Biomarkers of DNA damage response improve in vitro micronucleus assays by revealing genotoxic mode of action and reducing the occurrence of irrelevant positive results

Abstract

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 (MoA; 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 hours. 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 h, and micronuclei were scored at 24 h. 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 analysed with PROAST Benchmark Dose (BMD) software in order to calculate potency metrics for each endpoint, and ToxPi software was used to synthesise the resulting lower and upper bound 90% confidence intervals into visual profiles. The BMD/ToxPi combination was found to represent a powerful strategy for synthesising multiple BMD confidence intervals, as the software output provided MoA information as well as insights into genotoxic potency.

Application of a new approach methodology (NAM)-based strategy for genotoxicity assessment of data-poor compounds

“…A positive call was indicated by a 2.50-fold increase in %MN and a statistically significant ( p < 0.05) %MN increase relative to solvent controls in at least one non-cytotoxic concentration ( Avlasevich et al, 2021 ). Statistical significance was determined using a Poisson regression with a Holm-Sidak multiple correction procedure.…”

“…To model the continuous concentration-response data, a single 5-parameter exponential model (y = a*[c (1−exp(−x/b)^d) ]) was applied ( White et al, 2020 ). For the MicroFlow ® assay, the % induction of MN relative to test chemical concentration was modeled; a BMR of 1.0 (i.e., 100% increase in response relative to control) was applied ( Long et al, 2018 ; White et al, 2020 ; Avlasevich et al, 2021 ). For the MultiFlow assay, the fold change in response for each biomarker (e.g., 4H and 24H p53; 4H and 24H ɣH2AX) relative to test chemical concentration was modeled.…”

“…For the MultiFlow assay, the fold change in response for each biomarker (e.g., 4H and 24H p53; 4H and 24H ɣH2AX) relative to test chemical concentration was modeled. A BMR of 0.5 (i.e., 50% increase) was applied ( Avlasevich et al, 2021 ). The lowest BMC of the MultiFlow biomarkers was selected as the overall BMC.…”

“…Published case studies have explored the integration of the aforementioned assays to assess the genotoxicity of prototypical compounds with promising results ( Buick et al, 2020 ; Smart et al, 2020 ; Avlasevich et al, 2021 ). Buick et al (2020) applied the TGx-DDI biomarker in parallel with the MicroFlow ® assay in HepaRG™ cells ( Buick et al, 2020 ).…”

“… Buick et al (2020) applied the TGx-DDI biomarker in parallel with the MicroFlow ® assay in HepaRG™ cells ( Buick et al, 2020 ). Avlasevich et al (2021) evaluated the performance of the in vitro MicroFlow ® and MultiFlow ® genotoxicity endpoints ( Avlasevich et al, 2021 ). These case studies demonstrate that integrating several in vitro genotoxicity assays provides a more robust and accurate hazard assessment, with a limited number of irrelevant positives.…”