title: Activation of P53 in HepG2 cells as surrogate to detect mutagens and promutagens in vitro.
abstract: The current genotoxicity tests of the standard in vitro battery, especially those using mammalian cells, are limited by their low specificity and highlight the importance of new in vitro tools. This study aimed to evaluate the suitability of HepG2 cells for assaying mutagens and promutagens. We determined P53 activity as surrogate genotoxicity endpoint in HepG2 cells. Our results revealed a significant P53-induction by actinomycin D, methyl methanesulfonate and etoposide. Prior to the investigation of promutagens we characterized HepG2 cells by analyzing the expression of 45 genes involved in xenobiotic metabolism and measuring the activity of selected Cytochrome-P450 (CYP) enzymes. We determined a limited metabolic capacity prompting us to employ a co-treatment with rat liver S9 as metabolic activation system (MAS) for promutagens. While cyclophosphamide showed an elevation of activated P53 in the presence of S9, 7,12-dimethylbenz[a]anthracene and aflatoxin B(1) responded without the MAS. Inhibition of cellular CYP3A4 or CYP1A/1B suppressed the aflatoxin B(1)- and dimethylbenz[a]anthracene-mediated P53 response, respectively, indicating that HepG2 cells are capable of metabolizing these compounds in a CYP1A/B/3A4-dependent manner. In summary, our results indicate that P53 activation in HepG2 cells combined with a MAS can be used for the identification of new (pro)genotoxicants.
authors.affiliation: Merck KGaA, Merck Serono, Toxicology, 64293 Darmstadt, Germany.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Boehme
authors.forename: Yasmin
authors.initials: Y
authors.lastname: Dietz
authors.forename: Philip
authors.initials: P
authors.lastname: Hewitt
authors.forename: Stefan O
authors.initials: SO
authors.lastname: Mueller
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics; D051544:Cytochrome P-450 CYP3A / Q000096:biosynthesis / Q000235:genetics; D065692:Cytochrome P-450 CYP3A Inhibitors; D004249:DNA Damage; D020869:Gene Expression Profiling; D056945:Hep G2 Cells; D006801:Humans; D008658:Inactivation, Metabolic / Q000235:genetics; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000633:toxicity*; D051381:Rats; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
chemical_list: D015415:Biomarkers; D065692:Cytochrome P-450 CYP3A Inhibitors; D009153:Mutagens; D016159:Tumor Suppressor Protein p53; D019363:Cytochrome P-450 CYP1A1; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human