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 Caroline A. Metosh-Dickey,* ,1 Ronald P. Mason,† and Gary W. Winston* ,2 *Department of Biochemistry, Louisiana State University, Baton Rouge, Louisiana 27695; and †Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 Received July 15, 1998; accepted September 23, 1998 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. Metosh-Dickey, C. A., Mason, R. P., and Win- ston, G. W. (1999). Toxicol. Appl. Pharmacol. 154, 126 –134. Studies of the enzymatic properties of cell-free extracts pre- pared from overnight cultures of the normal, and nitroreductase- deficient and -enriched strains of Salmonella typhimurium, de- signed 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) spec- troscopy. 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 ex- tracts 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 4NQOby 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 CuSO 4 and PCMB, albeit a twofold or higher concen- tration of both inhibitors was required to inhibit the signal pro- duced by extract from the nitroreductase-deficient strain than that produced by the other strains. The results indicate that the ni- troreductase-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. © 1999 Academic Press Nitroarenes are ubiquitous contaminants in the environment (McCartney et al., 1986; Tokiwa et al., 1987). As such, they frequently react with low molecular weight compounds and enzymes within organisms which, through reductive metabo- lism, can activate them to more carcinogenic and mutagenic endpoints. The importance of enzymatic reduction of nitro compounds led to the development of nitroreductase-deficient and -enriched strains in bacterial mutagenicity assays which allow for more detailed understanding of the role of these enzymes in the activation of various compounds to mutagenic endpoints. There remains controversy over which enzymes are actually responsible for nitroarene reduction in vivo (Rosen- kranz et al., 1982; Kinouchi and Ohnishi, 1983; Bryant et al., 1984; Oda et al., 1992). The nitro reductase-deficient strain widely used in the Ames assay, TA98NR, was isolated for nitrofuran resistance and is not mutable by niridazole, nitronaphthalenes, and nitroflu- orenes (McCoy et al., 1981). However, it is responsive to some nitropyrenes, suggesting that the enzyme lacking or deficient in the system is not an obligatory requirement for activation to its penultimate mutagenic metabolites (Rosenkranz and Mermel- stein, 1985). Hajos and Winston (1991) studied the interaction of NAD(P)H-quinone oxidoreductase (DT-diaphorase, DTD) with 1,3-, 1,6-, and 1,8-dinitropyrene (DNP) in TA98, a stan- dard reductase-proficient strain, and in nitroreductase-deficient TA98NR of the Ames assay. DTD, which transfers two elec- trons to quinones and other acceptors including nitro group- containing substrates (Prochaska and Talalay, 1986; Lind et al., 1982), could contribute to the formation of hydroxylamine metabolites of nitroaromatic substrates thought to be penulti- mate mutagenic endpoints of nitroarene metabolism (Biaglow et al., 1977; Kato et al., 1970; Tada, 1981; Rosenkranz and Mermelstein, 1983). Based upon electrochemical reduction measurements of DNP, the 1,3-DNP isomer was purported to be a preferential one-electron acceptor, and 1,6- and 1,8-DNP preferential two-electron acceptors (Howard et al., 1987; Klop- man et al., 1984). The mutagenicity of 1,3-DNP in TA98NR was suppressed as relative to the normal nitro reductase-pro- 1 Present address: Applied Microbial Systems, Inc., Baton Rouge, LA 70802. 2 To whom correspondence should be addressed. Toxicology and Applied Pharmacology 154, 126 –134 (1999) Article ID taap.1998.8573, available online at http://www.idealibrary.com on 126 0041-008X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.