title: Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells.
abstract: In the present study an automated image analysis assisted in vitro micronucleus assay was developed with the rodent cell line CHO-k1 and the human hepatoma cell line HepG2, which are both commonly used in regulatory genotoxicity assays. The HepG2 cell line was chosen because of the presence in these cells of a functionally active p53 protein, a functionally competent DNA-repair system, active enzymes for phase-I and -II metabolism, and an active Nrf2 electrophile responsive system. These properties may result in an assay with a high predictivity for in vivo genotoxicity. The assays with CHO-k1 and HepG2 cells were both evaluated by testing a set of compounds recommended by the European Centre for the Validation of Alternative Methods (ECVAM), among which are in vivo genotoxins and non-genotoxins. The CHO-k1 cell line showed a high sensitivity (percentage of genotoxic compounds that gave a positive result: 80%; 16/20) and specificity (percentage of non-genotoxic compounds that came out negative: 88%; 37/42). Although the sensitivity of the HepG2 cell line was lower (60%; 12/20), the specificity was high (88%; 37/42). These results were confirmed by testing an additional series of 16 genotoxic compounds. For both the CHO-k1 and the HepG2 cell line it was possible to size-classify micronuclei, enabling distinguishing aneugens from clastogens. It is concluded that two high-throughput micronucleus assays were developed that can detect genotoxic potential and allow differentiation between clastogens and aneugens. The performance scores of the CHO-k1 and HepG2 cell lines for in vivo genotoxicity were high. Application of these assays in the early discovery phase of drug development may prove to be a useful strategy to assess genotoxic potential at an early stage.
authors.affiliation: Toxicology and Drug Disposition, Merck Sharpe & Dohme, PO Box 20, 5342 CC Oss, The Netherlands. westeriw@gmail.com
authors.forename: Walter M A
authors.initials: WM
authors.lastname: Westerink
authors.forename: Tom J J
authors.initials: TJ
authors.lastname: Schirris
authors.forename: G Jean
authors.initials: GJ
authors.lastname: Horbach
authors.forename: Willem G E J
authors.initials: WG
authors.lastname: Schoonen
mesh_terms: D024422:Aneugens; D000818:Animals; D001711:Biotransformation; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D056945:Hep G2 Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D015162:Micronucleus Tests / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D024422:Aneugens