Mutagen formation on nitrite treatment of indole compounds derived from indole-glucosinolate

Mutation Research, 250 (1991) 169-174

169

© 1991 Elsevier Science Publishers B.V. All rights reserved 002%5107/91/$03.50 ADONIS 0027510791001752 MUT 02516

Mutagen formation on nitrite treatment of indole compounds derived from indole-glucosinolate Chiaki Sasagawa and Taijiro Matsushima Department of Molecular Oncology, Institute of Medical Science, Unicersity of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 (Japan) (Accepted 5 April 1991)

Keyword~: Indole compounds; Indole-glucosinolate; Nitrite treatment

Summary The mutagenicities of 8 indole compounds (indole-3-acetonitrile, indole-3-carbinol, indole-3-acetamide, indole-3-acetic acid, 3-methylindole, indole-3-aldehyde, indole-3-carboxylic acid and indole) derived from indole glucosinolate were studied by mutation tests on Salmonella typhimurium TA98 and TA100 and Escherichia coli WP2 uvrA/pKM101 with and without $9 mix. None of the 8 indole compounds were mutagenic, but they became mutagenic on these 3 tester strains when treated with nitrite at pH 3. The nitrite-treated indole compounds were mutagenic without metabolic activation system ($9 mix), and their mutagenicities were decreased by the addition of $9 mix.

Direct-acting mutagens formed from nitrosable precursors in foods by reaction with nitrite at acidic pH in the stomach may be stomach carcinogens. Epidemiological studies have shown a correlation between nitrite/nitrate ingestion and the incidence of gastric cancer (Mirvish, 1983; Correa, 1988). Moreover, several indole derivatives have been isolated as nitrosable precursors in foods. Yang et al. (1984) reported the isolation of 4-chloro-6-methoxyindole from fava beans, which are consumed in a high-risk area of gastric cancer in Colombia (Correa et al., 1983). Wakabayasi et al. (1985, 1986) reported the presence of indole-3-acetonitrile, 4-methoxyindole-3-acetonitrile and 4-methoxyindole-3-aldehyde in Chi-

Correspondence: Ms. C. Sasagawa, Department of Molecular Oncology, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108 (Japan).

nese cabbage as nitrosable mutagen precursors. Many indole compounds become mutagenic on nitrite treatment at acidic pH (Valin et al., 1985; Ochiai et al., 1986). Several indole compounds are formed in plants from indole glucosinolate by the action of myrosinase or acidic and alkaline pH, as shown in Fig. 1 (McDanell et al., 1988). Indole glucosinolate is present in large amounts in cruciferous plants, such as cabbage, Brussels sprouts, broccoli and radish, and is a storage precursor form of auxins (indole-3-acetic acid and indole-3-acetonitrile), which are plant growth factors. These growth factors are present at high levels in young and growing plant tissues and they are also produced after tissue damage and during storage or cooking processes (Fenwick et al., 1983). Although indole glucosinolates and their hydrolysis products induce mixed-function oxygenases and modulate chemical carcinogenesis

170

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(Wattenberg, 1979), some nitrosated indole compounds have mutagenic and carcinogenic potentials. Therefore, we studied the formation of direct mutagens by nitrite treatment of indole compounds derived from indole glucosinolate. Salmonella typhimurium TA98 and TA100, and Escherichia coli WP2 uvrA/pKM101 were used in mutagenicity tests. Materials and methods

Chemicals Indole-3-carbinol hydrate, indole-3-acetamide, indole-3-acetic acid, indole-3-aldehyde sodium bisulfate addition compound and indole were obtained from Aldrich Chemical Co. (Milwaukee, WI, U.S.A.). Indole-3-acetonitrile and indole-3carboxylic acid were from Tokyo Kasei Kogyo Co. (Tokyo, Japan) and 3-methyl-indole was from Wako Pure Chemical Industry (Tokyo, Japan).

