Environmental and Molecular Mutagenesis 17:258-263 (1991)

Evaluation of the SOS Chromotest for the Detection of Antimutagens Takahiko Sato, Kazuhiko Chikazawa, Hidetomo Yamamori, Youki Ose, Hisamitsu Nagase, and Hideaki Kit0 Department of Public Health, Gifu Pharmaceutical University, Gifu, Japan The SOS chromotest was applied for the deteciion of antimutagens. To raise SOS induction, the bacteria were treated with the mutagens, UV, 4-nitroquinoline N-oxide (4NQO), Nmethyl-N’-nitro-N-nitroso-guanidine (MNNG), or benzo[a]pyrene (B[a]p).The inhibitory effects of t-ascorbic acid, glutathione,vanillin, 5-fluoro-

uracil (5-FU), 5-chlorouracil (5-CU), cobaltous chloride, sodium selenite and sodium arsenite, which are known as antimutagens, were investigated with their addition either simultaneously or post treatment time. It became clear that the SOS chromotest was very useful for the detection of antimutagens.

Key words: ascorbic acid, glutathiane, vanillin, 5-fluorouracil, 5-chlorouracil

INTRODUCTION

MATERIALS

To investigate the antimutagenic effect of the sample, the frequency of mutation was increased by ultraviolet (UV) irradiation or treatment with mutagens, and the sample was added to this system and the effect on mutation frequency was measured. For the investigation of mutagenicity, various mutagen tests such as Ames test etc. have been used. Recently, the SOS chromotest and the umu test were developed for the investigation of mutagenicity [Oda et al., 1985; Quillardet and Hofnung, 19851 and it has become clear that most SOS inducing agents are mutagenic [Quillardet et al., 1985; Nakamura et al., 19871. The principles of both tests are the same, except that SOS chromotest uses Escherichia coli and umu test uses Salmonella typhimurium. In this report, the inhibitory effects of various antimutagens on SOS induction were investigated with the SOS chromotest and the possibility of application of the SOS chromotest for the study of antimutagens was researched. Kada et al. [ 19851 classified antimutagens into two kinds, desmutagens and bioantimutagens. Desmutagens cause chemical or biochemical modifications of mutagens outside cells, while bioantimutagens interfere with cellular functions which produce genetically stable informative genes from primary damage to DNA. The authors investigated SOS inhibition with the addition either simultaneously or post treatment time, because, although desmutagens act at simultaneous treatment, they may be actually bioantimutagen-like substances (such as inhibitor of the process from DNA damage to P-galactosidase translation initiated by SOS induction) which would be active with addition at post treatment time.

Mutagens

0 1991 Wiley-Liss, Inc.

4-nitroquinoline N-oxide (4NQO) and N-methy1-N’nitro-N-nitroso-guanidine (MNNG) were purchased from Nakarai Tesque Co. and B[a]P from Wako Chemical Co. For UV irradiation, a sterilization lamp GL-10, 10 watt (Toshiba or National Corp.) was used. Antimutagens

L-ascorbic acid, 5-fluorouracil (5-FU), and sodium selenite (Na,SeO,) were purchased from Nakarai Tesque Co. Cobaltous chloride (CoCl,), vanillin, and sodium arsenite (NaAsO,) were purchased from Kisida Chemical Co. 5Chlorouracil (5-CU) and glutathione (reduced type) were obtained from Tokyo Kasai and Wako Chemical Co., respectively.

METH0DS The modification effect on SOS induction was tested by introduction of antimutagens into the SOS chromotest by Quillardet and Hofnung [ 19851. The methods are as follows. An aliquot of the original culture of Escherichiu coli PQ Received March 14, 1990: revised and accepted January 9, 1991 Address reprint requests to Takahiko Sato, Department of Public Health, Gifu Pharmaceutical University, 5-6- 1, Mitahora-higashi, Gifu. 502 Japan. Youki Ose’s Present address is Gifu City Women’s College, 2693, Nagara-Hukumitsu, Gifu SO2 Japan.

