代谢
V79中国仓鼠细胞系被基因工程改造,用于稳定表达人NADPH-细胞色素P450氧化还原酶(CYPOR)单独或与人细胞色素P450 1A2(CYP1A2)联合表达。通过免疫印迹法确定,后者细胞系中细胞色素P450 1A2的表达水平与之前构建的仅表达细胞色素P450 1A2的V79细胞系相同。V79细胞中NADPH-细胞色素P450氧化还原酶的异源表达导致对醌型细胞毒素(如 duroquinone 和 menadione)的敏感性增加,这些毒素主要通过在还原氧化循环过程中产生活性氧物种来发挥其毒性。表达NADPH-细胞色素P450氧化还原酶和细胞色素P450 1A2的细胞系在脱烷基化和脱乙基化7-烷氧基荧光素以及三嗪衍生物 ametrine 和 terbutryne 的磺氧化方面的代谢特性进行了研究,与仅表达细胞色素P450 1A2的细胞系进行了比较。增加的NADPH-细胞色素P450氧化还原酶活性损害了依赖于细胞色素P450 1A2的荧光素resorufin分析,可能是通过将8-烷氧基荧光素和resorufin转化为它们的一电子还原的半醌亚胺形式。增加NADPH-细胞色素P450氧化还原酶活性适度增强了细胞色素P450 1A2对三嗪衍生物ametryne和terbutryne的代谢。有趣的是,与N-脱乙基化相比,NADPH-细胞色素P450氧化还原酶过表达时,磺氧化增加了2-3倍,而N-脱乙基化仅增加了1.3-1.9倍。因此,NADPH-细胞色素P450氧化还原酶的水平不仅影响细胞色素P450 1A2的活性速率,还影响细胞色素P450 1A2特异性代谢物谱中代谢物的相对比例。
V79 Chinese hamster cell lines were genetically engineered for the stable expression of human NADPH-cytochrome P450 oxidoreductase (CYPOR) alone or for the combined expression of NADPH-cytochrome P450 oxidoreductase and human cytochrome P450 1A2 (CYP1A2). As determined by immunoblotting, the expression level of cytochrome P450 1A2 in the latter cell line was found to be the same as in a previously constructed V79 cell line expressing cytochrome P450 1A2 only. The heterologous expression of NADPH-cytochrome P450 oxidoreductase in V79 cells resulted in increased sensitivity to quinone-type cytotoxins, eg. duroquinone and menadione, that exert their toxicity primarily through the production of reactive oxygen species during redox cycling. The metabolic properties of the cell line expressing both NADPH-cytochrome P450 oxidoreductase and cytochrome P450 1A2 were characterized regarding dealkylation and deethylation of 7-alkoxyresorufins and sulfoxidation of the triazine derivatives ametryne and terbutryne, in comparison with the cell line expressing only cytochrome P450 1A2. Increased NADPH-cytochrome P450 oxidoreductase activity impaired the cytochrome P450 1A2-dependent fluorometric resorufin assay, presumably by conversion of the 8-alkoxyresorufins and resorufin to their one-electron-reduced semiquinoneimine forms. The cytochrome P450 1A2-dependent metabolism of the triazine derivatives ametryne and terbutryne was moderately enhanced by increased NADPH-cytochrome P450 oxidoreductase activity. Interestingly, with NADPH-cytochrome P450 oxidoreductase overexpression sulfoxidation was increased 2-3-fold, compared with N-deethylation, with a 1.3-1.9-fold increase. Thus, the level of NADPH-cytochrome P450 oxidoreductase not only had an influence on cytochrome P450 1A2 activity rates but also affected the relative proportions of metabolites in cytochrome P450 1A2-specific metabolite profiles.
来源:Hazardous Substances Data Bank (HSDB)