title: Evaluation of the LacZ reporter assay in cryopreserved primary hepatocytes for In vitro genotoxicity testing.
abstract: Assessment of genotoxic potential is an important step in the safety evaluation of chemical substances. Under most regulatory jurisdictions, the first tier of testing comprises a standard battery of in vitro genotoxicity tests in bacterial and mammalian cells. However, the mammalian cell tests commonly used exhibit a relatively high rate of misleading positive results, which may lead to unnecessary in vivo testing. We previously established a proof-of-concept for the LacZ reporter assay in proliferating primary hepatocytes as a promising alternative genotoxicity test. Here, cryopreserved instead of freshly isolated hepatocytes were used and the assay was evaluated in more detail. We examined the effect of cryopreservation on phenotype and metabolic capacity of the LacZ hepatocytes, and assessed the predictive performance of the assay by testing a set of substances comprising true positive, true negative, and misleading positive substances. Additionally, a historical negative control database was created and the type of mutations induced was analyzed for two of the substances tested. Our findings indicate that proliferating cryopreserved primary hepatocytes derived from LacZ plasmid mice retain their hepatocyte-specific characteristics and metabolic competence. Furthermore, we demonstrate that both gene mutations and genome rearrangements due to large deletions can be detected with the LacZ reporter assay. The assay seems to have a lower rate of misleading positive test results compared to the assays currently used. Together, our findings strongly support the use of the LacZ reporter assay in cryopreserved primary hepatocytes as follow-up to the standard in vitro test battery for genotoxicity testing. Environ. Mol. Mutagen. 57:643-655, 2016. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
authors.forename: Mirjam
authors.initials: M
authors.lastname: Luijten
authors.affiliation: Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
authors.forename: Edwin P
authors.initials: EP
authors.lastname: Zwart
authors.affiliation: Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
authors.forename: Martijn E T
authors.initials: ME
authors.lastname: Dollé
authors.affiliation: Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
authors.forename: Maaike
authors.initials: M
authors.lastname: de Pooter
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Julie A
authors.initials: JA
authors.lastname: Cox
authors.affiliation: Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, Canada.
authors.forename: Paul A
authors.initials: PA
authors.lastname: White
authors.affiliation: Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands.
authors.forename: Jan
authors.initials: J
authors.lastname: van Benthem
mesh_terms: D000826:Animal Testing Alternatives; D000818:Animals; D015415:Biomarkers; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D015925:Cryopreservation; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D007763:Lac Operon; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity; D010957:Plasmids; D061251:Primary Cell Culture; D012680:Sensitivity and Specificity; D019076:Transgenes
chemical_list: D015415:Biomarkers; D009153:Mutagens; D019363:Cytochrome P-450 CYP1A1
keywords: DNA damage; LacZ plasmid; chromosome aberration; mutagenicity; regulatory implementation; risk assessment