In vitro genotoxicity tests are extensively used by regulatory agencies worldwide to identify chemicals that induce genetic damage. Since chemicals can be converted into DNA reactive metabolites in vivo, current guidelines from the U.S. Food and Drug Administration (FDA) (FDA, 2007), the Organization for Economic Co-operation and Development (OECD) (OECD, 2016c), the International Council for Harmonization (ICH) (ICH, 2011), and the International Cooperation on Harmonization of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) (VICH, 2014) require the incorporation of a source of metabolic activation for all in vitro mammalian cell assays. Accordingly, chemical-induced liver S9 fractions from rodents, along with co-factors, have been routinely used in the battery of standard in vitro genotoxicity tests as a metabolic activation system (Hashizume et al., 2010).

Due to some well-recognized limitations of using exogenous rodent liver S9 for metabolism, human cell models expressing various phase I or phase II drug-metabolizing enzymes have been increasingly adopted for in vitro genotoxicity testing (Pfuhler et al., 2021; Seo et al., 2019; Seo et al., 2023b). Since its establishment in the late 1970s, the human lymphoblastoid TK6 cell line has been widely used for conducting genotoxicity assays, a major application of its use is in conducting the in vitro TK and HPRT gene mutation assays (OECD TGs 476 and 490) (OECD, 2015, OECD, 2016a). In addition, TK6 cells are one of the standard cell lines for conducting the in vitro chromosome aberration test (OECD TG 473) (OECD, 2016b), micronucleus (MN) test (OECD TG 487) (OECD, 2023), and comet assay. Whole genome sequencing analysis indicated negligible genetic variability between TK6 and the human reference genome (Revollo et al., 2016). TK6 cells, however, exhibit negligible expression of major drug metabolizing enzymes responsible for chemical biotransformation. To address this limitation, tissue homogenates, typically rat liver S9, are added to assays for metabolizing test substances that require metabolic activation. Recently, experts at the 7th International Workshop on Genotoxicity Testing (IWGT) evaluated the state-of-the-science with respect to (1) technologies and innovations for improving existing assays using human lymphoblastoid TK6 cells, and (2) novel and emerging technologies and approaches for in vitro mammalian cell mutagenicity test systems (Gollapudi et al., 2019). They recommended that any novel test system should be metabolically competent, thereby eliminating the need for exogenous metabolic activation (Evans et al., 2019).

Cytochrome P450 (CYP) enzymes were first discovered in 1954, and since then, numerous CYP proteins have been identified and found to be widespread throughout the human body (McDonnell and Dang, 2013). A review article reported that five CYPs (1A2, 2C9, 2C19, 2D6, and 3A4) account for over 75 % of P450-mediated drug metabolism in humans, while six CYPs (1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) contribute to approximately 77 % of P450 activation reactions for chemicals classified as carcinogens (Rendic and Guengerich, 2021). Additionally, five CYPs (1A1, 1A2, 2A6, 2E1, and 3A4) are predominantly responsible for generating activated metabolites from natural products. Earlier studies identified twelve CYPs (1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) as being involved in xenobiotic metabolism, with five of them (1A1, 1A2, 1B1, 2A6, and 2E1) primarily responsible for procarcinogen activation, while CYP2B6 and 2D6 have minor roles (He and Feng, 2015). Furthermore, seven CYPs (1A1, 1A2, 1B1, 2A6, 2A13, 2E1, and 3A4) have been identified as major enzymes involved in the activation of environmental carcinogens, as demonstrated by the umu genotoxicity assay and the Ames test (Shimada, 2017).

In response to the IWGT's recommendation, we have previously developed fourteen TK6-derived cell lines that individually express one of the 14 human CYPs (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, and 3A7), and demonstrated that this system is effective at bioactivating various indirect-acting compounds into genotoxicants and mutagens without the addition of a rodent liver S9 fraction (Li et al., 2020a; Li et al., 2020b; Li et al., 2023). In addition, since TK6 cells have negligible endogenous CYP expression, we are able to identify the specific CYP enzymes that account for the bioactivation of pro-genotoxicants, providing critical mechanistic information (Li et al., 2020b; Li et al., 2024b). Although these cell lines are valuable for in vitro genotoxicity screening, one limitation is that testing chemicals in each individual CYP-expressing cell line is labor-intensive. Therefore, it would be advantageous to develop metabolically competent TK6-derived cell lines that co-express multiple phase I enzymes. As such, we propose to further develop metabolically competent TK6 cell lines that co-express multiple human CYPs.

In this study, we created a TK6-derived cell line that simultaneously expresses four CYPs, including CYP2A6, 2E1, 2C19, and 3A4. The mRNA and protein expression, as well as enzymatic activity of each CYP in this cell line were confirmed and compared to the parental TK6 cells, and all were comparable with those in each corresponding single-CYP cell line. The performance of genotoxicity detection was further verified by several commonly used assays on four pro-genotoxicants that have been identified to be metabolized by CYP enzymes.