Adverse outcome pathway-driven identification of rat liver tumorigens in short-term assays

Chemicals induce liver cancer in rodents through well characterized adverse outcome pathways (AOPs). We hypothesized that measurement of molecular initiating events (MIEs) and downstream key events (KEs) in liver cancer AOPs in short-term assays will allow early identification of chemicals and their associated doses that are likely to be tumorigenic in the liver in two-year bioassays. We tested this hypothesis using the rat in vivo TG-GATES study data to measure MIEs (genotoxicity, cytotoxicity, AhR, CAR, ER, PPARα) and associated KEs (oxidative stress, cell proliferation, liver to body weights) across 77 chemicals that could be linked to doses with previously established effects on rat liver tumor induction. Gene expression biomarkers for MIEs generally considered to be rodent specific and human irrelevant (CAR, PPARα) and for MIEs that would be considered of greater risk at human relevant exposures (ER, AhR) were built using microarray comparisons from the livers of rats treated with prototypical activators of the receptors. The genotoxicity biomarker, also a potentially human relevant MIE, was comprised of 7 p53-responsive genes known to be induced upon DNA damage. The ability of the biomarkers to accurately predict MIE activation ranged from 91% to 98%. The Toxicological Priority Index (ToxPi) was used to rank chemicals based on their ability to activate MIEs/KEs. Chemicals administered at tumorigenic doses clearly gave the highest ranked scores. Our AOP-directed approach could be used in short term assays to identify chemicals and their doses that would be predicted to cause liver tumors in rats.