Development of a TK6-derived cell line expressing four human cytochrome P450s for genotoxicity testing

Abstract

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 2¹⁰-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.

Keywords: Biotransformation; Chromosomal damage; Cytochrome P450; DNA damage; TK6 cells.

Fig. 1.

Lentiviral transduction pipeline to generate TK6 cells expressing four CYPs.

Fig. 2.

Comparison of mRNA level, protein expression, and enzymatic activity in wild-type TK6 cells and TK6-4CYP cells. (A) mRNA levels of each CYP in TK6-4CYP cells were measured by quantitative real-time PCR. All gene expression values were normalized to the level of GAPDH. (B) Protein levels of CYP isoforms in TK6-4CYP cells were detected by Western blotting using GAPDH (37 kDa) as a loading control. (C) Enzymatic activities of various CYPs in the TK6-4CYP cells were determined by mass spectrometric analysis after a 24 h incubation with each substrate.

Fig. 3.

Longitudinal analysis of mRNA expression for the four transduced CYPs. The stability of mRNA expression levels for each CYP in TK6-4CYP cells was determined at the indicated timepoints during a 20-day culture period by qPCR. All gene expression values were normalized to GAPDH levels. Data points represent the means ± SD from at least three independent experiments.

Fig. 4.

Induction of micronuclei by pro-genotoxicants in TK6-4CYP cells. TK6-4CYP cells were treated with different concentrations of four chemicals bioactivated by different CYPs, for 24 h. Micronucleus frequency was determined as the percentage of micronuclei relative to intact nuclei (left y-axis and open bars). Cytotoxicity was calculated as the percentage of relative nuclei count compared to control (right y-axis and red line). Data points represent the means ± SD from at least three independent experiments and * indicates p < 0.05 for a treatment group vs. the corresponding control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5.

Induction of G2/M phase cell cycle arrest by pro-genotoxicants in TK6-4CYP cells. TK6-4CYP cells were treated with different chemicals for 24 h. (A) Representative images are shown cell cycle distribution from one experiment analyzed by flow cytometry. (B) Histograms show cell cycle changes at the indicated concentrations of each chemical, represented as the percentage of cells in each phase. Data were expressed as means ± SD from at least three independent experiments. * indicates p < 0.05 comparing treated groups to the corresponding control.

Fig. 6.

Induction of DNA damage by pro-genotoxicants in TK6-4CYP cells. TK6-4CYP cells were exposed to different chemicals at the indicated concentrations for 24 h. (A) Representative arrayed comet images following the treatment in the CometChip assay. (B) DNA damage was measured as the percentage of DNA in the tail. Data are expressed as means ± SD from at least three independent experiments. * indicates p < 0.05 comparing treated groups to the corresponding control.