title: Proliferating primary hepatocytes from the pUR288 lacZ plasmid mouse are valuable tools for genotoxicity assessment in vitro.
abstract: Safety assessments of substances with regard to genotoxicity are generally based on a combination of in vitro and in vivo tests. These tests are performed according to a (tiered) test strategy whereby a positive result in vitro usually triggers further testing in vivo. A low specificity and high frequency of irrelevant positive results associated with most in vitro mammalian cell genotoxicity assays necessitates the design and validation of suitable alternatives. As such, we examined the feasibility of culturing primary hepatocytes from the pUR288 lacZ reporter mouse, and moreover, using established cultures to reliably assess genotoxic activity in vitro. Initial studies characterizing the metabolic capacity of proliferating lacZ primary hepatocytes indicated that these cells retained at least some activities important for xenobiotic metabolism: cytochrome P450 1A1 enzyme activities were markedly increased in the hepatocytes after exposure to benzo[a]pyrene, and also UDP-glucuronosyl transferase and glutathione-S-transferase activities, both Phase II enzymes, were detected. Increasing levels of phosphorylated p53 at residue serine 389 after ultraviolet treatment indicated a properly functioning p53, one of the criteria for an effective new test system. Four genotoxic substances with different mechanisms of genotoxicity, i.e., benzo[a]pyrene, bleomycin, etoposide, and cyclophosphamide, were tested in the lacZ rescue assay. For etoposide and cyclophosphamide, the induction of mutant colonies was rather low. Exposure to benzo[a]pyrene and bleomycin, however, yielded a clear concentration-dependent induction of the lacZ mutant frequency. Based on our preliminary observations, proliferating lacZ primary hepatocytes are a promising new tool for the assessment of genotoxic hazard.
authors.affiliation: Laboratory for Health Protection Research, National Institute for Public Health and the Environment, Bilthoven, The Netherlands.
authors.forename: Edwin P
authors.initials: EP
authors.lastname: Zwart
authors.forename: Mirjam M
authors.initials: MM
authors.lastname: Schaap
authors.forename: Myrthe W
authors.initials: MW
authors.lastname: van den Dungen
authors.forename: Hedwig M
authors.initials: HM
authors.lastname: Braakhuis
authors.forename: Paul A
authors.initials: PA
authors.lastname: White
authors.forename: Harry
authors.initials: H
authors.lastname: van Steeg
authors.forename: Jan
authors.initials: J
authors.lastname: van Benthem
authors.forename: Mirjam
authors.initials: M
authors.lastname: Luijten
mesh_terms: D000818:Animals; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007763:Lac Operon; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009152:Mutagenicity Tests; D009153:Mutagens / Q000493:pharmacokinetics / Q000633:toxicity; D010957:Plasmids
chemical_list: D009153:Mutagens; D019363:Cytochrome P-450 CYP1A1