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. 1995 Nov;61(11):3781–3787. doi: 10.1128/aem.61.11.3781-3787.1995

Bioactivation of cysteine conjugates of 1-nitropyrene oxides by cysteine conjugate beta-lyase purified from Peptostreptococcus magnus.

K Kataoka 1, T Kinouchi 1, S Akimoto 1, Y Ohnishi 1
PMCID: PMC167679  PMID: 8526486

Abstract

To determine the role of cysteine conjugate beta-lyase (beta-lyase) in the metabolism of mutagenic nitropolycyclic aromatic hydrocarbons, we determined the effect of beta-lyase on the mutagenicities and DNA binding of cysteine conjugates of 4,5-epoxy-4,5-dihydro-1-nitropyrene (1-NP 4,5-oxide) and 9,10-epoxy-9,10-dihydro-1-nitropyrene (1-NP 9,10-oxide), which are detoxified metabolites of the mutagenic compound 1-nitropyrene. We purified beta-lyase from Peptostreptococcus magnus GAI0663, since P. magnus is one of the constituents of the intestinal microflora and exhibits high levels of degrading activity with cysteine conjugates of 1-nitropyrene oxides (1-NP oxide-Cys). The activity of purified beta-lyase was optimal at pH 7.5 to 8.0, was completely inhibited by aminooxyacetic acid and hydroxylamine, and was eliminated by heating the enzyme at 55 degrees C for 5 min. The molecular weight of beta-lyase was 150,000, as determined by fast protein liquid chromatography. S-Arylcysteine conjugates were good substrates for this enzyme. As determined by the Salmonella mutagenicity test, 5 ng of beta-lyase protein increased the mutagenicity of the cysteine conjugate of 1-NP 9,10-oxide (10 nmol per plate) 4.5-fold in Salmonella typhimurium TA98 and 4.1-fold in strain TA100. However, beta-lyase had little effect on the cysteine conjugate of 1-NP 4,5-oxide (10 nmol per plate). Both conjugates exhibited only low levels of mutagenicity with nitroreductase-deficient strain TA98NR. In vitro binding of 1-NP oxide-Cys to calf thymus DNA was increased by adding purified beta-lyase or xanthine oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)

