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Updated: Jun 27, 2026
Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation
Published on: June 17, 2015
Development of a TK6-derived cell line expressing four human cytochrome P450s for genotoxicity testing
Xilin Li1, Yuhan Wang1, Hannah Xu1
1National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, AR 72079, USA.
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.
Insights
A new TK6 cell line co-expressing four human cytochrome P450 enzymes (TK6-4CYP) effectively metabolizes pro-genotoxicants, showing promise for in vitro genotoxicity testing without S9 fractions.
Area of Science:
- Toxicology
- Genetics
- Biochemistry
Background:
- Metabolism is critical for accurate in vitro genotoxicity testing.
- Previous work established TK6 cell lines expressing single human cytochrome P450 (CYP) enzymes for bioactivation.
- A need exists for models that mimic complex metabolic activation of genotoxicants.
Purpose of the Study:
- To develop and validate a TK6 cell line co-expressing four key human CYP enzymes (CYP2A6, CYP2E1, CYP2C19, CYP3A4) for genotoxicity assessment.
- To evaluate the metabolic capacity of the engineered TK6-4CYP cell line using known CYP-metabolized pro-genotoxicants.
- To establish proof-of-concept for a multi-enzyme expressing cell model in genotoxicity testing.
Main Methods:
- Sequential lentiviral transduction of TK6 cells to co-express CYP2A6/2E1 and CYP2C19/3A4.
- RNA sequencing to confirm selective CYP mRNA upregulation.
- Protein expression and enzymatic activity assays.
- Genotoxicity assays (micronucleus, cell cycle, comet) using NDEA, NDMA, NNP, and riddelliine.
Main Results:
- TK6-4CYP cells exhibited significantly increased mRNA and protein expression for all four targeted CYPs.
- The cell line demonstrated metabolic activation of NDEA, NDMA, and NNP, leading to increased micronuclei induction, G2/M arrest, and DNA damage.
- Riddelliine showed partial activation, inducing micronuclei and G2/M arrest but not DNA strand breaks in the comet assay.
- The model successfully predicted genotoxicity for three out of four tested pro-genotoxicants.
Conclusions:
- A TK6 cell line co-expressing four human CYP enzymes (TK6-4CYP) has been successfully developed.
- This model effectively metabolizes and activates several pro-genotoxicants, demonstrating its utility in genotoxicity testing.
- The TK6-4CYP cell line offers a promising alternative to rodent S9 fractions for in vitro metabolic activation studies.
