Identification of estrogenic and DNA damaging chemicals using a combined morphological and genotoxic biomarker assay

One challenge for current chemical risk assessment is the integration of different in vitro based new approach methodologies (NAMs) to establish a concept for Next-Generation Risk Assessment (NGRA). It is particularly difficult to assess activities of chemicals associated with carcinogenicity that is not mediated by direct effects on genome integrity, known as non-genotoxic carcinogens (NGTxC). NGTxC activities may be induced through various mechanisms and cellular pathways. Thus, it is important to combine readouts, reflecting distinct mechanisms in a single test system, to limit the total number of tests needed. In this study, the previously described E-Morph screening assay (ESA), based on human MCF-7 breast cancer cells, for detection of ER-agonistic chemicals, was expanded for a simultaneous detection of DNA damage, to facilitate the distinction of genotoxic and non-genotoxic nuclear-receptor mediated carcinogens. For assessing the assay performance, we assembled a chemical library including chemicals with genotoxic or NGTxC mode of action and monitor their ER-agonistic and genotoxic activity after application of six different concentrations starting from 100 µM. The same set of chemicals was tested in a human liver cell line (HepG2) with the γH2AX/pH3 genotoxicity method. We observed a strong correlation between the two cell lines for the detection of genotoxic effects. Additionally, we observed a strong correlation between γH2AX induction and ER-agonistic activity in MCF-7 cells and aneugenicity in HepG2 cells. Consequently, the extended ESA is a promising new NAM that might play an important role in future NGRA for identifying and characterizing carcinogenic hazards.