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Selected References

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  1. Buckberry L. D., Blagbrough I. S., Bycroft B. W., Shaw P. N. Kynurenine aminotransferase activity in human liver: identity with human hepatic C-S lyase activity and a physiological role for this enzyme. Toxicol Lett. 1992 May;60(3):241–246. doi: 10.1016/0378-4274(92)90281-n. [DOI] [PubMed] [Google Scholar]
  2. Commandeur J. N., Boogaard P. J., Mulder G. J., Vermeulen N. P. Mutagenicity and cytotoxicity of two regioisomeric mercapturic acids and cysteine S-conjugates of trichloroethylene. Arch Toxicol. 1991;65(5):373–380. doi: 10.1007/BF02284259. [DOI] [PubMed] [Google Scholar]
  3. Dekant W., Vamvakas S., Anders M. W. Bioactivation of nephrotoxic haloalkenes by glutathione conjugation: formation of toxic and mutagenic intermediates by cysteine conjugate beta-lyase. Drug Metab Rev. 1989;20(1):43–83. doi: 10.3109/03602538908994144. [DOI] [PubMed] [Google Scholar]
  4. Dekant W., Vamvakas S., Berthold K., Schmidt S., Wild D., Henschler D. Bacterial beta-lyase mediated cleavage and mutagenicity of cysteine conjugates derived from the nephrocarcinogenic alkenes trichloroethylene, tetrachloroethylene and hexachlorobutadiene. Chem Biol Interact. 1986 Oct 15;60(1):31–45. doi: 10.1016/0009-2797(86)90015-3. [DOI] [PubMed] [Google Scholar]
  5. Djurić Z., Coles B., Fifer E. K., Ketterer B., Beland F. A. In vivo and in vitro formation of glutathione conjugates from the K-region epoxides of 1-nitropyrene. Carcinogenesis. 1987 Dec;8(12):1781–1786. doi: 10.1093/carcin/8.12.1781. [DOI] [PubMed] [Google Scholar]
  6. El-Bayoumy K., Hecht S. S., Sackl T., Stoner G. D. Tumorigenicity and metabolism of 1-nitropyrene in A/J mice. Carcinogenesis. 1984 Nov;5(11):1449–1452. doi: 10.1093/carcin/5.11.1449. [DOI] [PubMed] [Google Scholar]
  7. Elmhirst T. R., Chipman J. K., Ribeiro O., Hirom P. C., Millburn P. Metabolism and enterohepatic circulation of benzo(a)pyrene-4,5-epoxide in the rat. Xenobiotica. 1985 Nov;15(11):899–906. doi: 10.3109/00498258509045043. [DOI] [PubMed] [Google Scholar]
  8. Finegold S. M., Attebery H. R., Sutter V. L. Effect of diet on human fecal flora: comparison of Japanese and American diets. Am J Clin Nutr. 1974 Dec;27(12):1456–1469. doi: 10.1093/ajcn/27.12.1456. [DOI] [PubMed] [Google Scholar]
  9. Fricke U. Tritosol: a new scintillation cocktail based on Triton X-100. Anal Biochem. 1975 Feb;63(2):555–558. doi: 10.1016/0003-2697(75)90379-6. [DOI] [PubMed] [Google Scholar]
  10. Goldman P. Biochemical pharmacology of the intestinal flora. Annu Rev Pharmacol Toxicol. 1978;18:523–539. doi: 10.1146/annurev.pa.18.040178.002515. [DOI] [PubMed] [Google Scholar]
  11. Hirose M., Lee M. S., Wang C. Y., King C. M. Induction of rat mammary gland tumors by 1-nitropyrene, a recently recognized environmental mutagen. Cancer Res. 1984 Mar;44(3):1158–1162. [PubMed] [Google Scholar]
  12. Howard P. C., Beland F. A. Xanthine oxidase catalyzed binding of 1-nitropyrene to DNA. Biochem Biophys Res Commun. 1982 Jan 29;104(2):727–732. doi: 10.1016/0006-291x(82)90697-0. [DOI] [PubMed] [Google Scholar]
  13. Howard P. C., Heflich R. H., Evans F. E., Beland F. A. Formation of DNA adducts in vitro and in Salmonella typhimurium upon metabolic reduction of the environmental mutagen 1-nitropyrene. Cancer Res. 1983 May;43(5):2052–2058. [PubMed] [Google Scholar]
  14. Kataoka K., Kinouchi T., Ohnishi Y. Species differences in metabolic activation and inactivation of 1-nitropyrene in the liver. Cancer Res. 1991 Aug 1;51(15):3919–3924. [PubMed] [Google Scholar]
  15. Kinouchi T., Kataoka K., Miyanishi K., Akimoto S., Ohnishi Y. Biological activities of the intestinal microflora in mice treated with antibiotics or untreated and the effects of the microflora on absorption and metabolic activation of orally administered glutathione conjugates of K-region epoxides of 1-nitropyrene. Carcinogenesis. 1993 May;14(5):869–874. doi: 10.1093/carcin/14.5.869. [DOI] [PubMed] [Google Scholar]
  16. Kinouchi T., Morotomi M., Mutai M., Fifer E. K., Beland F. A., Ohnishi Y. Metabolism of 1-nitropyrene in germ-free and conventional rats. Jpn J Cancer Res. 1986 Apr;77(4):356–369. [PubMed] [Google Scholar]
  17. Kinouchi T., Nishifuji K., Ohnishi Y. Biliary excretion of glutathione conjugates of 4,5-epoxy-4,5-dihydro-1- nitropyrene and 9,10-epoxy-9,10-dihydro-1-nitropyrene in rats administered 1-nitropyrene orally and their further metabolism in the intestinal tract. Carcinogenesis. 1990 Aug;11(8):1381–1387. doi: 10.1093/carcin/11.8.1381. [DOI] [PubMed] [Google Scholar]
  18. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  19. Larsen G. L., Larson J. D., Gustafsson J. A. Cysteine conjugate beta-lyase in the gastrointestinal bacterium Fusobacterium necrophorum. Xenobiotica. 1983 Nov;13(11):689–700. doi: 10.3109/00498258309052230. [DOI] [PubMed] [Google Scholar]
  20. Larsen G. L., Stevens J. L. Cysteine conjugate beta-lyase in the gastrointestinal bacterium Eubacterium limosum. Mol Pharmacol. 1986 Jan;29(1):97–103. [PubMed] [Google Scholar]
