Use of in vitro alternative methods in the study of emerging contaminants for identifying mechanisms of action in non-genotoxic carcinogenesis

Chemical carcinogenesis driven by non-genotoxic compounds is a challenging and dynamic research area. Unlike genotoxic carcinogens, non-genotoxic agents such as di-(2-ethylhexyl) phthalate (DEHP) and perfluorooctane sulfonic acid (PFOS) induce cancer via indirect mechanisms, often involving receptor-mediated pathways. This thesis investigated the carcinogenic potential of DEHP and PFOS in the BALB/c 3T3 A31-1-1 cell model using the Cell Transformation Assay (CTA) and explored their molecular mechanisms with a focus on PPARα activation, a pathway implicated in rodent tumorigenesis. DEHP was assessed through CTA coupled with transcriptomics to identify post-exposure molecular alterations, offering insights into its mechanism of action. The results showed that DEHP did not induce cellular transformation in vitro. Comparative analysis with other CTA studies highlighted variations in model responses, highlighting the critical importance of characterizing compound metabolism. PFOS, which is known for its receptor-mediated effects, was evaluated by co-treatment with GW6471, a PPARα antagonist. Although PFOS induced cellular transformation, the involvement of PPARα alone was insufficient to explain its carcinogenicity in the BALB c/ 3T3 A31-1-1 cell model, suggesting the participation of alternative pathways. Key findings from this study demonstrated that although both compounds can activate PPARα, they exhibit different mechanisms of action. This study underscores the complexity of non-genotoxic carcinogenesis, in which receptor-mediated events, metabolic alterations, and disruptions in signaling pathways interact to promote tumorigenesis. The integration of CTA with additional mechanistic approaches, such as transcriptomics for DEHP and the use of a PPARα inhibitor for PFOS, has allowed for deeper investigation into the mechanisms of action underlying the cellular effects observed. These findings promote the enhancement of CTA within Integrated Approaches to Testing and Assessment (IATA) for non-genotoxic compounds, emphasizing the need for refined in vitro models to better replicate human responses and improve chemical risk assessment for public health.