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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE123584 | GEO | 2018/12/11

REPOSITORIES: GEO

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Project description:Control of intracellular heme levels by extracellular scavenger proteins and intracellular heme oxygenases are essential functions during disease states with enhanced extracellular heme release. During severe hemolysis or rhabdomyolysis uncontrolled heme exposure can cause acute kidney injury and endothelial damage. The cytotoxic activity of heme has been primarily attributed to its pro-oxidative potential. However, the mechanisms of heme toxicity have never been systematically explored. Besides its redox reactivity, heme could also adversely alter cellular functions through its broad binding affinity to multiple non-hemoproteins. Such interactions may impair protein functions and support heme toxicity. In this study we mapped the gene expression profile of Hb triggered acute kidney injury in old blood transfused guinea pigs by serial analysis of gene expression (SAGE). Additionally, the toxic heme response of mouse embryo fibroblasts was systematically characterized on the gene and protein expression levels by gene array experiments and quantitative mass-spectrometry of stable isotope labeled cells. In all these studies, in addition to oxidative stress signals, the most significant signals were reproducibly found for biologic networks related to altered protein degradation, which ultimately triggers the response to unfolded proteins and apoptosis. These screening data could be mechanistically explained by heme-proteasome interactions and a proteasome inhibitor activity of heme. Proteasome inhibition drastically reduced the threshold of cellular toxicity during heme exposure. We therefore propose a novel model of heme toxicity whereby proteasome inhibition by the porphyrin fuels a vicious cycle of oxidative protein modification, accumulation of damaged proteins, cell damage and apoptosis. A two color common reference design was chosen with 2-4 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled). To compensate for dye bias control arrays with competitively hybridized Cy3- and Cy5-labeled non-treated reference samples were used. The latter allowed for a very robust statistical analysis with pair-wise comparison of treatment array replicates versus the corresponding control array replicates.

Project description:Control of intracellular heme levels by extracellular scavenger proteins and intracellular heme oxygenases are essential functions during disease states with enhanced extracellular heme release. During severe hemolysis or rhabdomyolysis uncontrolled heme exposure can cause acute kidney injury and endothelial damage. The cytotoxic activity of heme has been primarily attributed to its pro-oxidative potential. However, the mechanisms of heme toxicity have never been systematically explored. Besides its redox reactivity, heme could also adversely alter cellular functions through its broad binding affinity to multiple non-hemoproteins. Such interactions may impair protein functions and support heme toxicity. In this study we mapped the gene expression profile of Hb triggered acute kidney injury in old blood transfused guinea pigs by serial analysis of gene expression (SAGE). Additionally, the toxic heme response of mouse embryo fibroblasts was systematically characterized on the gene and protein expression levels by gene array experiments and quantitative mass-spectrometry of stable isotope labeled cells. In all these studies, in addition to oxidative stress signals, the most significant signals were reproducibly found for biologic networks related to altered protein degradation, which ultimately triggers the response to unfolded proteins and apoptosis. These screening data could be mechanistically explained by heme-proteasome interactions and a proteasome inhibitor activity of heme. Proteasome inhibition drastically reduced the threshold of cellular toxicity during heme exposure. We therefore propose a novel model of heme toxicity whereby proteasome inhibition by the porphyrin fuels a vicious cycle of oxidative protein modification, accumulation of damaged proteins, cell damage and apoptosis. A two color common reference design was chosen with 2-8 independent biological replicates of each condition. Each experimental sample (Cy5 labeled) was hybridized against a non-treated reference sample (Cy3 labeled). To compensate for dye bias control arrays with competitively hybridized Cy3- and Cy5-labeled non-treated reference samples were used. The latter allowed for a very robust statistical analysis with pair-wise comparison of treatment array replicates versus the corresponding control array replicates.

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

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