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Cellular and Molecular Effects of Prolonged Low-Level Sodium Arsenite Exposure on Human Hepatic HepaRG Cells.

ABSTRACT: Inorganic arsenic is a human carcinogen associated with several types of cancers, including liver cancer. Inorganic arsenic has been postulated to target stem cells, causing their oncogenic transformation. This is proposed to be one of the key events in arsenic-associated carcinogenesis; however, the underlying mechanisms for this process remain largely unknown. To address this question, human hepatic HepaRG cells, at progenitor and differentiated states, were continuously treated with a noncytotoxic concentration of 1 ?M sodium arsenite (NaAsO2). The HepaRG cells demonstrated active intracellular arsenite metabolism that shared important characteristic with primary human hepatocytes. Treatment of proliferating progenitor-like HepaRG cells with NaAsO2 inhibited their differentiation into mature hepatocyte-like cells, up-regulated genes involved in cell growth, proliferation, and survival, and down-regulated genes involved in cell death. In contrast, treatment of differentiated hepatocyte-like HepaRG cells with NaAsO2 resulted in enhanced cell death of mature hepatocyte-like cells, overexpression of cell death-related genes, and down-regulation of genes in the cell proliferation pathway, while biliary-like cells remained largely unaffected. Mechanistically, the cytotoxic effect of arsenic on mature hepatocyte-like HepaRG cells may be attributed to arsenic-induced dysregulation of cellular iron metabolism. The inhibitory effect of NaAsO2 on the differentiation of progenitor cells, the resistance of biliary-like cells to cell death, and the enhanced cell death of functional hepatocyte-like cells resulted in stem-cell activation. These effects favored the proliferation of liver progenitor cells that can serve as a source of initiation and driving force of arsenic-mediated liver carcinogenesis.

SUBMITTER: Dreval K

PROVIDER: S-EPMC5889046 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Cellular and Molecular Effects of Prolonged Low-Level Sodium Arsenite Exposure on Human Hepatic HepaRG Cells.

Dreval Kostiantyn K Tryndyak Volodymyr V Kindrat Iryna I Twaddle Nathan C NC Orisakwe Orish Ebere OE Mudalige Thilak K TK Beland Frederick A FA Doerge Daniel R DR Pogribny Igor P IP

Toxicological sciences : an official journal of the Society of Toxicology 20180401 2

Inorganic arsenic is a human carcinogen associated with several types of cancers, including liver cancer. Inorganic arsenic has been postulated to target stem cells, causing their oncogenic transformation. This is proposed to be one of the key events in arsenic-associated carcinogenesis; however, the underlying mechanisms for this process remain largely unknown. To address this question, human hepatic HepaRG cells, at progenitor and differentiated states, were continuously treated with a noncyto ...[more]

PMID: 29301061

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Project description:Inorganic arsenic, a ubiquitous environmental contaminant of food and drinking water, is a human carcinogen associated with lung, liver, prostate, renal, and bladder cancers. It has been postulated that inorganic arsenic targets stem cells or partially differentiated progenitor cells, causing their oncogenic transformation. This is proposed to be one of the key mechanisms in arsenic-associated carcinogenesis; however, the underlying mechanisms for this process remain largely unknown. To address this question, human liver HepaRG cells, at progenitor and differentiated states, were continuously treated with a non-cytotoxic concentration of 1 μM sodium arsenite (NaAsO2). Briefly, in Experiment 1, three days after the initial seeding, 1 μM NaAsO2 was added to the media and the cells were maintained in the NaAsO2-containing media for an additional 25 days. In Experiment 2, fourteen days after the initial seeding, 1 μM NaAsO2 was added to the media and the cells were maintained in the NaAsO2-containing media for an additional 14 days. In Experiment 1 and Experiment 2, control and NaAsO2-treated cells were harvested on the 28th day after the initial seeding. In Experiment 3, twenty-eight days after the initial seeding, the terminally-differentiated cells were treated continuously with 1 μM NaAsO2 for an additional 14 days and then harvested. Transcriptomic analysis of NaAsO2-treated progenitor-like HepaRG cells (Experiment 1 and Experiment 2) identified 743 and 639 differentially expressed genes, respectively, among which 343 genes were expressed in common. Pathway analysis of common differentially expressed genes demonstrated a substantial inhibition of cellular metabolic pathways, mainly lipid and xenobiotic metabolism, and cell death pathways. In contrast, cell proliferation, cell survival, and inflammation, were substantially activated. Treatment of differentiated HepaRG cells with NaAsO2 (Experiment 3) resulted in prominent gene expression changes, with a total of 1058 transcripts being significantly different from the control HepaRG cells. Pathway analysis of differentially expressed genes in NaAsO2-treated cells differentiated HepaRG cells showed activation of cellular death-associated pathways and inhibition of cell survival and cell proliferation.