Sodium nitrite was obtained from Wako ammonium sulfamatc from Koso Chemical Co. (Tokyo, Japan) and dimethyl sulfoxide (DMSO), HPLC grade, from Aldrich.

Nitrite treatment lndole compounds were dissolved at 10 mM concentration in citrate-phosphate buffer (100 mM, pH 3) with ultra-sonification. Volumes of 2 ml of the solutions were mixed with 50/zl of 4 M sodium nitrite, adjusted to pH 3 with a small amount of HCI and incubated for 1 h at 37 ° C in the dark. The reaction with nitrite was stopped by adding 50 /.tl of 4 M ammonium suifamate and the insoluble products were solubilized by adding DMSO. Then the mixtures were sterilized by passage through an FH filter (0.45/xm, Milliporc Corp., Bedford, MA, U.S.A.) and used for mutation tests.

171 in the test room to avoid photo-decomposition of the nitrite-treated products. $9 was prepared from the liver of male Sprague-Dawley rats pretreated with sodium phenobarbital and 5,6-benzoflavone (Matsushima et al., 1976). $9 mix contained 4 mM concentration of NADPH and NADH, and other additions.

Mutation test Mutation tests were carried out by the preincubation method (30 ° C, 30 min) with and without $9 mix using S. typhimurium TA98, TA100 and E. coli WP2 uvrA/pKM101. Oxoid nutrient broth was used for pre-culture of tester strains, and Oxoid No. 1 agar and Difco Bacto agar were used in the 30 ml of minimal glucose agar and 2 ml of top agar, respectively. Top agar layers containing 0.5 mM concentrations of both t.-histidine hydrochloride and biotin, and 0.5 mM Ltryptophan were used for tests on S. typhimurium and E. coli, respectively. Yellow lamps were used

Results

None of the untreated test compounds showed any mutagenicity on S. typhimurium TA98 or TA100 and E. coli WP2 uvrA/pKM101 with and

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without $9 mix. But after treatment with nitrite, they showed mutagenicity on these tester strains without $9 mix (Fig. 2). The specific mutagenic activities (number of revertant colonies//x mole of precursor) of these nitrite-treated compounds are shown in Table 1. After nitrite treatment, indole-3-acetic acid and indole-3-acetonitrile showed strong mutagenicity on all 3 strains, but indole-3-carboxylic acid, indole-3-carbinol, indole and indole-3-aldehyde had preferential mutagenic effects on TA98. Of these 4 compounds, indole-3-carboxylic acid had the strongest mutagenic effect on TA98.

3-Methylindole showed stronger mutagenicity on TA100 than on the other 2 strains and indole-3acetamidc showed stronger mutagenicity on E. coli WP2 u v r A / p K M 1 0 1 than on the other strains. The mutagenicity of all these nitrite-treated compounds except indole-3-acetamide on all 3 strains decreased on addition of a metabolic activation system ($9 mix). Some weak direct mutagens became non-mutagenic in the presence of $9 mix. However, indole-3-acetamide showed higher mutagenicity on E. coil WP2 u v r A / p K M 1 0 1 with $9 mix, equal mutagenicity on TA98 with and

173 TABLE 1 SPECIFIC M U T A G E N I C I T I E S O F N I T R I T E - T R E A T E D I N D O L E C O M P O U N D S ON S. typhimurium TA98 A N D TAI00, A N D E. coli WP2 u v r A / p K M 1 0 1 W I T H A N D W I T H O U T $9 MIX Number

1 2 3 4 5 6 7 8

Nitrite-treated indole c o m ~ m n d

I ndole-3-acetonitrile lndole-3-carbinol Indole-3-acetamide Indole-3-acetic acid 3-Methylindolc I ndole-3-aldehyde Indole-3-carboxylic acid indolc

Specific mutagenicity ( r e v . / ~ m o l e precursor) - $9 mix

+ $9 mix

TA98

TAI(X)

