Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes

We examined which human CYP450 forms contribute to carbon tetrachloride (CC l4) bioactivation using hepatic microsomes, heterologously expressed enzyme s, inhibitory antibodies and selective chemical inhibitors. CCl4, metabolis m was determined by measuring chloroform formation under anaerobic conditio ns. Pooled human microsomes metabolized CCl4 with a K-m of 57 mu M and a V- max of 2.3 nmol CHCl3/min/mg protein. Expressed CYP2E1 metabolized CCl4 wit h a K-m of 1.9 mu M and a V-max of 8.9 nmol CHCl3/min/nmol CYP2E1. At 17 mu M CCl4, a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl4 metabolism in three separate human microsomal samples, indicating tha t at low CCl4 concentrations, CYP2E1 was the primary enzyme responsible for CCl4 metabolism. At 530 mu M CCl4, anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl4 metabolism, suggesting that other CYP450s may have a signif icant role in CCl4, metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not co ntribute significantly to CCl4 metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CY P3A) were examined in the liver microsome sample that was inhibited only 36 % by anti-CYP2E1 at 530 mu M CCl4. Clotrimazole inhibited CCl4 metabolism b y 23% but the other chemical inhibitors were without significant effect. Ov erall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl4 bioactivation at lower, environmentally relevant levels. At highe r CCl4 levels. CYP3A and possibly other CYP450 forms may contribute to CCl4 metabolism. (C) 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.