the surviving fraction of cells treated with EMS alone, that of cells treated simultaneously with EMS and AsA rose markedly. In post-treatments of AsA, however, no such increase was observed in surviving fractions of EMS-treated cells. Table 2 shows the effect of AsA on EMS-induced 6TG-resistant mutations in V79 cells. EMS strongly induced 6TG-resistant mutations in the absence of AsA. AsA markedly reduced 6TG-resistant mutations induced by EMS in simultaneous treatments. The mutagenic activity of EMS at 1000 p,g/ml was reduced to 28.2% by the addition of 100 p,g/ml AsA. With all concentrations of EMS used, the induced mutation frequencies reduced about one third or one fourth. When mixtures of EMS and AsA which had been prein-
88
cubated for 2 h were used to treat cells for 3 h, 6TG-resistant mutations were also reduced markedly. In pre- and post-treatments of cells with AsA for 3 h, however, no significant reduction of EMS-induced 6TG-resistant mutations was observed. Fig. 2 shows EMS-induced chromosome aberrations in the absence or the presence of AsA in V79 cells. AsA had a marked effect on the reduction of the number of cells with chromosome aberrations induced by EMS in simultaneous treatments. In the post-treatment with AsA, however, EMS-induced chromosome aberrations did not decrease, but were slightly increased. Table 3 shows the frequencies of chromosome aberrations induced by EMS in the absence or the presence of AsA. In the presence of AsA, EMS-induccd chromosome aberrations markedly decreased in all types of aberrations. In the posttreatments with AsA, however, the number of cells with EMS-induccd chromosome aberrations increased markedly (Fig. 2, Table 3). The =lumber of chromosome aberrations increased to twice or more that in the absence of AsA in control cultures.
ao
,= O
20
=D
E O
,.=
10
0~
~
1
o
-i-I--''~
400
I
!
800
13oo
Concentration of EMS (#g/mI) Fig. 2, Effect of AsA on chromosome aberrations of cells treated with EMS. o, treatment with EMS alone; El, simultaneous treatment with EMS and AsA; 4 , post-treatment with AsA after treatment with EMS. AsA was used at a concentration of 100 ~.g/ml.
Discussion
There are a number of reports on carcinogenesis and mutagenesis induced by various chemicals. it was shown that AsA inhibited the gene mutations induced by dimethylnitrosamine (DMN) (Lo and Stich, 1978), N-methyl-N'-nitro.
N-nitrosoguanidin¢ (MNNG) (Outtenplan, 1977; $aruno et al., 1979; Vojtekova and Miertus, 1986), aflatoxin B I (AFB) (Raina and Gurtoo, 1985), malonaldehyde and /3-propiolactone (Sham-
TABLE 2 COMPARISON OF VARIOUS TREATMENTS WITH AsA ON 6TG-RESISTANT MUTATIONS INDUCED BY EMS IN CHINESE HAMSTER V79 CELLS Concentration
Induced mutation frequency (per l0 s survivors)"
of EMS (/~g/ml)
No treatment
Simultaneous treatment ~'
Pre-treatment h
Post-treatment b
Pre-incubated mixtures i~
0 100 200 400 600 800 1000
0.0 3,0 I 1.0 19.5 36.0 43.0 87,5
0.0 2,5 1.2 7.7 12.3 12.2 24.7
0,2 ! 2.0 7,0 19,0 45,0 69.0 6%0
0.4 10.6 18.0 30,9 61,0 77,7 92.3
0.0 2.2 4.9 15.! 20.3 30.6 26.0
;' Treatment procedures arc shown in the legend to Fig. 1. t, AsA was used at a concentration of 100 #g/ml.
