Assessment of 3-nitrobenzanthrone reductase activity in mammalian tissues by normal-phase HPLC with fluorescence detection

3-Nitrobenzanthrone (3-NBA) is a potent mutagen and possible human carcinogen present in diesel exhaust and airborne particulate matter. Nitroreduction is believed to play a crucial role in nitroarene activation and mutagenicity; however, quantification of nitroreduction rate in mammalian samples has proved difficult. In this study, we present a sensitive method to quantify 3-nitrobenzanthrone reductase activity in murine tissues via normal-phase HPLC with fluorescence detection of the reduced product 3-aminobenzanthrone (3-ABA). Calibration linearity was obtained for pure 3-ABA concentrations of 1–500 ng/ml (r2 > 0.99), with a detection limit of 0.25 ng/ml (S/N = 3). Incubation time, substrate concentration, and protein concentration in the reaction mixture were optimized, and the detection limit of the enzyme assay is 0.97 pmol/min/mg protein. The apparent Km and Vmax for post-mitochondrial supernatant from Muta™Mouse liver (i.e., liver S9) were 23.9 μM and 70.2 pmol/min/mg protein, respectively. Analysis of replicate samples of Muta™Mouse liver and lung S9 yielded mean activity values of 39.0 ± 3.0 and 61.1 ± 4.3 pmol/min/mg, respectively. ANOVA revealed significant effects of tissue type and incubation condition (i.e., with or without N2). The results show significantly higher activity in lung, and, in contrast to that observed for 1-nitropyrene, incubation in open air (i.e., without N2 bubbling) causes only a marginal decrease in activity. Quantification of 3-NBA nitroreductase activity in murine tissues will provide insight into the published tissue-specific mutagenic activity of 3-NBA.