Murray M, Butler A M
Department of Medicine, University of Sydney, Westmead Hospital, NSW, Australia.
Chem Res Toxicol. 1994 Nov-Dec;7(6):792-9. doi: 10.1021/tx00042a012.
In vitro studies have established that cytochrome P450 (P450) is deactivated by the electrophilic sulfur atom released during the enzymic oxidation of parathion to paraoxon. However, in vivo studies in rats have been unable to demonstrate significant P450 loss. This study evaluated the possibility that there may be alternate pathways of parathion biotransformation in liver, other than those mediated by P450 and supported by NADPH. Initial experiments confirmed that parathion administration did not decrease microsomal P450 or testosterone hydroxylation activities. Subsequent in vitro experiments identified an NADH-dependent pathway of parathion biotransformation, and MS was used to confirm that paraoxon and 4-nitrophenol were the products of both the NADH- and NADPH-dependent reactions. The Michaelis constants of the NADH-dependent formation of paraoxon and 4-nitrophenol (26 +/- 6 microM and 53 +/- 10 microM, respectively) were approximately 3-fold greater than those for the NADPH-supported reactions (9 +/- 1 microM and 18 +/- 3 microM, respectively). Induction of male rats with phenobarbital and dexamethasone, but not beta-naphthoflavone, produced similar increases in the rates of NADH- and NADPH-mediated parathion metabolism. Rates of NADH- and NADPH-dependent metabolism were highly correlated in linear relationships. An anti-NADPH-cytochrome P450 reductase (NADPH-P450 reductase) antibody partially inhibited microsomal parathion oxidation mediated by either cofactor, and the P450 inhibitor clotrimazole was similarly effective against the NADH- and NADPH-supported oxidation of parathion. Finally, a reconstituted system containing P450 2B1, NADPH-P450 reductase, and phospholipid supported parathion oxidation mediated by NADH.(ABSTRACT TRUNCATED AT 250 WORDS)
体外研究已证实，细胞色素P450（P450）可被对硫磷酶促氧化生成对氧磷过程中释放的亲电子硫原子失活。然而，对大鼠的体内研究未能证明P450有显著损失。本研究评估了肝脏中可能存在除由P450介导并由NADPH支持之外的对硫磷生物转化替代途径的可能性。初步实验证实，给予对硫磷不会降低微粒体P450或睾酮羟化活性。随后的体外实验确定了一条依赖NADH的对硫磷生物转化途径，质谱分析用于确认对氧磷和4-硝基苯酚是依赖NADH和NADPH反应的产物。依赖NADH生成对氧磷和4-硝基苯酚的米氏常数（分别为26±6微摩尔和53±10微摩尔）比对硫磷由NADPH支持的反应（分别为9±1微摩尔和18±3微摩尔）约大3倍。用苯巴比妥和地塞米松而非β-萘黄酮诱导雄性大鼠，可使依赖NADH和NADPH的对硫磷代谢速率产生类似增加。依赖NADH和NADPH的代谢速率在线性关系中高度相关。抗NADPH-细胞色素P450还原酶（NADPH-P450还原酶）抗体部分抑制了由任一辅因子介导的微粒体对硫磷氧化，P450抑制剂克霉唑对依赖NADH和NADPH的对硫磷氧化同样有效。最后，一个包含P450 2B1、NADPH-P450还原酶和磷脂的重组系统支持了由NADH介导的对硫磷氧化。（摘要截于250字）
