Toxicology and applied pharmacology

Nitroarene reduction and generation of free radicals by cell-free extracts of wild-type, and nitroreductase-deficient and -enriched Salmonella typhimurium strains used in the umu gene induction assay.

PMID 9925796


Studies of the enzymatic properties of cell-free extracts prepared from overnight cultures of the normal, and nitroreductase-deficient and -enriched strains of Salmonella typhimurium, designed for use in the umu gene induction assay of Oda et al. (1992), were undertaken in an effort to clarify the nature of nitroreductase deficiency in relation to mutagenicity. The ability of these strains to promote oxygen consumption and free radical intermediates of representative nitroarene substrates was measured, respectively, by oxygen polarography and electron spin resonance (ESR) spectroscopy. The substrates 4-nitropyridine N-oxide (4NPO) and 4-nitroquinoline N-oxide (4NQO) stimulated the rate and extent of NADH-dependent oxygen consumption catalyzed by cell-free extracts prepared from wild-type, and nitroreductase-deficient and -enriched strains. The extent of oxygen consumption was greater than stoichiometric with respect to the amount of nitroaromatic substrate, which implied one-electron reduction of 4NQO by these bacterial extracts and subsequent redox cycling with oxygen. ESR spectroscopy confirmed the production of free radical metabolites of the nitroarene substrates, which were inferred by the oxygen consumption studies. At equal protein concentrations the cell-free extracts of each strain catalyzed univalent reduction of 4NPO yielding the 59 line signal characteristic of the 4NPO nitro anion radical. This ESR signal was potently inhibited by the flavoprotein inhibitors CuSO4 and PCMB, albeit a twofold or higher concentration of both inhibitors was required to inhibit the signal produced by extract from the nitroreductase-deficient strain than that produced by the other strains. The results indicate that the nitroreductase-deficient strain of Salmonella typhimurium developed for use in the umu gene induction assay is not deficient in either one-electron nitro group or quinone reductase activity.

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4-Nitropyridine N-oxide, 97%