Biomarkers of DNA Damage Response Improves In Vitro Micronucleus Assays by Revealing Genotoxic Mode of Action and Reducing the Occurrence of Irrelevant Positive Results.

We have previously described two flow cytometry-based in vitro genotoxicity tests: micronucleus (MN) scoring (MicroFlow ®) and a multiplexed DNA damage response biomarker assay (MultiFlow ®). Here, we describe a strategy for combining the assays in order to efficiently supplement MN analyses with a panel of biomarkers that comment on cytotoxicity (i.e., relative nuclei count, relative increased nuclei count, cleaved PARP-positive chromatin, and ethidium monoazide-positive chromatin) and genotoxic mode of action (i.e., γH2AX, phospho-histone H3, p53 activation, and polyploidy). For these experiments, human TK6 cells were exposed to each of 32 well-studied reference chemicals in 96-well plates for 24 continuous hr. The test chemicals were evaluated over a range of concentrations in the presence and absence of a rat liver S9-based metabolic activation system. MultiFlow assay data were acquired at 4 and 24 hr, and MN were scored at 24 hr. Testing 32 chemicals in two metabolic activation arms translated into 64 a priori calls: 42 genotoxicants and 22 non-genotoxicants. The MN assay showed high sensitivity and moderate specificity (90% and 68%, respectively). When a genotoxic call required significant MN and MultiFlow responses, specificity increased to 95% without adversely affecting sensitivity. The dose response data were analyzed with PROAST Benchmark Dose (BMD) software in order to calculate potency metrics for each endpoint, and ToxPi software was used to synthesize the resulting lower and upper bound 90% confidence intervals into visual profiles. The BMD/ToxPi combination was found to represent a powerful strategy for synthesizing multiple BMD confidence intervals, as the software output provided MoA information as well as insights into genotoxic potency.