Cytochrome P-450-mediated denitrification of 2-nitropropane in mouse liver microsomes.
Enzymatic denitrification of 2-nitropropane (2NP) was investigated in an NADPH-dependent hepatic microsomal system from male CD1 mice. The involvement of cytochrome P-450 (P-450) as the catalyst in 2NP denitrification was revealed by the induction of nitrite-releasing activity following phenobarbital (PB) pretreatment, by a decrease in activity with carbon tetrachloride pretreatment, by the inhibition of the reaction with classical P-450 inhibitors, and by the observation of a type I binding spectrum. Under optimal conditions, two pH-dependent peaks of activity were observed at pH 7.6 and pH 8.8, each with its own optimal substrate concentration. Inhibition of the reaction by metyrapone and carbon monoxide (CO) (among others) produced differential responses dependent on pH. These results, along with two pH optima and two substrate optima, suggested the involvement of multiple P-450 isozymes. Average specific activities were 8.05 nmoles of nitrite released per minute per milligram microsomal protein at pH 7.6 and 6.44 nmoles of nitrite released per minute per milligram microsomal protein at pH 8.8. Acetone was identified as the second product of the reaction by gas chromatography/mass spectrometry (GC/MS). Stoichiometry studies indicated that the acetone production was slightly less than expected (about 70%) from nitrite release. Up to 25% residual activity was observed under anaerobic conditions. These results suggested that though the predominant reaction mechanism was oxidative, oxygen-independent metabolism of 2NP also occurred to some extent. In contrast to the reported lack of activity in untreated rat, the observed denitrification in uninduced mouse liver microsomes was significant and suggested that major species-specific differences exist in the in vitro metabolism of 2NP.
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Department of Environmental and Industrial Health, School of Public Health, University of Michigan, Ann Arbor 48109-2029.
Kulkarni
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D000332:Aerobiosis; D000473:Alkanes / Q000378:metabolism*; D000693:Anaerobiosis; D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D008297:Male; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology* / Q000378:metabolism; D009249:NADP / Q000494:pharmacology; D009573:Nitrites / Q000378:metabolism*; D009595:Nitroparaffins / Q000378:metabolism*; D011407:Propane / Q000031:analogs & derivatives* / Q000378:metabolism
D000473:Alkanes; D009573:Nitrites; D009595:Nitroparaffins; D009249:NADP; D003577:Cytochrome P-450 Enzyme System; C016617:2-nitropropane; D011407:Propane