Use of an established human hepatoma cell line with endogenous bioactivation for gene mutation studies

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 10(6) thioguanine-resistant HepG2 cells (average plating efficiency = 60-80%); 2) a thioguanine concentration in selection dishes of 10(-4) M with a maximum seeding density of 2.5 x 10(5) 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.

Pathways for the mutagenesis of 1-nitropyrene and dinitropyrenes in the human hepatoma cell line HepG2.

“…Dearfield et al (17) demonstrated that the HepG2 was capable of activating cyclophosphamide to induce sister chromatid exchanges (SCEs), and demonstrated that the content of cytochrome P450 is very low in the HepG2 cells (a phenomenon that has been reported by several laboratories). Other compounds that have been reported to be activated by the HepG2 include benzo-[a] pyrene (18,19), 7,1 2-dimethylbenz- [a]anthracene (20,21), aflatoxin B1, (22,23), several N-nitroso compounds (24), benzidine (23,25), acetylbenzidine (23) (21).…”

Protective Effect of Soybean Saponins and Major Antioxidants Against Aflatoxin B1-Induced Mutagenicity and DNA-Adduct Formation

“…HepG2, the human hepatoma cells used in this study, retain many of the functions of a normal liver cell, such as secretion of plasma lipoproteins and the synthesis of albumin and glycogen. 23,24 Also, HepG2 cells express an inducible cytochrome P-450 system, which makes them an ideal model to investigate the metabolism of genotoxins. A synthetic antioxidant, BHT, was reported to affect a number of phase II enzymes including glutathione transferase, and therefore to inhibit the binding of genotoxins to cellular macromolecules.…”

Evaluation of Cytotoxicity of Food in Human Hepatoma HepG2 Cells: Comet Assay Coupled to the MTT Assay

“…In recent years, considerable effort has been made to characterize, identify and quantitate these compounds in human foods and to elucidate their potential health risks [3,4] .Heterocyclic aromatic amines have been tested extensively in microbial in vitro assays such as Ames Salmonella typhimurium test [5] and are potent mutagens in these test procedures [6] however, literature survey has indicated that almost none of the studies have simultaneously employed two in vitro genocytotoxicity assays, the MTT assay and the comet assay (single cell gel electrophoresis assay) to authenticate the genotoxicity of food. further, it is well documented that HepG2 cell lines retain the activity of certain phase I enzymes involved in metabolism of genotoxic carcinogens such as cytochrome P450 cYP1A1, cYP1A2, cYP2B, cYP2E1 as well as phase II enzymes including glutathione-S-transferases, sulfotransferases, n-acteyltransferases and glucoranosyltransferases, which reflect the metabolism of heterocyclic aromatic amines [7] in mammals better than other in vitro models which require addition of exogenous activation mixtures [8]. The potential hepatotoxicity of heterocyclic aromatic amines is supported by experimental evidence [9].…”