  21. MacFarlane M., Foster J. R., Gibson G. G., King L. J., Lock E. A. Cysteine conjugate beta-lyase of rat kidney cytosol: characterization, immunocytochemical localization, and correlation with hexachlorobutadiene nephrotoxicity. Toxicol Appl Pharmacol. 1989 Apr;98(2):185–197. doi: 10.1016/0041-008x(89)90224-x. [DOI] [PubMed] [Google Scholar]
  22. Maron D. M., Ames B. N. Revised methods for the Salmonella mutagenicity test. Mutat Res. 1983 May;113(3-4):173–215. doi: 10.1016/0165-1161(83)90010-9. [DOI] [PubMed] [Google Scholar]
  23. McCoy E. C., Rosenkranz H. S., Mermelstein R. Evidence for the existence of a family of bacterial nitroreductases capable of activating nitrated polycyclics to mutagens. Environ Mutagen. 1981;3(4):421–427. doi: 10.1002/em.2860030403. [DOI] [PubMed] [Google Scholar]
  24. Monks T. J., Anders M. W., Dekant W., Stevens J. L., Lau S. S., van Bladeren P. J. Glutathione conjugate mediated toxicities. Toxicol Appl Pharmacol. 1990 Oct;106(1):1–19. doi: 10.1016/0041-008x(90)90100-9. [DOI] [PubMed] [Google Scholar]
  25. Rosenkranz H. S., Mermelstein R. Mutagenicity and genotoxicity of nitroarenes. All nitro-containing chemicals were not created equal. Mutat Res. 1983 Apr;114(3):217–267. doi: 10.1016/0165-1110(83)90034-9. [DOI] [PubMed] [Google Scholar]
  26. Rowland I. R., Mallett A. K., Wise A. The effect of diet on the mammalian gut flora and its metabolic activities. Crit Rev Toxicol. 1985;16(1):31–103. doi: 10.3109/10408448509041324. [DOI] [PubMed] [Google Scholar]
  27. Saunders B. C. Glutathione. Its reaction with alkali and some N and S derivatives. Biochem J. 1934;28(6):1977–1981. doi: 10.1042/bj0281977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Scheline R. R. Metabolism of foreign compounds by gastrointestinal microorganisms. Pharmacol Rev. 1973 Dec;25(4):451–523. [PubMed] [Google Scholar]
  29. Stevens J. L., Robbins J. D., Byrd R. A. A purified cysteine conjugate beta-lyase from rat kidney cytosol. Requirement for an alpha-keto acid or an amino acid oxidase for activity and identity with soluble glutamine transaminase K. J Biol Chem. 1986 Nov 25;261(33):15529–15537. [PubMed] [Google Scholar]
  30. Stevens J., Jakoby W. B. Cysteine conjugate beta-lyase. Mol Pharmacol. 1983 May;23(3):761–765. [PubMed] [Google Scholar]
  31. Tateishi M. Methylthiolated metabolites. Drug Metab Rev. 1983;14(6):1207–1234. doi: 10.3109/03602538308991428. [DOI] [PubMed] [Google Scholar]
  32. Tateishi M., Suzuki S., Shimizu H. Cysteine conjugate beta-lyase in rat liver. A novel enzyme catalyzing formation of thiol-containing metabolites of drugs. J Biol Chem. 1978 Dec 25;253(24):8854–8859. [PubMed] [Google Scholar]
  33. Tatsumi K., Inoue A., Yoshimura H. Mode of reactions between xanthine oxidase and aromatic nitro compounds. J Pharmacobiodyn. 1981 Feb;4(2):101–108. doi: 10.1248/bpb1978.4.101. [DOI] [PubMed] [Google Scholar]
  34. Tatsumi K., Koga N., Kitamura S., Yoshimura H., Wardman P., Kato Y. Enzymic cis-trans isomerization of nitrofuran derivatives: isomerizing activity of xanthine oxidase, lipoyl dehydrogenase, DT-diaphorase and liver microsomes. Biochim Biophys Acta. 1979 Mar 16;567(1):75–87. doi: 10.1016/0005-2744(79)90174-8. [DOI] [PubMed] [Google Scholar]
  35. Tokiwa H., Ohnishi Y. Mutagenicity and carcinogenicity of nitroarenes and their sources in the environment. Crit Rev Toxicol. 1986;17(1):23–60. doi: 10.3109/10408448609037070. [DOI] [PubMed] [Google Scholar]
  36. Tomisawa H., Ichihara S., Fukazawa H., Ichimoto N., Tateishi M., Yamamoto I. Purification and characterization of human hepatic cysteine-conjugate beta-lyase. Biochem J. 1986 Apr 15;235(2):569–575. doi: 10.1042/bj2350569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Tomisawa H., Suzuki S., Ichihara S., Fukazawa H., Tateishi M. Purification and characterization of C-S lyase from Fusobacterium varium. A C-S cleavage enzyme of cysteine conjugates and some S-containing amino acids. J Biol Chem. 1984 Feb 25;259(4):2588–2593. [PubMed] [Google Scholar]
  38. Vamvakas S., Berthold K., Dekant W., Henschler D. Bacterial cysteine conjugate beta-lyase and the metabolism of cysteine S-conjugates: structural requirements for the cleavage of S-conjugates and the formation of reactive intermediates. Chem Biol Interact. 1988;65(1):59–71. doi: 10.1016/0009-2797(88)90031-2. [DOI] [PubMed] [Google Scholar]
  39. Wei C., Macy J. M., Hsieh D. P. Biotransformation of aflatoxin B1 and its conjugated metabolites by rat gastrointestinal microfloras. Appl Environ Microbiol. 1981 Feb;41(2):549–551. doi: 10.1128/aem.41.2.549-551.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wislocki P. G., Bagan E. S., Lu A. Y., Dooley K. L., Fu P. P., Han-Hsu H., Beland F. A., Kadlubar F. F. Tumorigenicity of nitrated derivatives of pyrene, benz[a]anthracene, chrysene and benzo[a]pyrene in the newborn mouse assay. Carcinogenesis. 1986 Aug;7(8):1317–1322. doi: 10.1093/carcin/7.8.1317. [DOI] [PubMed] [Google Scholar]
  41. el-Bayoumy K., Rivenson A., Johnson B., DiBello J., Little P., Hecht S. S. Comparative tumorigenicity of 1-nitropyrene, 1-nitrosopyrene, and 1-aminopyrene administered by gavage to Sprague-Dawley rats. Cancer Res. 1988 Aug 1;48(15):4256–4260. [PubMed] [Google Scholar]

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