Li Xilin, Wang Yuhan, Xu Hannah, He Xiaobo, Chen Si, Guo Xiaoqing, Manjanatha Mugimane G, Zhou Tong, Bonzo Jessica, Mei Nan
National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA.
Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, MD 20855, USA.
Toxicol In Vitro. 2025 Oct;108:106085. doi: 10.1016/j.tiv.2025.106085. Epub 2025 May 26.
Metabolism is essential for in vitro genotoxicity testing. We previously developed a panel of TK6 cell lines, each expressing one of 14 human cytochrome P450 (CYP) enzymes, demonstrating their ability to effectively bioactivate indirect genotoxicants without relying on a rodent liver S9 fraction. In the present study, we extended this work by developing a TK6 cell line co-expressing four human CYP enzymes, including CYP2A6, CYP2E1, CYP2C19, and CYP3A4 (designated as TK6-4CYP), and subsequently assessed its capability to metabolize and activate pro-genotoxicants. Human lymphoblastoid TK6 cells were sequentially transduced with lentiviral vectors carrying CYP2A6/2E1 and CYP2C19/3A4, resulting in more than a 2-fold increase in mRNA expression levels for each CYP compared to parental cells. RNA sequencing revealed selective upregulation of the four CYPs. Their protein expression and enzymatic activities were also confirmed. TK6-4CYP cells were subsequently tested with four CYP-metabolized pro-genotoxicants, including N-nitroso-diethylamine (NDEA) metabolized by CYP2A6, N-nitroso-dimethylamine (NDMA) by CYP2E1, N-nitroso-propranolol (NNP) by CYP2C19, and riddelliine by CYP3A4, in the micronucleus assay, cell cycle analysis, and comet assay. Significant increases were observed in the percentage (%) of micronuclei induction, G2/M phase arrest, and % DNA in tails with all compounds except riddelliine, which showed increases in % micronuclei induction and G2/M phase arrest but no positive response in the comet assay. This study establishes proof-of-concept for using a TK6 cell model co-expressing multiple drug-metabolizing enzymes for genotoxicity evaluation.
代谢对于体外遗传毒性测试至关重要。我们之前开发了一组TK6细胞系，每个细胞系表达14种人类细胞色素P450（CYP）酶中的一种，证明了它们在不依赖啮齿动物肝脏S9组分的情况下有效生物活化间接遗传毒性剂的能力。在本研究中，我们通过开发一种共表达四种人类CYP酶（包括CYP2A6、CYP2E1、CYP2C19和CYP3A4，命名为TK6-4CYP）的TK6细胞系扩展了这项工作，随后评估了其代谢和活化前遗传毒性剂的能力。用人淋巴细胞样TK6细胞依次用携带CYP2A6/2E1和CYP2C19/3A4的慢病毒载体进行转导，与亲代细胞相比，每种CYP的mRNA表达水平增加了2倍以上。RNA测序显示这四种CYP有选择性上调。它们的蛋白质表达和酶活性也得到了证实。随后在微核试验、细胞周期分析和彗星试验中，用四种由CYP代谢的前遗传毒性剂对TK6-4CYP细胞进行测试，包括由CYP2A6代谢的N-亚硝基二乙胺（NDEA）、由CYP2E1代谢的N-亚硝基二甲胺（NDMA）、由CYP2C19代谢的N-亚硝基普萘洛尔（NNP）以及由CYP3A4代谢的里德灵。除里德灵外，所有化合物的微核诱导百分比（%）、G2/M期阻滞和尾部%DNA均显著增加，里德灵显示微核诱导%和G2/M期阻滞增加，但在彗星试验中无阳性反应。本研究建立了使用共表达多种药物代谢酶的TK6细胞模型进行遗传毒性评估的概念验证。
