Department of Comparative Biomedicine and Food Science, University of Padua, Viale Dell'Università 16, Legnaro, 35020, Padua, Italy.
University of Eastern Finland, A.I. Virtanen Institute for Molecular Sciences, Neulaniementie 2, 70211, Kuopio, Finland.
Cell Biol Toxicol. 2024 Mar 26;40(1):18. doi: 10.1007/s10565-024-09856-7.
The cytochrome P450 1A (CYP1A) subfamily of xenobiotic metabolizing enzymes (XMEs) consists of two different isoforms, namely CYP1A1 and CYP1A2, which are highly conserved among species. These two isoenzymes are involved in the biotransformation of many endogenous compounds as well as in the bioactivation of several xenobiotics into carcinogenic derivatives, thereby increasing the risk of tumour development. Cattle (Bos taurus) are one of the most important food-producing animal species, being a significant source of nutrition worldwide. Despite daily exposure to xenobiotics, data on the contribution of CYP1A to bovine hepatic metabolism are still scarce. The CRISPR/Cas9-mediated knockout (KO) is a useful method for generating in vivo and in vitro models for studying xenobiotic biotransformations. In this study, we applied the ribonucleoprotein (RNP)-complex approach to successfully obtain the KO of CYP1A1 in a bovine foetal hepatocyte cell line (BFH12). After clonal expansion and selection, CYP1A1 excision was confirmed at the DNA, mRNA and protein level. Therefore, RNA-seq analysis revealed significant transcriptomic changes associated with cell cycle regulation, proliferation, and detoxification processes as well as on iron, lipid and mitochondrial homeostasis. Altogether, this study successfully generates a new bovine CYP1A1 KO in vitro model, representing a valuable resource for xenobiotic metabolism studies in this important farm animal species.
细胞色素 P450 1A（CYP1A）亚家族的外源物质代谢酶（XMEs）由两种不同的同工酶组成，即 CYP1A1 和 CYP1A2，它们在物种间高度保守。这两种同工酶参与许多内源性化合物的生物转化，以及将几种外源物质生物转化为致癌衍生物，从而增加肿瘤发展的风险。牛（Bos taurus）是最重要的食品生产动物物种之一，是全球重要的营养来源。尽管每天都接触外源物质，但关于 CYP1A 对牛肝脏代谢的贡献的数据仍然很少。CRISPR/Cas9 介导的基因敲除（KO）是一种用于生成体内和体外模型以研究外源物质生物转化的有用方法。在这项研究中，我们应用核糖核蛋白（RNP）复合物方法成功地获得了牛胎肝细胞系（BFH12）中 CYP1A1 的 KO。经过克隆扩增和选择，在 DNA、mRNA 和蛋白质水平上证实了 CYP1A1 的缺失。因此，RNA-seq 分析揭示了与细胞周期调控、增殖和解毒过程以及铁、脂质和线粒体稳态相关的显著转录组变化。总之，这项研究成功地在体外生成了一种新的牛 CYP1A1 KO 模型，为该重要农场动物物种的外源物质代谢研究提供了有价值的资源。
