Benchmark Dose Analysis of DNA Damage Biomarker Responses Provides Compound Potency and Adverse Outcome Pathway Information for the Topoisomerase II Inhibitor Class of Compounds

Abstract

Genetic toxicology data have traditionally been utilized for hazard identification to provide a binary call for a compound's risk. Recent advances in the scientific field, especially with the development of high-throughput methods to quantify DNA damage, have influenced a change of approach in genotoxicity assessment. The in vitro MultiFlow ® DNA Damage Assay is one such method which multiplexes γH2AX, p53, phospho-histone H3 biomarkers into a single-flow cytometric analysis : Environ Mol Mutagen 57:546-558). This assay was used to study human TK6 cells exposed to each of eight topoisomerase II poisons for 4 and 24 hr. Using PROAST v65.5, the Benchmark Dose approach was applied to the resulting flow cytometric datasets. With "compound" serving as covariate, all eight compounds were combined into a single analysis, per time point and endpoint. The resulting 90% confidence intervals, plotted in Log scale, were considered as the potency rank for the eight compounds. The in vitro MultiFlow data showed a maximum confidence interval span of 1Log, which indicates data of good quality. Patterns observed in the compound potency rank were scrutinized by using the expert rule-based software program Derek Nexus, developed by Lhasa Limited. Compound sub-classification and structural alerts were considered contributory to the potencies observed for the topoisomerase II poisons studied herein. The Topo II poison Adverse Outcome Pathway was evaluated with MultiFlow endpoints serving as Key Events. The step-wise approach described herein can be considered as a foundation for risk assessment of compounds within a specific mode of action of interest. Environ. Mol. Mutagen. 61:396-407, 2020.

The use of benchmark dose uncertainty measurements for robust comparative potency analyses

“…Researchers have used BMD CIs of chemical classes of interest to plot compound genotoxic and/or carcinogenic potency in rank order (Hernández et al, 2011; Soeteman‐Hernández et al, 2015a; Soeteman‐Hernández et al, 2015b; Wills et al, 2016, 2017). Some prominent examples where BMD CIs were used in comparative genotoxicity potency analyses include studies by Allemang et al (2018) and Wheeldon et al (2020). Allemang et al (2018) performed BMD analyses to evaluate the relative genotoxic potency of 15 pyrrolizidine alkaloids (PAs) via in vitro micronuclei formation in HepaRG cells.…”

“…Allemang et al (2018) performed BMD analyses to evaluate the relative genotoxic potency of 15 pyrrolizidine alkaloids (PAs) via in vitro micronuclei formation in HepaRG cells. Wheeldon et al (2020) performed BMD analyses to evaluate the comparative genotoxic potency of 8 Topoisomerase II poisons studied in human lymphoblastoid TK6 cells using the MultiFlow DNA damage response assay. Both publications identified the utility of BMD CIs in compound comparative genotoxicity potency analyses to support read across and MoA determination for a limited number of compounds of interest.…”

Application of Benchmark Dose Analysis to in vitro Genotoxicity Data for Compound Risk Characterisation

“…In the case of Topo-II Poison mechanism of action, replication fork collapse is the plausible explanation for the increase in Bscl2-GFP expression observed in these experiments. The BMD confidence intervals plots reported in this chapter (sections 5.3.3 and 5.3.4.2) and published in Wheeldon et al (2020) were the first instances where Topo-II…”

“…The findings presented in this thesis have added valuable contributions to the literature with regards to the utility of the BMD approach to compound potency ranks from dose-response datasets from multiplexed in vitro genotoxicity biomarker responses. The additions to the literature include demonstrating that compound potency from multiplexed DNA damage biomarkers can inform potency in a standard battery endpoint such as the in vitro micronucleus (Dertinger et al, 2019); that statistically robust potency comparisons across different treatment conditions can be made using BMD confidence intervals from in vitro DNA damage response biomarkers (Wheeldon et al, 2021); and that BMD robust potency comparisons can be made for a compound class of interest with supporting mechanistic and compound structural information (Wheeldon et al, 2020). Some individuals have suggested the development of in vitro to in vivo extrapolation methods, where in vitro genotoxicity tests could replace in vivo alternatives (White et al, 2020); however, this remains a contentious topic due to pharmacodynamic and pharmacokinetic differences observed between in vitro and in vivo systems.…”

“…Firstly, DNA strand separation is required to overcome the negatively supercoiled nature of the DNA double helix. The linear movement of tracking enzymes such as helicases and polymerases causes compression of the double helix into a shorter region, leading to increasingly overwound sections in the DNA making it 7 The data presented in this results chapter was published in "Benchmark Dose Analysis of DNA Damage Biomarker Responses Provides Compound Potency and Adverse Outcome Pathway Information for the Topoisomerase II Inhibitor Class of Compounds" (Wheeldon et al, 2020) and some passages in this chapter have been quoted verbatim from the same article. A copy of the research article is available in Appendix 3.…”