Evaluation of SOS Chromotest

37, which was stored in -8O"C, was inoculated to 5 ml of La-medium and cultured at 37°C for 15 hr. 0.1 ml of the culture was diluted to 5 ml by La-medium and incubated at 37°C for 2 hr by shaking. 0.5-5 ml of the culture (the volume was decided by the appearance of bacteria concentration) was diluted to 10 ml by L-medium. In the case of UV, 1.5 ml of the diluted culture was poured into a Petri plate (3 cm in diameter) and was irradiated with UV from a 22 cm distance for 2 min. Antimutagen was added simultaneously at UV irradiation for simultaneous treatment, and antimutagen was added after UV irradiation for post treatment. The culture was transferred to a test tube and incubated at 37°C for 2 hr by shaking. Then 0.3 ml aliquots of the culture were put into four test tubes. Two test tubes were used for the assay of P-galactosidase and the others were used for the assay of alkaline phosphatase. In the case of the chemical mutagens, 1.5 ml of diluted culture was transferred to a test tube and 150 ~1 of mutagen was added. For simultaneous treatment, the antimutagen was added simutaneousl y and the culture was incubated at 37°C for 2 hr by shaking. After centrifugation (ca. 2,OOOg, 20 min, 4°C) was performed to remove the mutagen, it was resuspended to La-medium and the enzyme assays were performed. For post treatment, 150 ~1 of mutagen was added to 1.5 nil of diluted culture and it was incubated at 37°C for 20 min by shaking. After centrifugation and resuspension, the antimutagen was added and the culture was reincubated at 37°C for 2 hr by shaking, and the enzyme assays were performed. o-Nitrophenyl-P-D-galactopyranoside(ONPG) was used for the measurement of P-galactosidase activity and alkaline phosphatase was measured with p-nitrophenyl phosphate disodium [Quillardet and Hofnung, 19851. After enzyme units of P-galactosidase and alkaline phosphatase were calculated [Quillardet and Hofnung, 19851, the ratio of P-galactosidase unit to alkaline phosphatase unit (R) and factor of modification on SOS induction [modification factor, F(c)] were calculated as follows

where R(c) is R at concentration of antimutagen c and R(0) is R at concentration of antimutagen 0. Quillardet and Hofnung [198S] used the term induction factor I(c), which is R(c) divided by R(0). Both terms seem the same, but their R(c) is a ratio at concentration of mutagen c, not antimutagen. To avoid confusion, we introduce the new term F(c).

RESULTS A N D DISCUSSION

L-Ascorbic Acid There are many reports that ascorbic acid showed antimutagenicity activity. Shenberger [ 19841 has reviewed genetic

259

toxicology and the antimutagenic effect of ascorbic acid. By his review, ascorbic acid had an antimutagenic effect on many kinds of mutagens such as hexavalent chromium, ferrous ions, several nitroso compounds, radioactive irradiation, malonaldehyde, soy bean sauce, etc. with bacterial tests. Also, ascorbic acid had antimutagenic as well as anticarcinogenic effects on mammalian cells. L-ascorbic acid showed SOS inhibition with simultaneous UV or 4NQ0 treatment. The enzyme units and modification factors with UV treatment are shown in Figure 1. If alkaline phosphatase activity decreases due to the killing effect, the modification factor F(c) increases, even if P-galactosidase activity is constant, and it is mistaken to be the stimulation of SOS induction. It is important to interpret the data from both enzyme units and modification factor. However, SOS inhibition was not observed with post treatment of ascorbic acid after UV or 4NQ0 treatment. Also, by simultaneous treatment for MNNG and benzo [alpyrene (B[a]p), ascorbic acid had no inhibitory effect.

Glutathione There are many articles which have reported the antimutagenicity of glutathione. Rosin and Stich [ 19791 reported that the mutagenicity of MNNG was inhibited by glutathione. Also, glutathione decreased the mutagenicity of 2-naphthylamine [Bock-Henning et al., 19821, N-acetoxyacetylaminofluorene [Rosin and Stich, 19781, and mutagens produced by chlorination of water [Cheh et al., 19801. The SOS inhibition of glutathione was observed by simultaneous treatments with 4 N Q 0 and MNNG. The enzyme units and modification factors at MNNG treatment are shown in Figure 2. SOS inhibition was not observed with either simultaneous or post treatments with UV irradiation. Post treatment with 4NQ0 or simultaneous treatment with B[a]p did not show the effect.

Van illin Watanabe et al. [1988] reported that vanillin had the antimutagenicity for 4NQ0 in Escherichia coli WP2s uvrA trpE. The enzyme units and modification factors with 4NQ0 treatment are shown in Figure 3. It was clear that the P-galactosidase unit increased from above SO pg of vanillin in spite of the decrease of the alkaline phosphatase unit. It was determined beforehand that vanillin itself did not cause SOS induction and these results suggested that vanillin increased the mutagenicity of 4NQO. The same phenomena were observed with simultaneous treatment with UV and MNNG as well as post treatment with UV. Sasaki et al. [1987] reported that vanillin decreased the chromosome aberration by post treatment, but increased the frequency of sister-chromatid exchange with mitomycin C-treated cells.

