Guengerich F P, Mason P S, Stott W T, Fox T R, Watanabe P G
Cancer Res. 1981 Nov;41(11 Pt 1):4391-8.
The metabolism of [1,2-14C]vinyl bromide (VBR) to products irreversibly bound to DNA and protein was examined in rat liver microsomes, reconstituted cytochrome P-450 systems, and isolated hepatocytes. A role for cytochrome P-450 was confirmed using inhibition and reconstitution experiments. The major form of cytochrome P-450 involved in VBR metabolism does not appear to be either of the major isozymes induced by phenobarbital or beta-naphthoflavone, as determined by induction, reconstitution, and antibody inhibition studies. 2-Bromoethylene oxide and 2-bromoacetaldehyde, suspected metabolites of VBR, were synthesized and found to be substrates for rat liver epoxide hydrolase and equine liver alcohol dehydrogenase, respectively. These enzymes were used to probe the roles of the two possible metabolites in the irreversible binding of products of VBR to protein and DNA. Alcohol dehydrogenase was more effective than epoxide hydrolase in inhibiting the binding of VBR metabolites to protein in microsomal incubations. Epoxide hydrolase was effective in inhibiting the binding of VBR or vinyl chloride metabolites to calf thymus DNA added to such systems, but alcohol dehydrogenase was not. Similar results were obtained for binding of VBR metabolites to DNA in a reconstituted enzyme system. Reduced glutathione blocked nonenzymatic binding of 2-bromo[1,2-14C]acetaldehyde to protein but not DNA. Binding of vinyl chloride and VBR metabolites to protein was blocked by reduced glutathione, but binding to DNA was not. These results are consistent with the view that 2-haloethylene oxides are the major alkylating agents bound to DNA, and 2-haloacetaldehydes are the major alkylating agents bound to protein in these experimental systems. Studies with labeled 2-bromoacetaldehyde indicate that the slow kinetics of DNA binding by this compound is responsible in part for this phenomenon. Studies with isolated rat hepatocytes suggest that a significant portion of the total and reactive metabolites are able to leave these cells. In these systems, binding of metabolites of vinyl chloride to DNA outside the hepatocytes could be partially blocked by epoxide hydrolase or by alcohol dehydrogenase, implying that, as target farther away from sources of reactive species are considered, the stabilities of these species become more important for reaction with nucleophilic sites.
在大鼠肝微粒体、重组细胞色素P - 450系统和分离的肝细胞中，研究了[1,2 - 14C]溴乙烯（VBR）代谢为与DNA和蛋白质不可逆结合产物的过程。通过抑制和重组实验证实了细胞色素P - 450的作用。通过诱导、重组和抗体抑制研究确定，参与VBR代谢的细胞色素P - 450的主要形式似乎既不是苯巴比妥或β - 萘黄酮诱导的主要同工酶。合成了疑似VBR代谢产物的2 - 溴环氧乙烷和2 - 溴乙醛，发现它们分别是大鼠肝环氧化物水解酶和马肝醇脱氢酶的底物。这些酶被用于探究这两种可能的代谢产物在VBR产物与蛋白质和DNA的不可逆结合中的作用。在微粒体孵育中，醇脱氢酶比环氧化物水解酶更有效地抑制VBR代谢产物与蛋白质的结合。环氧化物水解酶有效地抑制了VBR或氯乙烯代谢产物与添加到此类系统中的小牛胸腺DNA的结合，但醇脱氢酶则不然。在重组酶系统中，VBR代谢产物与DNA的结合也得到了类似结果。还原型谷胱甘肽可阻断2 - 溴[1,2 - 14C]乙醛与蛋白质的非酶结合，但不能阻断与DNA的结合。氯乙烯和VBR代谢产物与蛋白质的结合被还原型谷胱甘肽阻断，但与DNA的结合未被阻断。这些结果与以下观点一致：在这些实验系统中，2 - 卤代环氧乙烷是与DNA结合的主要烷基化剂，2 - 卤代乙醛是与蛋白质结合的主要烷基化剂。用标记的2 - 溴乙醛进行的研究表明，该化合物与DNA结合的缓慢动力学部分导致了这一现象。对分离的大鼠肝细胞的研究表明，总代谢产物和活性代谢产物中有很大一部分能够离开这些细胞。在这些系统中，环氧化物水解酶或醇脱氢酶可部分阻断肝细胞外氯乙烯代谢产物与DNA的结合，这意味着，当考虑离活性物质来源更远的靶点时，这些物质的稳定性对于与亲核位点的反应变得更加重要。
