Application of Biological Modifiers to a Multiplexed, Human Cell-Based DNA Damage Assay Provides Mechanistic Information on Genotoxicity and Molecular Targets.

Abstract

This laboratory has reported that the combined use of In Vitro MicroFlow and MultiFlow assays provides information regarding chemicals' genotoxic mode of action (MoA). In an effort to go beyond MoA assessments, we incorporated a panel of biological response modifiers that elicit specific effects on the assays' biomarker response profiles. This was done to pursue our hypothesis that such perturbation signatures would reveal information on clastogenic mechanisms and molecular targets. For this proof-of-concept study, we exposed TK6 cells to 20 known clastogens. Cells were exposed in 96-well plates in the presence and absence of each of four modifying agents at one optimized concentration: talazoparib (PARP inhibitor), MK-8776 (CHK1 inhibitor), AZD-7648 (DNA-PK inhibitor), or a cocktail of reactive oxygen species scavengers. In parallel, cells were also exposed to each of the test chemicals for 4 h, at which time cells were washed and allowed to recover for an additional 20 h. For each of these treatment conditions, sample processing and flow cytometric analyses were performed using standard In Vitro MicroFlow and MultiFlow procedures to measure micronuclei, γH2AX, phosphohistone-H3 (p-H3), p53 activation, and relative nuclei counts. The resulting biomarker response data were processed with PROAST benchmark dose (BMD) software, with modifying agent as a covariate. Unsupervised hierarchical clustering of the collective potency metrics for various combinations of biomarkers showed that clastogens with similar genotoxic mechanisms grouped together. Overall, this study shows that in combination with biological response modifiers, MultiFlow and In Vitro MicroFlow biomarkers can provide mechanistic insights into chemical-induced genotoxicity.