6500 8 360 140 11360 1 190 5 490 11 200 7680

467{I 3 800 920 9240 5 290 810 1 040 1 7{•)

without $9 mix, and lower mutagenicity on TAI(X) with $9 mix. Discussion

Indole glucosinolate is widely distributed in cruciferous plants and its level is especially high in brassica vegetables such as Brussel sprouts, cabbage, Chinese cabbage, broccoli and cauliflower. After damage of plant tissues, activated myrosinase or acidic and alkaline pH shift releases indole derivatives from indole glucosinolate (McDanell et al., 1988). Tiedink et al. (1988) reported that the amounts of N-nitroso compounds formed after nitrite treatment correlated well with the levels of glucosinolate in cruciferous vegetables. In this work we tested the mutagenicity of 8 indole compounds derived from indole glucosinolate. None of these indole compounds themselves were mutagenic with and without metabolic activation, but they became mutagenic without metabolic activation after nitrite treatment at acidic pH. Addition of a metabolic activation system ($9 mix) decreased the mutagenicities of these nitrite-treated indole compounds. Nitrite-treated indole-3-acetic acid showed the highest mutagenic activity, having similar activities on all 3 strains tested. Nitrite-treated indole3-acetonitrile had a slightly lower mutagenicity, but also had similar effects on all 3 strains. Wakabayashi et al. (1985) reported that a l-nitroso

WP2 u v r A / pKMI()I

TA98

TAIO0

5600 3 200 2 6110 12960 7811 13(I 1 250 3220

730 520 140 4560 340 1 680 2 310 1 300

630 (1 370 2000 770 0 (1 751)

WP2 u v r A / pKM101 1020 300 4 040 9800 31X) 0 1711 560

derivative was formed on nitrite treatment of indole-3-acetonitrile. The same type of 1-nitroso derivative may be produced by nitrite treatment of indole-3-acetic acid. After nitrite treatment, indole-3-carboxylic acid, indole-3-aldehyde, indole and 3-indolemethanol showed higher mutagenicities on S. typhimurium TA98 than on TA100 or E. coli WP2 uvrA/pKM101 possibly due to the formation of frameshift-type mutagens. Nitrite-treated indole-3-acetamide showed higher mutagenicity on E. coli WP2 uvrA/pKM101 than on the other 2 strains, possibly due to formation of an AT-oriented base-pair change-type mutagen, differing from the mutagens in the other nitrite-treated products. Thus the mutagens formed from nitrosable precursors showed different mutagenic actions depending on differences in structure of their substituted groups at the 3 position. 1-Nitrosoindole-3-acetonitrile causes DNA damage (DNA single-strand scissions) in cells of the stomach mucosa of rats when given orally (Furihata et al., 1989) and also induces cell proliferation (replicative DNA synthesis and ornithine decarboxylase activity) in the stomach mucosa (Furihata et al., 1987). All these mutagenic nitrosated indole compounds may have the same activity on stomach mucosa as 1-nitrosoindole-3acetonitrile and are highly likely to be stomach carcinogens. The incidence of stomach cancer is high in Japan, where people consume larger amounts of cruciferous vegetables than in the

