The environmental carcinogen benzo[a]pyrene (BaP) is presumed to exert its genotoxic effects after metabolic activation by cytochrome P450 (CYP) enzymes. However, studies using the Hepatic Reductase Null (HRN) mouse model, in which cytochrome P450 oxidoreductase (POR), the electron donor to CYP enzymes, is deleted specifically in hepatocytes, have shown that loss of hepatic POR-mediated CYP function leads to greater BaP-DNA adduct formation in livers of these mice than in wild-type (WT) mice. Here, we used CRISPR/Cas9 technology to knockout (KO) POR expression in mouse hepatoma Hepa1c1c7 cells to create an in vitro model that can mimic the HRN mouse model. Western blotting confirmed the deletion of POR in POR KO Hepa1c1c7 cells whereas expression of other components of the mixed-function oxidase system including cytochrome b5 (Cyb5) and NADH:cytochrome b5 reductase (which can also serve as electron donors to CYP enzymes), and CYP1A1 was similar in BaP-exposed WT and POR KO Hepa1c1c7 cells. BaP exposure caused cytotoxicity in WT Hepa1c1c7 cells but not in POR KO Hepa1c1c7 cells. In contrast, CYP-catalysed BaP-DNA adduct levels were ~10-fold higher in POR KO Hepa1c1c7 cells than in WT Hepa1c1c7 cells, in concordance with the presence of higher levels of BaP metabolite (e.g. BaP-7,8-dihydrodiol) in the medium of cultured BaP-exposed POR KO Hepa1c1c7 cells. As was seen in the HRN mouse model, these results suggest that Cyb5 contributes to the bioactivation of BaP in POR KO Hepa1c1c7 cells. These results indicate that CYP enzymes may play a more important role in the detoxication of BaP, as opposed to its bioactivation.

中文翻译：

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删除细胞色素P450氧化还原酶可增强Hepa1c1c7细胞中苯并[a] re的代谢和DNA加合物的形成。

据推测，环境致癌物苯并[a] py（BaP）在被细胞色素P450（CYP）酶代谢激活后发挥了遗传毒性作用。但是，使用肝还原酶无效（HRN）小鼠模型的研究表明，肝细胞中CYP酶的电子供体细胞色素P450氧化还原酶（POR）被特异性删除，表明肝POR介导的CYP功能丧失会导致更大的损失。与野生型（WT）小鼠相比，这些小鼠的肝脏中BaP-DNA加合物的形成。在这里，我们使用了CRISPR / Cas9技术在小鼠肝癌Hepa1c1c7细胞中敲除（KO）POR表达，以创建可以模拟HRN小鼠模型的体外模型。Western blotting证实POR KO Hepa1c1c7细胞中POR缺失，而混合功能氧化酶系统其他成分的表达包括细胞色素b5（Cyb5）和NADH：细胞色素b5还原酶（也可以用作CYP酶的电子供体），而CYP1A1在暴露于BaP的WT和POR KO Hepa1c1c7细胞中相似。BaP暴露在WT Hepa1c1c7细胞中引起细胞毒性，但在POR KO Hepa1c1c7细胞中未引起细胞毒性。相反，CYP催化的BaP-DNA加合物水平在POR KO Hepa1c1c7细胞中比在WT Hepa1c1c7细胞中高约10倍，这与高水平BaP代谢产物（例如BaP-7,8-dihydrodiol）的存在相一致。培养的BaP暴露的POR KO Hepa1c1c7细胞的培养基。如在HRN小鼠模型中所见，这些结果表明Cyb5有助于POR KO Hepa1c1c7细胞中BaP的生物激活。这些结果表明，CYP酶可能在BaP的解毒中起更重要的作用，与其生物激活相反。