Sato et al.

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Fig. 2. The modification of SOS induction with glutathione on MNNG treatment simultaneously. 7.5 pg of MNNG was added. 0,enzyme unit of P-galactosidase; 0 , enzyme unit of alkaline phosphatase; A modification factor.

5-FU and 5-CU 5-FU decreased SOS induction in all experiments, such as s'mdtaneous and Post treatments with uv and 4NQO7 and simultaneous treatment with MNNG. The enzyme units and modification factors with UV treatment are shown in Figure 4. Ohta et al. [1986] also rePorted that 5-FU inhibited UV-induced SOS induction. 'For 5-CU, SOS inhibition has not been reported. But its

chemical structure is similar to that of 5-FU and the SOS inhibition test was performed. 5-CU showed SOS inhibition only with simultaneous treatment with u v . F~~MNNG, a slight decrease in mutagenicity was observed at 2,500 pg,

coc12,

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Kada et al. have reported that CoCI, inhibited many mutagens such as M " G [Kada and Kanematsu, 19781,

Evaluation of SOS Chromotest

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y-rays [Kada et al., 19791, and Trp-p-1 [Mochizuki and Kada. 19821, and they considered that cobalt corrected the error-proneness of DNA replicating enzyme by improving its fidelity in DNA synthesis [Inoue et al., 19811. There are reports concerning the antimutagenicity of selenium with various mutagens, such as 7,12-dimethylbenz [alanthracene [Adams et a]., 19801, sodium nitrate [Adams et al., 19801, 2-acetylaminofluorene [Jacobs et al., 19771, acridine orange [Martin et al., 19811, and methyl methane-

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sulfonate [Ray et al., 19781. Also, it was reported that the addition of selenium to the diet decreased the mutagenicity of 7,12-dimethylbenz[a]anthracene [Schillaci et al., 19821 and 1, l-dimethylhydrazine [Beije et al., 19841. Nunoshiba and Nishioka [I9871 reported that sodium arsenite had an antimutagenic effect on UV, 4NQ0, 2(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), and methylmethanesulfonate (MMS), as well as spontaneous mutation with E. coli WP2 uvrNpkM101.

262

Sat0 et

al.

TABLE I. Summarv of the Modification of SOS Induction

uv

4NQ0

Simultaneous

Post

Simultaneous

Post

MNNG

B[ a ]

L-ascorbic acid Glutathione Vanillin

0 X

X 0 Xa 0 O? X X X

X X

0 0 X X X

0 0 Xa 0 X X X X

X X

5-FU

X X Xa 0 X X X X

5-cu

coc12 NazSe03 NaAsOz

Xa

0

Abbreviations: 0. decrease; X, no change; Xa, increase; O?, decrease, but uncertain.

We investigated the inhibitory effects of CoCI,, Na,SeO,, and NaAsO, on SOS induction by simultaneous and post treatments with UV and simultaneous treatment with 4NQ0 and MNNG. But, the inhibition was not observed.

CONCLUSIONS All results are summarized in Table I. Various antimutagens showed the inhibitory effect on SOS induction and the SOS chromotest may be very useful for screening many antimutagens, because the test is simple and strict disinfection is not necessary. But, some results of SOS chromotest were different with those of other mutagen tests such as the Ames test. The substances which do not have SOS inhibition may not act as directly as desmutagens (at simultaneous treatment) or on SOS repair function (at post treatment). But, there are few reports which clarified the mechanism of antimutagens, and these data of the modification of SOS induction may be useful for researching the mechanisms of antimutagens.

ACKNOWLEDGMENTS The authors are grateful to Prof. J. Kitamura, Department of Microbiology, Gifu Pharmaceutical University, for supplying Escherichia coli PQ37.

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Shenberger RJ (1984): Genetic toxicology of ascorbic acid. Mutat Res 133 135- 159. Watanabe K. Ohta T, Shirasu Y (1988): Antimutagenic effects of benzaldehyde and its derivatives on mutagenesis induced by 4-nitroquinoline I-oxide in Escherichia coli. Agric Biol Chein 52: 1041-1 045.

Accepted by-

E.R. Nestman