Comparison of the Genotoxic Potential of Three Microcystin Variants (-LR, -RR, and -LF) by High Content Analysis and Micronucleus Assay in a Human Hepatic Cell Line

Microcystins (MCs) are cyclic hepatotoxins produced by various species of cyanobacteria that inhibit serine/threonine protein phosphatases. Their structure includes two variable amino acids, leading to 329 variants. MC-LR was considered the most toxic variant and has been extensively investigated, other variants, differing in hydrophobicity, could possess unique behavior and toxicity. The aim of the current study was to compare the toxicity and genotoxicity of 3 MC variants (MC-LR, -RR and -LF), in human liver HepaRG cells. Both undifferentiated and differentiated HepaRG cells were treated for 24 h with concentrations up to 50 μM for MC-LR and -RR and up to 6.25 μM for MC-LF. Using a high content analysis-based approach, cell count and apoptosis (active caspase-3) were quantified, in addition to three markers of DNA damage: γH2AX, p53 phospho S15 and ATM phospho S1981. A significant increase in all these markers was observed in differentiated HepaRG cells, correlating with the onset of cytotoxicity. The potency of the three variants to induce micronuclei (MN) was further assessed in differentiated HepaRG cells after 24 h of exposure to concentrations up to 25 μM, revealing a concentration-dependent increase with all variants. The responses in HepaRG cells treated with MC-LF were consistently observed at lower concentrations compared to MC-LR and MC-RR across all endpoints investigated, including micronucleus formation and DNA damage markers. An increase of pH 3 staining was also observed with the 3 variants, suggesting an aneugenic effect. The higher in vitro genotoxic potency of MC-LF compared to MC-LR needs to be confirmed in vivo.