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Adverse outcome pathways (AOPs) provide a framework to organize and weigh the evidence linking a toxicant’s initial interactions with molecules in the cell to adverse outcomes of regulatory concern. AOPs are constructed in modules that include key events (KEs) and key event relationships (KERs). Quantitative understanding of the KERs is critical for the development of predictive toxicological models. The objective of this project was to investigate the ability to define the quantitative associations of the KERs upstream, and contained in, an existing AOP (#296): “Oxidative DNA Damage Leading to Chromosomal Aberrations and Mutations”. The data supporting quantitative associations between these KERs was gathered through literature review and experimental methods. I first used systematic literature review tools to develop and apply a pragmatic and transparent method to search the literature for AOP evidence. A broad search, covering all of the KERs of interest, was initially conducted. This search, which retrieved more than 230 thousand articles, demonstrates the data-rich nature of the AOP. An artificial intelligence informed prioritization of the top 100 articles were then examined in detail. This approach identified 39 articles containing qualitative empirical support for the AOP, but limited quantitative evidence of the KERs. A second search was conducted to address the need for quantitative evidence as well as the lack of evidence for the KER between and increase in reactive oxygen species (ROS) and oxidative DNA damage. The second search retrieved 12 articles that could be used to define a quantitative relationship between cellular ROS and oxidative DNA damage. To begin to address gaps in quantitative understanding, I then conducted experiments in the laboratory to measure oxidative DNA damage, DNA strand breaks, chromosomal aberrations, and mutations in TK6 cells after exposure to a range of concentrations of 4-Nitroquinoline 1-oxide (4NQO: a prototype ROS producing agent). An increase in both oxidative DNA damage and DNA strand breaks was observed after 2, 4, and 6 h exposures with the high throughput comet assay (CometChip). An increase in the incidence of micronuclei was observed after a 24 h exposure to a low concentration of 4NQO, as measured with the flow cytometry micronucleus assay, while high cytotoxicity was found at higher concentrations. Lastly an increase in mutation frequency was observed with Duplex Sequencing, an error-corrected sequencing technology. Additionally, an increase in the proportion of C>A transversions was observed, consistent with the expected mutations following oxidative DNA lesions. Overall, my work contributes to the quantitative understanding of AOP #296 and this project serves as a key example of AOP-informed study design, highlighting notable challenges in characterizing quantitative relationships.

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