89 berger et al., 1979) in microbial assay systems and tetracycline hydrochloride (TC) (Bhattacharjee and Pal, 1982) in mammalian assay systems. A s A reduces the c h r o m o s o m e aberrations ind u c e d by trenimon, cyclophosphamide ( C P A ) ( G e b h a r t , 1984), 7,12-dimethylhenzoanthracene ( D M B A ) (Shamberger et al., 1973) and H202 (Dailapiccola et al., 1985), and SCEs (sister-chromatid exchanges) induced by M N N G (Galloway and Painter, 1979), CPA, mitomycin C (Krishna et al., 1986), thiotepa, L-ethionine (Lialiaris et al., 1987) and oxygen radicals (Weitberg and Weitzman, 1985), and micronueleus formation induced by d i i o d o h y d r o x y q u i n o l i n e ( G h a s k a d b i a n d Vaidya, 1989) and aminopyrine plus nitrite (Pienkowska et al., 1985). O n the o t h e r hand, t h e r e is evidence to show that AsA enhances the gene mutations induced by M N N G (Galloway and Painter, 1979), N-
hydroxy-2-acetylaminofluorene (N-OH-AAF) (Andrews et al., 1978; Wirth and Thorgeirsson, 1980), 3-hydroxyamino- 1-methyl-5 H-pyrido[4,3b]indole (N-OH-Trp-P-2) (Mita et al., 1982) and benzo[a]pyrene (Alzieu et al., 1987), and SCEs induced by thiotepa and L-ethionine (Lialiaris et ai., 1987). In the present study, it was shown that the cytotoxicity, 6TG-resistant mutations and chromosome aberrations induced by E M S in Chinese hamster V79 cells decreased markedly by simultaneous t r e a t m e n t with AsA and t r e a t m e n t with preincubated mixtures of EMS and AsA. Several compounds with antioxidant properties, such as vitamin E (Saruno et al., 1979; S h a m b e r g e r et al., 1979; G e b h a r t , 1984), cysteine (Saruno et al., 1979; Osawa et al., 1980), butylhydroperoxide ( B H T ) and selenium (Shamberger et al., 1973; 1979), also have similar antimutagenic activity to
TABLE 3 COMPARISON OF VARIOUS TREATMENTS WITH AsA ON CHROMOSOME ABERRATIONS INDUCED BY EMS IN CHINESE HAMSTER V79 CELLS Concentration of EMS (p,g/ml)
Concentration of AsA (#g/ml)
Number of cells examined
Cells with aberration,~" (~)
Number of aberrations per cell cdg cdb icdg icdb b
cdx
Totals
I) 401}
0 0
I00 IIH}
ILl} 2.0
2 3
0 I
0 II
0 I
0 0
0 5
~lll)
0 I)
I IHI I(H)
4,1} 9.I)
7 9
3 8
0 I)
0 2
2 I}
12
0
I(H)
16.0
12
16
0
5
2
19 35
100 I00 11}0 IO0 I00 I00
II.0 1.0 0.0 1.0 2.0 5.0
2 0 2 2
0 I 0 0
0 I 0 0
0 0 0 I
0 0 4) 0
2 2 2 3
3
2
0
0
0
5
3
4
0
I
0
8
I) I()) I O0
I O0 I00 I O0
I ,I) 0.0 5.0
2 4 7
I 4) 5
{I 0 I
0 0 0
(l 0 4
3 4 17
IO0 I00 Ill0
I O0 I{}0 100
7.0 14,(I 28.0
13 16 25
8 17 43
2
I
0
I
0
3
3 13 14
27 47 85
8011 1200
Sinlldtallt, olis tredtnle#ll with A+,4 c
0 0 400 600 800 1200
0 lifo I00 II}O I0{} I00
Post-treatment with AxA c
(I II
400 600 801} 1 200
cdg, chromatid gap; cdb, chromatid break: icdg, isochromatid gap: icdb. isochromatid break: cdx+chromatid exchange. " Cells with gaps alone were excluded. I, Acentric fragments not associated with dicentrics and rings were scored as isochromatid breaks. c AsA was used at a concentration of 100 ktg/ml.
