Chlorzoxazone hydroxylation in microsomes and hepatocytes from cytochrome P450 oxidoreductase-null mice.
Previous studies have demonstrated that the NADH-dependent cytochrome b(5) electron transfer pathway can support some cytochrome P450 monooxygenases in vitro in the absence of their normal redox partner, NADPH-cytochrome P450 oxidoreductase. However, the ability of this pathway to support P450 activity in whole cells and in vivo remains unresolved. To address this question, liver microsomes and hepatocytes were prepared from hepatic cytochrome P450 oxidoreductase-null mice and chlorzoxazone hydroxylation, a reaction catalyzed primarily by cytochrome P450 2E1, was evaluated. As expected, NADPH-supported chlorzoxazone hydroxylation was absent in liver microsomes from oxidoreductase-null mice, whereas NADH-supported activity was about twofold higher than that found in normal (wild-type) liver microsomes. This greater activity in oxidoreductase-null microsomes could be attributed to the fourfold higher level of CYP2E1 and 1.4-fold higher level of cytochrome b(5). Chlorzoxazone hydroxylation in hepatocytes from oxidoreductase-null mice was about 5% of that in hepatocytes from wild-type mice and matched the results obtained with wild-type microsomes, where activity obtained with NADH was about 5% of that obtained when both NADH and NADPH were included in the reaction mixture. These results argue that the cytochrome b(5) electron transfer pathway can support a low but measurable level of CYP2E1 activity under physiological conditions.
Li
Li
L
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY, 40536-0082, USA.
Porter
Todd D
TD
D000818:Animals; D002753:Chlorzoxazone / Q000378:metabolism* / Q000494:pharmacology; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000378:metabolism*; D006900:Hydroxylation; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008862:Microsomes, Liver / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature; D013997:Time Factors; D019076:Transgenes
D009249:NADP; D015786:Cytochromes b5; D019392:Cytochrome P-450 CYP2E1; D009251:NADPH-Ferrihemoprotein Reductase; D002753:Chlorzoxazone