Transcriptional Insights into Liver Toxicity from Mixture Effects of Pesticides and Natural Toxins: A case study of Propyzamide and Microcystins.

Humans are simultaneously exposed to multiple substances through diet and environmental sources, yet rigorously assessing the health risks of these combined exposures is challenging. New Approach Methodologies (NAMs) provide supporting tools, such as transcriptional analysis of cell models. Among relevant contaminants, pesticides and natural toxins, such as microcystins (MCs), may be of concern due to their potential co-occurrence in food. We investigated binary mixtures of two MCs (MC-LA, MC-LR) with the hepatotoxic pesticide propyzamide (PRO) using gene expression analysis in differentiated HepaRG cells. Marker genes for activity of the nuclear receptors AhR, CAR, PXR and NRF2 were analysed to model mixture effects. RT2 Profiler PCR Arrays and Ingenuity Pathway Analysis (IPA) were used to evaluate gene expression profiles and liver toxicity-related pathways. Individual MCs down-regulated the marker genes (i.e., CYP1A2, CYP2B6, CYP3A4, GPX2). IPA linked transcriptional patterns of MC-LR to cholestasis and hepatocarcinogenesis. In contrast, PRO induced the same nuclear receptors and affected liver-enriched pathways linked to inflammation, cholestasis, carcinogenesis, and oxidative stress. Based on marker genes, mixtures of MC congeners showed dose addition, whereas mixtures of MCs with PRO exhibited a functional antagonism, likely reflecting opposing effects on nuclear receptor signaling. Despite the antagonism on nuclear receptor level, pathway analysis indicated consistent or enhanced associations with liver toxicity endpoints including cholestasis, inflammation and hepatocellular carcinoma. Our findings indicate that co-exposure to pesticides and MCs may cause complex interactions including antagonism and helps to gain mechanistic knowledge on mixture toxicity.