90 AsA. When mutagens induce damage in DNA and protein, antioxidants may protect against or reduce the production of the mutational endpoint. As an action mechanism of AsA, AsA is composed of indole-3-methanol anion at physiological pH, which causes alkylation at nucleophilic sites of DNA and protein (Edger, 1974). Competition for alkylation of AsA and ethylation of EMS (Dion et al., 1982) may result in a decrease of the cytotoxicity and mutagenicity of EMS. Treatment with preincubated mixtures of AsA and EMS also reduced the 6GT-resistant mutations induced by EMS. These results suggest that the action of AsA on EMS may be desmutagenic. There were some differences in the reduction of mutations between the simultaneous treatments and treatments with preincubated mixtures of AsA and EMS. These differences may be due to some bio-antimutagenic activity of AsA incorporated into cells during the simultaneous treatment with AsA and EMS. On the other hand, AsA is an important vitamin to support redox reactions in vivo. The possibility of its connection with repair enzymes of DNA cannot be excluded. The effects of AsA on EMS-induced mutations were examined in proand post.treatments with AsA. The results (Table 2) of both treatments with AsA indicated that the frequencies of EMS.induced mutations were not reduced but increased slightly. This suggests that AsA may not protect DNA against mutagenie agents, and it may not be concerned with DNArepair mechanisms. The possibility that AsA enhanced the mutagenicity due to damaged DNA and protein may be considered, These results support the reports of Galloway and Painter (1979) and Mira et al. (1982). Bhattacharjce and Pal (1982)suggested, however, that AsA slightly decreased the frequency of 8azaguanine (8AG)-resistant mutations in Chinese hamster cells induced by TC in simultaneous treatments with AsA, and also significantly reduced the frequency in pre- and post-treatments with AsA. They considered that AsA shielded the genetic material from mutagens. These results are contrary to those in the present study, The mechanism of induction of mutations by EMS may be different from that by TC. It is known
that TC inhibits protein synthesis (Bhattacharjee and Pal, 1982). A concentration of AsA used in the present experiments was 100 /.~g/ml. At this concentration AsA has little cell to,~icity. High concentrations of AsA were not tested in simultaneous treatment of cells with EMS. Many results using various mutagens and methods are different from each other. Comparison of the results of antimutagenic effects of AsA indicates that the action of AsA varies significantly among mutagens used, mutation endpoints, kinds of DNA damages, and assay systems with different organisms. More investigations are needed to understand the mechanism of activity of AsA on chemical-induced mutagenicity. Acknowledgements
We acknowledge the helpful advice of K. Shinkawa (Tokyo University), H. Tezuka, A. Yokoiyama and Dr. T. Kada (National Institute of Genetics). We also wish to thank Miss Y. Takada for her technical assistance during this work. References
Alzieu, P,, P. Cassand,C. Colin,P. Grolier and J.F. Narbonne (1987) Effect of vitamin A, C and glutathione on the mutagenicityof benzo[a]pyrenemediatedby $9 fromvitamin A-deficientrats, Mutation Res., 192,227-231. Andrews, L.S., J.A. Hinson and J.R. Gillette (1978) Studies on the mutagenieityof N-hydro~.2-acetylaminofluorene in the Ames-Salmonellamutagenesistest system,Biochem. Pharmacol.,27, 2399-2408. Bhattacharjee, S.B., and B. Pal (1982) Tetracycline induced mutationin cultured Chinesehamstercells, MutationRes., 101,329-338. Cohen, M.M., and K. l-!irschhorn(1971) Cytogeneticstudies in animals, in: A. |-|o!laender(Ed.), Chemic,~lMutagens, Principles and Methods for Their Detection, Vol. 2, Plenum, New York, pp. 515-534. Dallapiccola, B,, B, Porfiro, V, Mokini, G. Alimena, G. lsacchi and E, Gandini (1985) Effect of oxidants and antioxidants on chromosomalbreakage in Fanconi anemia iymphocytes,Hum. Genet., 69, 62-65. Dion, P.W., E.B. Bryght-See,C.C. Smith and W.R. Bruce (1982)The effect of dietaryascorbicacid and a-tocopherol on fecal mutagenicity,Mutation Res., 102, 27-37. Edger, J.&. (1974) Ascorbie acid and biological aikylating agents, Nature (London),248, 136.
