Mechanistic investigation of the hepatotoxicity and potential carcinogenicity of microcystin-LR, -LA, and -RR in the HepaRG cell line model.

Microcystins (MCs) are cyanobacterial toxins found in water and food and are suspected to pose significant health risks, particularly to the liver. To gain insight into the mechanisms of hepatotoxicity and the potential carcinogenicity of MCs, this study investigated the effects of MC-LR, -LA, and -RR (congeners differing in cellular uptake and detoxification efficiency) on PP2A activity, cell proliferation, and gene expression profiling focused on toxicity pathways and carcinogenesis in HepaRG cells. The three MCs similarly inhibited PP2A activity; however, only MC-LR and -LA significantly increased cell proliferation. Gene expression profiles (mRNA) revealed strong, similar treatment effects induced by MC-LR and -LA (R2 = 0.66), whereas -RR affected fewer genes, showing weak correlations with the other two congeners (-LRx-RR - R2 = 0.31; -LAx-RR - R2 = 0.06). Moreover, MC-LR and -LA, but not -RR, affected several liver-enriched toxicity pathways, which correlate most closely to hepatocellular/liver carcinoma, cholestasis, and fibrosis. All congeners altered carcinogenesis-related miRNA expression, but no consistent pattern was observed among them. Overall, the present findings reinforce the congener-specific toxicity of MCs, likely driven by differences in toxicokinetics among congeners. Furthermore, the findings indicate that MC-induced liver effects also involve PP2A-independent mechanisms, as MC-LR/-LA and -RR elicited markedly different biological responses despite comparable PP2A inhibition. Finally, the study demonstrates the applicability of the NAM-based approach for evaluating liver effects (hepatocytes) in the MC family and, as structurally different congeners were investigated, provides data that could be used for read-across and grouping.