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Global transcriptomic profiling of microcystin-LR or -RR treated hepatocytes (HepaRG)

ABSTRACT: Microcystins (MC) are toxins found in harmful algal blooms. The canonical MC mode of action (MOA) is the inhibition of protein phosphatases, but complete characterization of its toxicity pathway has yet to be identified. The existence of 100s of MC structural congeners further complicates risk estimates. This work employed RNA-seq to provide an unbiased and comprehensive characterization of cellular targets and impacted cellular processes of hepatocytes exposed to either MC-LR or MC-RR congeners. The human hepatocyte cell line, HepaRG, was treated with three concentrations of MC-LR or RR (1000, 100, 10 g/L) for two hours. Significant reduction in cell survival was observed in the LR1000 and LR100 treatments. RNA-seq was performed on the control groups and all concentrations of MC-LR and -RR and differentially expressed genes (DEGs) were identified. Genes and pathways associated with oxidative and endoplasmic reticulum stress, and the unfolded protein response (UPR) were highly enriched by both congeners. Enrichment of inflammatory pathways was also evident. In the cytotoxic LR1000 treatment, apoptotic pathways and related genes were identified, suggesting cells pursue both inflammation and apoptosis simultaneously and that cellular fate may be determined by the activation kinetics of jun N-terminal kinase (JNK). We present a model of MC toxicity where MCs cause the production of ROS and oxidative stress leading to ER stress and activation of the UPR. Under non-cytotoxic conditions this leads to an inflammatory and pro-survival cellular response mediated through up-regulation and activation of NF- and its inhibition of JNK. With acute MC exposure, increased ER stress results in the cellular program switching to favor apoptosis by inhibiting NF- resulting in sustained JNK activation and through the activation of C/EBP homologous protein (CHOP) via the protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm of the UPR

ORGANISM(S): Homo sapiens

PROVIDER: GSE147999 | GEO | 2021/03/09

REPOSITORIES: GEO

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Project description:Microcystins (MCs) are cyclic hepatotoxins produced worldwide by various species of cyanobacteria. Their structure includes two variable amino acids (AAs) and most of the studies focused on the most toxic variant: the microcystin LR (MC-LR). However, more than 80 MC variants have been described to date. Despite ingestion being the major pathway of human exposure, few in vivo studies have demonstrated macroscopic effects on the gastro-intestinal tract, but no data are available on the affected pathways by several variants on intestinal cells. Here, using a non-selective method, we investigated for the first time the effect of MC-RR and MC-LR on the human intestinal cell line Caco-2 and compared their response at the pangenomic scale. The cells were incubated for 4 hrs or 24 hrs with the same range of sub-lethal concentrations of MC-RR or MC-LR. Low effects were observed for both variants after a short-term exposure. On the contrary, dose-dependent modulations of the genes transcription levels were noticed with MC-RR and MC-LR after 24 hrs. Furthermore, the genomic profiles induced by both variants were similar suggesting a common toxicity mechanism but with higher modulation following MC-LR than MC-RR exposure. However, the functional annotation revealed major differences between the variants effects. Indeed, the well-known MC-LR affected mainly two pathways, the oxidative stress response and the cell cycle regulation, which did not elicit significant alteration following MC-RR exposure. This work is the first comparative description of the MC-LR and MC-RR effects on a human intestinal cell model. It allowed us to suggest differences in the mechanism of toxicity for MC-RR and MC-LR. These results illustrate that the toxicity of MC variants remains a key point for risk assessment. Differentiated Caco-2 cells were exposed to microcystins in free FCS culture medium for either 4 or 24 hours. Sub-lethal concentrations of 10, 50 and 100 M-BM-5M of MC-LR or MC-RR were chosen for 4 hours, while 1, 5 and 10 M-BM-5M were selected for 24 hours. For each condition (including the controls), the solvent concentration was fixed to 2% EtOH for MC-LR and 1.5% of 80% MeOH for MC-RR. Four to five culture replicates per condition were done.

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Global transcriptomic profiling of microcystin-LR or -RR treated hepatocytes (HepaRG)

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