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Title: Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing

Abstract

Abstract Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.

Authors:
 [1];  [2];  [1];  [2];  [1];  [2];  [1];  [3];  [4];  [1]
  1. Division of Genetic and Molecular Toxicology
  2. Division of Biochemical Toxicology, National Center for Toxicological Research, U.S. Food and Drug Administration, Jefferson, Arkansas 72079
  3. Center for Veterinary Medicine, U.S. Food and Drug Administration, Rockville, Maryland 20855
  4. Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1630986
Alternate Identifier(s):
OSTI ID: 1630661
Resource Type:
Published Article
Journal Name:
Toxicological Sciences
Additional Journal Information:
Journal Name: Toxicological Sciences Journal Volume: 175 Journal Issue: 2; Journal ID: ISSN 1096-6080
Publisher:
Oxford University Press
Country of Publication:
United States
Language:
English

Citation Formats

Li, Xilin, Chen, Si, Guo, Xiaoqing, Wu, Qiangen, Seo, Ji-Eun, Guo, Lei, Manjanatha, Mugimane G., Zhou, Tong, Witt, Kristine L., and Mei, Nan. Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing. United States: N. p., 2020. Web. doi:10.1093/toxsci/kfaa035.
Li, Xilin, Chen, Si, Guo, Xiaoqing, Wu, Qiangen, Seo, Ji-Eun, Guo, Lei, Manjanatha, Mugimane G., Zhou, Tong, Witt, Kristine L., & Mei, Nan. Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing. United States. doi:https://doi.org/10.1093/toxsci/kfaa035
Li, Xilin, Chen, Si, Guo, Xiaoqing, Wu, Qiangen, Seo, Ji-Eun, Guo, Lei, Manjanatha, Mugimane G., Zhou, Tong, Witt, Kristine L., and Mei, Nan. Wed . "Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing". United States. doi:https://doi.org/10.1093/toxsci/kfaa035.
@article{osti_1630986,
title = {Development and Application of TK6-derived Cells Expressing Human Cytochrome P450s for Genotoxicity Testing},
author = {Li, Xilin and Chen, Si and Guo, Xiaoqing and Wu, Qiangen and Seo, Ji-Eun and Guo, Lei and Manjanatha, Mugimane G. and Zhou, Tong and Witt, Kristine L. and Mei, Nan},
abstractNote = {Abstract Metabolism plays a key role in chemical genotoxicity; however, most mammalian cells used for in vitro genotoxicity testing lack effective metabolizing enzymes. We recently developed a battery of TK6-derived cell lines that individually overexpress 1 of 8 cytochrome P450s (CYP1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, and 3A4) using a lentiviral expression system. The increased expression and metabolic function of each individual CYP in each established cell line were confirmed using real-time PCR, Western blotting, and mass spectrometry analysis; the parental TK6 cells and empty vector (EV) transduced cells had negligible CYP levels. Subsequently, we evaluated these cell lines using 2 prototypical polyaromatic hydrocarbon mutagens, 7,12-dimethylbenz[a]anthracene (DMBA) and benzo[a]pyrene (B[a]P), that require metabolic activation to exert their genotoxicity. DMBA-induced cytotoxicity, phosphorylation of histone H2A.X, and micronucleus formation were significantly increased in TK6 cells with CYP1A1, 1B1, 2B6, and 2C19 expression as compared with EV controls. B[a]P significantly increased cytotoxicity, DNA damage, and chromosomal damage in TK6 cells overexpressing CYP1A1 and 1B1 when compared with EV controls. B[a]P also induced micronucleus formation in TK6 cells expressing CYP1A2. These results suggest that our CYP-expressing TK6 cell system can be used to detect the genotoxicity of compounds requiring metabolic transformation.},
doi = {10.1093/toxsci/kfaa035},
journal = {Toxicological Sciences},
number = 2,
volume = 175,
place = {United States},
year = {2020},
month = {3}
}

Journal Article:
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DOI: https://doi.org/10.1093/toxsci/kfaa035

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