The development of RAD51C, Cystatin A, p53 and Nrf2 luciferase-reporter assays in metabolically competent HepG2 cells for the assessment of mechanism-based genotoxicity and of oxidative stress in the early research phase of drug development

Abstract

Four different mechanism-based high-throughput luciferase-reporter assays were developed in human HepG2 cells, which contain phase I and II metabolic activity and a functionally active p53 protein. The promoter regions of RAD51C and Cystatin A, as well as the responsive element of the p53 protein, were selected for the generation of the genotoxicity reporter assays. Moreover, a luciferase-based reporter assay was generated that measures the activation of the Nrf2 oxidative stress pathway. Validation with respect to the ECVAM compound list [D. Kirkland, P. Kasper, L. Muller, R. Corvi, G. Speit, Recommended lists of genotoxic and non-genotoxic chemicals for assessment of the performance of new or improved genotoxicity tests: a follow-up to an ECVAM workshop, Mutat. Res. 653 (2008) 99-108] resulted in an overall sensitivity of the HepG2 genotoxicity reporter assays for genotoxicity of 85% (17/20). The specificity and predictivity were high with 81% (34/42) and 82% (51/62), respectively. Various compounds had a positive score although metabolic activation was needed. The HepG2 reporter data were also compared with the available data on bacterial mutagenicity (Ames test), in vitro clastogenicity and in vivo clastogenicity for an additional set of 192 compounds. The predictivity for mutagenicity results was 74% (sensitivity, 61%, 30/49; specificity, 80%, 77/96) and for in vitro clastogenicity 59% (sensitivity, 45%, 35/78; specificity 83%, 38/46). The correlation between results from the HepG2 genotoxicity reporter assays and in vivo clastogenicity was much higher with 77% (sensitivity, 74%, 28/38; specificity 81%, 26/32). Results from the Nrf2 reporter assay showed that a large number of genotoxic compounds activated the Nrf2 oxidative stress pathway. In conclusion, four high-throughput mechanism-based reporter assays in the HepG2 cell line were developed, which can be applied for screening in the early research phase of drug development. The use of these assays in combination with the previously validated Vitotox and RadarScreen assays will certainly reduce the attrition rate due to genotoxicity in the developmental phase of drug development.