91 Ford, D.K., and G. Yerganian (1958) Observations on the chromosomes of Chinese hamster cells in tissue culture, J. Natl. Cancer Inst., 21, 393-425. Galloway, S.M., and R.B. Painter (1979) Vitamin C is positive in the DNA synthesis inhibition and sister-chromatid exchange tests, Mutation Res., 60, 321-327. Gebhart, E. (1984) The action of anticlastogens on chemically induced SCE, Basic Life Sci., 29A, 319-332. Ghaskadbi, S., and V.G. Vaidya (1989) In vivo antimutagenic effect of ascorbic acid against mutagenicity of the common antiamebic drug diiodohydroxyquinoline, Mutation Res., 222, 219-222. Guttenplan, J.B. (1977) Inhibition by L-ascorbat,~, of bacterial mutagenesis induced by two N-nitroso compounds, Nature (London), 268, 368-370. Krishna, G., J. Nath and T. Ong (1986) Inhibition of cyclophosphamide and mitomycin C-induced sister chromatid exchanges in mice by vitamin C, Cancer Res., 46, 2670-2674. Kuroda, Y. (1984) Dose-rate effects of chemicals on mutation induction in mammalian cells in culture, in: Y. Tazima, S. Kondo and Y. Kuroda (Eds.), Problems of Threshold in Chemical Mutagenesis, Environmental Mntagen Society of Japan, Mishima, Shizuoka, pp. 99-108. Kuroda, Y, (1986a) Genetic and chemical factors affecting chemical mutagenesis in cultured mammalian cells, in: D.M. Shankel, P.E. Hartman, T. Kada and A. Hollaender (Eds.), Antimutagenesis and Anticarcinogenesis: Mechanisms, Plenum, New York, pp. 359-375. Kuroda, Y, (1986b) Antimutagenic activity of vitamin C in cultured mammalian cells, Mutation Res., 164, 273. Kuroda, Y., A. Yokoiyama and T. Kada (1985) Assay for the induction of mutations to 6-thioguanine resistance in Chinese hamster V79 cells in culture, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B,H. Margolin, B.E. Matter and M.D. Shelby (Eds.), Evaluation of Short-Term Tests for Carcinogens, World Health Organization, Elsevier, Amsterdam, pp. 537-545. Lialiaris. T., D. Mourelatos and J.D. Vassiliades (1987) Enhancement and attenuation of ¢ytogenetic damage by vitamin C in cultured human lymphocytes exposed to Thiotepa or L-ethionine, Cytogenet. Cell Genet., 44, 209-214. Lo, L.W., and H.F. Stich (1978) The use of short-term test to
measure the preventive action of reducing agents on formation and activation of carcinogenic nitroso compounds, Mutation Res., 57, 57-67. Mita, S., Y. Yamazoe, T. Kamataki and R. Kato (1982) Effects of ascorbic acid on the nonenzymatic binding to DNA and the mutagenicity of N-hydroxylated metabolite of a tryptophan-pyrolysis product, Biochem. Biophys. Res. Commun., 105, 1396-1401. Osawa, T., H. Ishibashi, M. Namiki and T. Kada (1980) Desmutagenic actions of ascorbie acid and cystein on a new pyrole mutagen formed by the reaction between food additives: sorbic acid and sodium nitrite, Biochem. Biophys. Res. Commun., 95, 835-841. Pienkowska, K., H. Gajcy and J. Koziorowka (1985) Protective action of ascorbic acid against mutagenicity of aminopyrine plus nitrite, Pol. J. Pharmacol. Pharm., 37, 601-607. Raina, V., and H.L. Gurtoo (1985) Effects of vitamins A, C and E on aflatoxin Bi-induced mutagenesis in Salmonella typhimurium TA98 and TA100, Teratogen. Carcinogen. Mutagen., 5, 29-40. Saruno, R., M. Yoshida, F. Kato and A. Murata (1979) Screening of desmutagenic substances on mutagen NmethyI-N'-nitro-N-nitrosoguanidine, Hakkoogaku, 57, 8691. Shamberger, R.J., F.F. Banghman, S,L. Kalchert, C.E. Willis and G.C. Hoffman (1973)Carcinogen induced chromosomal breakage decreased by antioxidanls, Proc. Natl. Acad. Sci. (U.S.A.), 70, 1461-1463. Shamberger, R.J., C.L. Corlett, K.D. Beamam and B.L. Kasten (1979) Antioxidants reduce the mutagenic effect of malonaldehyde and g-pirolactone, Part IX, Antioxidants and cancer, Mutation Res., 66, 349-355. Vojtekova, H., and S. Miertus (1986) Influence of ascorbic acid on the mutagenicity of N-methyI-N=nitrosoguanidine and nitrofurans studied by SOS chromotest, Neoplasma, 33, 691-698. Weitherg, A,B., and S,A. Weitzman (1985) The effect of vitamin C on oxTgen radical-induced sister chromatid exchanges, Mutation Res,, 144, 23-26. Wirth, P,J., and S.S. Thorgeirsson (1980) Mechanism of N-hydroxv.2-acetyl-aminofluorene mutagenicity in the Salmonella test system, Mol. Pharmacol., 19, 337-344.