174

U.S.A., Canada or the U.K. Japanese cat large amounts of radish, Chinese cabbage and salted, fermcnted vegetables. The per capita consumption of some cruciferous vcgctables in Japan is about 790 g / w c c k , whereas in thc U.S.A., Canada and U.K. it is about 13(L 120 and 320 g / w c c k , respectively (Fenwick ct al., 1983). The actual production of mutagenic nitrosated indolc compounds from foods in the stomach must be studicd to evaluate the risk of stomach cancer from indolc glucosinolate in vegetables. Acknowledgement The authors are indebted to B.N. Ames, University of California, for generously supplying the Salmonella tester strains. References Correa, P. (1988) A human model of gastric carcinogenesis, Cancer Res., 48, 3554-3560. Correa, P., C. Cuello, L.F. Fajardo, W. Haenszel. O. Bolafios and B. de Ramirez (1983) Diet and gastric cancer: nutrition survey in a high-risk area, J. Natl. Cancer Inst., 7(I, 673-678. Fenwick, G.R., R.K. lleaney and W.J. Mullin (1.;83) Glucosinolates and their breakdown products in f ~ d and fi~d plants, C R C Crit. Rev. F1.~~d Sci. Nutr., 18, 123-201. Furihata, ('., Y. Sato, A. Yamakoshi, M. Takimoto and T. Matsushima (1987) Inductions of ornithine decarboxylase and D N A synthesis in rat stomach mucosa by 1-nitroso-indole-3-acetonitrile, Jpn. J. Cancer Res. (GannL 78. 432435. Furihata. C., M. Obara and T. Matsushima (1989) In viw~ short-term assay for identification of organ-specific carcinogens and tumor-promoters, J. U O E H , 11,641-652. Matsushima, T., M. Sawamura. K. Hara, and T. Sugimura 11.;76) A safe substitute fi~r polychlorinated biphenyls as

an inducer t+l metabolic activation system, in: FJ. de Serres. J.R. Fouts, J.R Bend and R.M. Philpot (Eds.,L I11 Vitro Metabolic Activation in Mutagenesis Testing. Flsevier, Amsterdam, pp. g5-88. McDanell. R, A.E.M. McLean, A.B. tlanley, R . K Hcane,, and (3.R Fenwick (l*;g8)Chemical and biological properties of indole glucosinolates (glucobrassieins): a review. Ft'~3d ('hem. Toxicol., 26.5*;-70. Mirvish. S.S.. (1.;83) The etiology of gastric cancer, lntragastric nitrosamide t~+rmation and other theories. J. Natl. ('ancer Inst., 71,629-647. Ochiai. M., K. Wakabayashi, T. Sugimura and M. Nagao {l*;g6) Mutagenicities of indole and 30 derivatives after nitrite treatment. Mutation Res., 172, 18.;-]*;7. l"icdink, I-t.(.+.M., J.A.R. Davies. S.W. van Broekhoven. tl..1. van der Kamp and W.M.F. Jongen (l*;8g) Formation ol mutagenic N-nitroso compounds in vegetable extracts upon nitrite treatment: a comparison with the glucosinolatc content. Fuod Chem. Toxicol.. 2b. 947-*;54. Valin. N.. D. Haybron, L. (;roves and I I.F. Mower ( 1985 ) The nitrosation of alcohol-induced metabolites produces mutagenicsul~stances, Mutation Res.. 15g, 15.; 168. Wakabayashi. K., M. Nagao. M. ()chiai, "]'. Tahira. Z. Yanlaiznnli and T. Sugimura (1';85) A mutagcn precursor in Chinese cabbage, indole-3-acetonitrile, which becomes mutagenic on nitrite treatment, Mutation Res., 143. 17 21. Wakabayashi. K.. M. Nagao, T. Tahira. Z Yamaizurni, M. Katayama. S. Marumo and T. Sugimura (1.;86) 4Methoxyindole derivatives as nitrosable precursors of 1111.1tagens in Chinese cabbage. Mutagenesis, 1. 423--42b. Wattenberg, I+.W. (197.;) Naturally occurring inhibitors of chemical carcinogenesis, in: E.C. Millet', J.A. Miller, 1. t lirono. T. Sugimura and S. Takayama (Eds.), Naturally ()courting Careinogens-Mutagens and Modulators of Carcinogenesis, Japan Scientific Societies Press. "Fokyo/University Park Press. Baltimore. MD. pp. 315-32.;. Yang. D., S.R Tannenbaum. (;. Bfiehi and (L('.M. Lee (l*;g4) 4-('hloro-6-methoxyindole b, the precursor of a potent mutagen (4-ch]oro-6-met hox2,-2-hydroxy- I -nit roso-indolin3-one oxime) that forms during nitrosation of the fava bean ( l'icm luha ), Carcinogenesis. 5, 1219- 1224.