Use of an established human hepatoma cell line with endogenous bioactivation for gene mutation studies |
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Authors: | George Chang David Jacobson-Kram Jerry R Williams |
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Institution: | (1) Radiobiology Laboratory, Johns Hopkins University Oncology Center, Baltimore, Maryland;(2) Radiobiology Laboratory 2-121, The Johns Hopkins Oncology Center, 600 North Wolfe Street, 21205 Baltimore, MD |
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Abstract: | Genetic toxicology assays that rely on S9 microsomal mixes are subject to artifacts related to the generation of mutagenic metabolites by acidic pHs, variation in individual isolations of microsomes and the failure of subcellular fractions to faithfully produce metabolites generated in intact cells. We have developed a gene mutation assay utilizing the human hepatoma cell line HepG2, which has been shown to metabolize a broad spectrum of promutagens. Optimal conditions for assaying the induction of 6-thioguanine-resistant mutants in this cell line include: 1) growth of colonies for three weeks on lethally irradiated feeder layers of 106 thioguanine-resistant HepG2 cells (average plating efficiency = 60–80%); 2) a thioguanine concentration in selection dishes of 10–4M with a maximum seeding density of 2.5 × 105 cells per 100 mm culture dish; and 3) a minimum expression time of 6 days. In addition to ultraviolet light C (254 nm), a cytochrome P450 (cyclophosphamide)-dependent and a cytochrome P448 (aflatoxin B1)-dependent promutagen were shown to induce cytotoxicity and mutations in this test system. The present studies, therefore, suggest that the HepG2 cell line may be useful for a variety of assays in genetic toxicology.Abbreviations HAT
hypoxanthine, aminopterin, thymidine
- TG
6-thioguanine |
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Keywords: | gene mutations human hepatoma cells metabolic activation |
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