A Pathway Analysis, Connectivity Mapping and Transcriptional Benchmark Dosing Based Exploration of Oxidative Stress Mediated Genetic Toxicology Modes of Actions

While current genetic toxicology practices can detect downstream genotoxicity effects, such as gene mutation and double strand breaks, they are unable to detect the underlying Mode of Action (MoA) of a chemical or differentiate between direct and indirect acting genotoxicants without additional modification. The Adverse Outcome Pathway (AOP) framework is a useful tool to critically identify and evaluate MOAs and can enable subsequent quantitative dose response assessments of genotoxicity endpoints. The recently developed AOP, “Oxidative DNA damage leading to chromosomal aberrations and mutations” ( https://aopwiki.org/aops/296), pertains to one common genetic toxicology relevant MOA: oxidative stress. ROS are key to regulating many biological processes, however, when disrupted, an excess of ROS can eventually lead to DNA damage and double-strand breaks. Here, we look at 18 compounds with a complete or mixed oxidative stress MOA and use a combination of genomic tools such as Pathway analysis, Connectivity mapping and Transcriptional benchmark dose modeling to investigate the diversity of mechanisms that contribute to genotoxicity. TK6 cells were treated with the 18 compounds for 4 hours and the resulting genomic data was analyzed in correlation to the downstream genotoxic endpoint, micronucleus formation. We provide both a qualitative and quantitative analysis of the contribution of oxidative stress MoAs towards the overall genotoxicity outcome of each chemical. These methods have the potential to evolve into Next Generation Risk Assessment (NGRA) tools that can be used for determining the contribution of the oxidative stress MoA in a predictive toxicology setting. Human TK6 cells were treated with the 18 compounds for 4 hours in triplicate. The solvents (Water or DMSO) for each compound were also tested and used as controls.