Development and validation of a high-content screening in vitro micronucleus assay in CHO-k1 and HepG2 cells

• Published in: Mutation research. Genetic toxicology and environmental mutagenesis 2011-09, Vol.724 (1-2), p.7-21
• Main Authors: Westerink, Walter M.A., Schirris, Tom J.J., Horbach, G. Jean, Schoonen, Willem G.E.J.
• Format: Article
• Language: English
• Subjects: Automated in vitro micronucleus assay; Biological and medical sciences; Cells; CHO-k1; DNA repair; Enzymes; Fundamental and applied biological sciences. Psychology; Genetics of eukaryotes. Biological and molecular evolution; Genotoxicity; HepG2; High throughput screening; Medical sciences; Medical screening; Rodents; Toxicology
• ISSN: 1383-5718; 1879-3592
• DOI: 10.1016/j.mrgentox.2011.05.007

► In drug development assays for the early detection of genotoxicity are needed. ► Two high content screening (HCS) micronucleus assays were developed and validated. ► Both HCS assays in HepG2 cells and CHO-k1 cells were able to detect genotoxic potential. ► The assays gave a low number of false-positive results. ► The assays allow differentiation into clastogens and aneugens. 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.