Zangar R C, Benson J M, Burnett V L, Springer D L
Molecular Biosciences Department, Battelle PNNL, Richland, WA, USA.
Chem Biol Interact. 2000 Mar 15;125(3):233-43. doi: 10.1016/s0009-2797(00)00149-6.
We examined which human CYP450 forms contribute to carbon tetrachloride (CCl(4)) bioactivation using hepatic microsomes, heterologously expressed enzymes, inhibitory antibodies and selective chemical inhibitors. CCl(4) metabolism was determined by measuring chloroform formation under anaerobic conditions. Pooled human microsomes metabolized CCl(4) with a K(m) of 57 microM and a V(max) of 2.3 nmol CHCl(3)/min/mg protein. Expressed CYP2E1 metabolized CCl(4) with a K(m) of 1.9 microM and a V(max) of 8.9 nmol CHCl(3)/min/nmol CYP2E1. At 17 microM CCl(4), a monoclonal CYP2E1 antibody inhibited 64, 74 and 83% of the total CCl(4) metabolism in three separate human microsomal samples, indicating that at low CCl(4) concentrations, CYP2E1 was the primary enzyme responsible for CCl(4) metabolism. At 530 microM CCl(4), anti-CYP2E1 inhibited 36, 51 and 75% of the total CCl(4) metabolism, suggesting that other CYP450s may have a significant role in CCl(4) metabolism at this concentration. Tests with expressed CYP2B6 and inhibitory CYP2B6 antibodies suggested that this form did not contribute significantly to CCl(4) metabolism. Effects of the CYP450 inhibitors alpha-naphthoflavone (CYP1A), sulfaphenazole (CYP2C9) and clotrimazole (CYP3A) were examined in the liver microsome sample that was inhibited only 36% by anti-CYP2E1 at 530 microM CCl(4). Clotrimazole inhibited CCl(4) metabolism by 23% but the other chemical inhibitors were without significant effect. Overall, these data suggest that CYP2E1 is the major human enzyme responsible for CCl(4) bioactivation at lower, environmentally relevant levels. At higher CCl(4) levels, CYP3A and possibly other CYP450 forms may contribute to CCl(4) metabolism.
我们使用肝微粒体、异源表达酶、抑制性抗体和选择性化学抑制剂，研究了哪些人类细胞色素P450（CYP450）亚型参与四氯化碳（CCl₄）的生物活化。通过在厌氧条件下测量氯仿生成量来确定CCl₄的代谢情况。混合的人类微粒体代谢CCl₄的米氏常数（Kₘ）为57微摩尔，最大反应速度（Vₘₐₓ）为2.3纳摩尔氯仿/分钟/毫克蛋白质。表达的CYP2E1代谢CCl₄的Kₘ为1.9微摩尔，Vₘₐₓ为8.9纳摩尔氯仿/分钟/纳摩尔CYP2E1。在17微摩尔CCl₄时，一种单克隆CYP2E1抗体在三个不同的人类微粒体样本中分别抑制了64%、74%和83%的总CCl₄代谢，这表明在低CCl₄浓度下，CYP2E1是负责CCl₄代谢的主要酶。在530微摩尔CCl₄时，抗CYP2E1抗体分别抑制了36%、51%和75%的总CCl₄代谢，这表明在该浓度下，其他CYP450亚型可能在CCl₄代谢中发挥重要作用。对表达的CYP2B6和抑制性CYP2B6抗体的测试表明，该亚型对CCl₄代谢的贡献不显著。在530微摩尔CCl₄时，抗CYP2E1仅抑制了36%的肝微粒体样本中，研究了CYP450抑制剂α-萘黄酮（CYP1A）、磺胺苯吡唑（CYP2C9）和克霉唑（CYP3A）的作用。克霉唑抑制了23%的CCl₄代谢，但其他化学抑制剂没有显著作用。总体而言，这些数据表明，在较低的、与环境相关的水平下，CYP2E1是负责CCl₄生物活化的主要人类酶。在较高的CCl₄水平下，CYP3A以及可能的其他CYP450亚型可能参与CCl₄代谢。
