HEPATIC CYTOCHROME P450 REDUCTASE-NULL MICE AS AN ANIMAL MODEL TO STUDY ELECTRON TRANSFER PATHWAYS IN CHOLESTEROL SYNTHESIS AND CYP2E1-MEDIATED DRUG METABOLISM

NADPH-cytochrome P450 reductase (CPR) is a flavoprotein containing both FAD and FMN and functions as the electron donor protein for several oxygenase enzymes found on the endoplasmic reticulum of eukaryotic cells, including cytochrome P450s involved in drug metabolism and cholesterol biosynthesis. As many as three enzymes in the cholesterol biosynthetic pathway have been demonstrated, or proposed, to use CPR as a redox partner: squalene monooxygenase, which converts squalene to 2,3-oxidosqualene; lanosterol demethylase, a cytochrome P450 (CYP51); and 7-dehydrocholesterol reductase, the final step in cholesterol synthesis. In yeast CPR can be replaced by the NADH-cytochrome b5 pathway, but this has not been demonstrated in animals or plants. My studies with hepatic cytochrome P450 reductase-null mice have revealed a second microsomal reductase for squalene monooxygenase that was not previously detected. Studies carried out with hepatocytes from CPR-null mice demonstrate that this second reductase is active in whole cells and leads to the accumulation of 24-dihydrolanosterol, indicating that lanosterol demethylation, catalyzed by CYP51, is blocked. These results demonstrate that this second reductase plays a significant role in supporting squalene monooxygenase but not cytochrome P450-mediated reactions. 7-Dehydrocholesterol reductase (E.C. 1.3.1.21) catalyzes the reduction of the 7-8 double bond of 7-dehydrocholesterol to yield cholesterol. It has been suggested that cytochrome-P450 reductase is required for this reaction. My studies show that 7-dehydrocholesterol reductase is enzymatically active in CPR-null microsomes, with activity equal to or greater than that found in preparations from wild-type mice. Mammalian cytochrome b5, which can accept electrons from either cytochrome P450 reductase or NADH-cytochrome b5 reductase, is known to be involved in augmenting some P450-dependent monooxygenase reactions. Cytochrome P450 2E1 has been found to exhibit reasonable rates of turnover via an NADHcytochrome b5 pathway in reconstituted enzyme systems and in heterologous hosts. Using microsomes from hepatic CPR-null mice, I have determined that NADH-dependent CYP2E1 activity in the absence of NADPH-dependent activity constituted approximately 10% of CYP2E1 activity observed in microsomal preparations with NADPH from wild-type mice. However, little or no CYP2E1 activity could be detected in primary hepatocytes isolated from CPR-null mice