Sensitive DsRed fluorescence-based reporter cell systems for genotoxicity and oxidative stress assessment

Abstract

Various in vitro test systems have been developed for genotoxic risk assessment in early drug development. However, these genotoxicity tests often show limited specificity, and provide limited insights into the mode of toxicity of the tested compounds. To identify genes that could serve as specific biomarkers for genotoxicity or oxidative stress, we exposed mouse embryonic stem (ES) cells to various genotoxic and oxidative stress-inducing compounds and performed genome-wide expression profiling. Differentially expressed genes were classified based on the fold-change of expression and their specificity for either genotoxic or oxidative stress. Promoter regions of four selected genes (Ephx1, Btg2, Cbr3 and Perp) were fused to a DsRed fluorescent reporter gene and stably integrated in mouse ES cells. Established stable reporter cell lines displayed significant induction of DsRed expression upon exposure to different classes of genotoxic and oxidative stress-inducing compounds. In contrast, exposure to non-genotoxic carcinogenic compounds did not induce DsRed expression even at cytotoxic doses. Expression of the Cbr3-DsRed reporter was more responsive to compounds that induce oxidative stress while the other three DsRed reporters reacted more specific to direct-acting genotoxic agents. Therefore, the differential response of the Btg2- and Cbr3-DsRed reporters can serve as indicator for the main action mechanism of genotoxic and oxidative stress-inducing compounds. In addition, we provide evidence that inhibition of DNA replication results in preferential activation of the Btg2-DsRed genotoxicity reporter. In conclusion, we have generated sensitive mouse ES cell reporter systems that allow detection of genotoxic and oxidative stress-inducing properties of chemical compounds and can be used in high-throughput assays.