Project description:Arsenic (As) exposure is a significant worldwide environmental health concern. Low dose, chronic arsenic exposure has been associated with higher risk of skin, lung, and bladder cancer, as well as cardiovascular disease and diabetes. While arsenic-induced biological changes play a role in disease pathology, little is known about the dynamic cellular changes due to arsenic exposure and withdrawal. In these studies, we seek to understand the molecular mechanisms behind the biological changes induced by chronic low doses of arsenic exposure. We used a comprehensive approach involving chromatin structural studies and mRNA microarray analyses to determine how chromatin structure and gene expression patterns change in response to chronic low dose arsenic exposure and its subsequent withdrawal. Our results show that cells exposed to low doses of sodium arsenite have distinct temporal and coordinated chromatin, gene expression and miRNA changes that are consistent with differentiation and activation of multiple biochemical pathways. Most of these temporal patterns in gene expression are reversed when arsenic was withdrawn. However, some of the gene expression patterns remained altered, plausibly as a result of an adaptive response by these cells. Additionally, these gene expression patterns correlated with changes in chromatin structure, further solidifying the role of chromatin structure in gene regulatory changes due to arsenite exposure. Lastly, we show that arsenite exposure influences gene regulation both at the transcription initiation as well as at the splicing level. Thus our results suggest that general patterns of alternative splicing, as well as expression of particular gene regulators, can be indicative of arsenite-induced cell transformation. A total of eight (8) samples with two biological replicates under four separate conditions: wild-type treated with deionized H2O for 36 days (NT); chronic low-dose arsenic exposure of 1 uM of sodium arsenite (iAs-T) for 36 days; chronic arsenic exposure of 1 uM of sodium arsenite for 26 days followed by removal of sodium arsenite for 10 days, measured at day 36 (iAs-Rev); and chronic arsenic exposure of 1 uM of sodium arsenite for 26 days, followed by removal of sodium arsenite exposure for 10 days, followed by 1 uM of chronic sodium arsenite exposure for 10 days (measured at day 46) (iAs-Rev-T).

Project description:Studying chemical disturbances during neural differentiation of mES cells has been established as an alternative in vitro testing approach for the identification of developmental toxicants. miRNAs represent a class of small regulatory RNA molecules, which bind to target mRNAs thereby repressing their translation. Many studies have shown an essential role of miRNAs in regulation of gene expression during development and ESC differentiation. Thus, neural differentiation of ESC in vitro allows investigating the role of miRNAs in chemical-mediated developmental toxicity. We analyzed the expression of miRNAs and transcriptomics changes during neural differentiation of mESC exposed to the developmental neurotoxicant sodium valproate (VPA). A total of 110 miRNAs and 377 mRNAs were identified differently expressed in neural differentiating mES cells under VPA treatment (300µM) compared to solvent control on day 16 of differentiation. Analysis of miRNA expression revealed that valproate switches the lineage specification from neural to myogenic differentiation (upregulation of muscle-enriched miRNAs mir-206, mir-133a and mir-10a and downregulation of neuro-specific miRNAs mir-124a, mir-128 and mir-137). The findings on the miRNA level could be confirmed on mRNA level (induction of expression of myogenic regulatory factors (MRFs) as well as muscle specific genes (Actc1, calponin, myosin light chain, asporin, decorin) and repression of genes involved in neurogenesis (Otx1 and 2, Zic3, 4, 5)) as well as morphologically by immunocytochemistry. The observed results were VPA specific and most probably due to inhibition of histone deacetylase (HDAC) activity of VPA for two reasons: (i) we did not observe any induction of muscle specific miRNAs in neural differentiating ES cells exposed to the unrelated developmental neurotoxicant sodium arsenite; (ii) expression of muscle specific mir-206 and muscle enriched mir-10a was similarly increased in cells exposed to a structurally different HDAC inhibitor, trichostatin A (TSA). Furthermore, using our in vitro cell system we could confirm an aberrant expression of known VPA target genes and genes involved in neural tube closure. We conclude that miRNA expression profiling is a suitable molecular endpoint for developmental neurotoxicity. Observed lineage shift into myogenesis, where miRNAs play a significant role, could be a major developmental neurotoxical mechanism of VPA. We used microarray approach to identify altered miRNA expression in neural differentiated mES cells exposed to two known developmental neurotxicants and epigenetic active substances, sodium valproate (VPA) and sodium arsenite (As)

Dataset Information

Cellular and Molecular Effects of Prolonged Low-Level Sodium Arsenite Exposure on Human Hepatic HepaRG Cells.

Publications

Cellular and Molecular Effects of Prolonged Low-Level Sodium Arsenite Exposure on Human Hepatic HepaRG Cells.

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