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 K(m) and V(max) for post-mitochondrial supernatant from Mutatrade markMouse liver (i.e., liver S9) were 23.9 microM and 70.2 pmol/min/mg protein, respectively. Analysis of replicate samples of Mutatrade markMouse 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.
Chen
Guosheng
G
Mutagenesis Section, Safe Environments Program, Health Canada, Tunney's Pasture 0803A, Ottawa, Ont., Canada.
Lambert
Iain B
IB
Douglas
George R
GR
White
Paul A
PA
D000818:Animals; D001551:Benz(a)Anthracenes / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005260:Female; D005453:Fluorescence; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008168:Lung / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic; D010088:Oxidoreductases / Q000378:metabolism*; D015203:Reproducibility of Results
C475568:3-aminobenzanthrone; D001551:Benz(a)Anthracenes; D010088:Oxidoreductases; C117220:3-nitrobenzanthrone