Advancing the Adverse Outcome Pathway for PPARγ Inactivation Leading to Pulmonary Fibrosis Using Bradford-Hill Consideration and the Comparative Toxicogenomics Database

Pulmonary fibrosis is regulated by transforming growth factor-β (TGF-β) and peroxisome proliferator-activated receptor-gamma (PPARγ). An adverse outcome pathway (AOP) for PPARγ inactivation leading to pulmonary fibrosis has been previously developed. To advance the development of this AOP, the confidence of the overall AOP was assessed using the Bradford-Hill considerations as per the recommendations from the Organisation for Economic Co-operation and Development (OECD) Users' Handbook. Overall, the essentiality of key events (KEs) and the biological plausibility of key event relationships (KERs) were rated high. In contrast, the empirical support of KERs was found to be moderate. To experimentally evaluate the KERs from the molecular initiating event (MIE) and KE1, PPARγ (MIE) and TGF-β (KE1) inhibitors were used to examine the effects of downstream events following inhibition of their upstream events. PPARγ inhibition (MIE) led to TGF-β activation (KE1), upregulation in vimentin expression (KE3), and an increase in the fibronectin level (KE4). Similarly, activated TGF-β (KE1) led to an increase in vimentin (KE3) and fibronectin expression (KE4). In the database analysis using the Comparative Toxicogenomics Database, 31 genes related to each KE were found to be highly correlated with pulmonary fibrosis, and the top 21 potential stressors were suggested. The AOP for pulmonary fibrosis evaluated in this study will be the basis for the screening of inhaled toxic substances in the environment.