Identification of key pathways involved in the toxic response of the cyanobacterial toxin cylindrospermopsin on human hepatic HepaRG cells

Cylindrospermopsin (CYN), a hepatotoxin produced by several cyanobacterial species, has been implied in human intoxications following ingestion. Although some pathways involved in CYN toxicity have been reported, the whole process is certainly more complex. In order to identify the mechanisms related to CYN toxicity on liver, we studied the transcriptomic profile of HepaRG human hepatic cells exposed to a low concentration of CYN (0.8 µM for 24 hrs). The affected pathways were further confirmed through the expression of key genes by qPCR, and the investigation of toxicity markers by high content analysis (HCA) on a broader CYN concentration range. Also CYP450 activities and cell redox homeostasis were investigated after acute and repeated exposure to CYN. Our results revealed that CYN induced up-regulation of genes implied in RNA modification and maturation, but also down-regulation of genes involved in xenobiotic metabolism and cell cycle progress. There was cell cycle disturbance characterised by an accumulation of G1/S and G2/M cells, and an increase of phospho-H3 positive cells. The cell cycle arrest was linked to the induction of DNA damage pointed out by an increase of γH2AX positive cells, but also an accumulation of sub G1 cells indicating apoptosis not involving caspase 3. While GSH content decreased strongly after acute exposure to CYN, it increased after a repeated exposure reflecting an adaptive response of cell redox homeostasis when exposure to CYN was prolonged. However, our data suggested also that CYN induced a down-regulation of phase I and II metabolism gene products, and CYP450 activities were affected following both acute and repeated exposure to CYN. Our study indicated that repeated exposure of liver cells to low concentrations of CYN may affect their detoxification capacities. Modulation of gene expression in differentiated HepaRG cells was measured at 24 hours after exposure to doses of 0 and 0.8 µM of cylindrospermopsin. Six independent experiments were performed, and 12 samples (6 controls and 6 exposed to cylindrospermopsin) had an RNA quality indicator ≥ 9 and were used for the microarray experiments.