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Epigenetic changes in p21 expression in renal cells after exposure to bromate.

ABSTRACT: This study tested the hypothesis that bromate (KBrO3)-induced renal cell death is mediated by epigenetic mechanisms. Global DNA methylation, as assessed by 5-methylcytosine staining, was not changed in normal rat kidney cells treated with acute cytotoxic doses of KBrO3 (100 and 200 ppm), as compared with controls. However, KBrO3 treatment did increase p38, p53 and histone 2AX (H2AX) phosphorylation, and p21 expression. Treatment of cells with inhibitors of DNA methyltransferase (5-azacytidine or 5-Aza) and histone deacetylase (trichostatin A or TSA) in addition to KBrO3 increased cytotoxicity, as compared with cells exposed to KBrO3 alone. 5-Aza and TSA co-treatment did not alter p38 or p53 phosphorylation, but slightly decreased H2AX phosphorylation and significantly decreased p21 expression. We also assessed epigenetic changes in cells treated under sub-chronic conditions with environmentally relevant concentrations of KBrO3. Under these conditions (0-10ppm KBrO3 for up to 18 days), we detected no increases in cell death or DNA damage. In contrast, slight alterations were detected in the phosphorylation of H2AX, p38, and p53. Sub-chronic low-dose KBrO3 treatment also induced a biphasic response in p21 expression, with lower concentrations increasing expression, but higher concentrations decreasing expression. Methylation-specific PCR demonstrated that sub-chronic KBrO3 treatment altered the methylation of cytosine bases in the p21 gene, as compared with controls, correlating to alterations in p21 protein expression. Collectively, these data show the novel finding that KBrO3-induced renal cell death is altered by inhibitors of epigenetic modifying enzymes and that KBrO3 itself induces epigenetic changes in the p21 gene.

SUBMITTER: Scholpa NE

PROVIDER: S-EPMC4271043 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Project description:BackgroundPotassium bromate (KBrO3), used in both the food and cosmetics industry, and a drinking water disinfection by-product, is a nephrotoxic compound and rodent carcinogen. To gain insight into the carcinogenic mechanism of action and provide possible biomarkers of KBrO3 exposure, the gene expression in kidneys from chronically exposed male F344 rats was investigated.MethodsMale F344 rats were exposed to KBrO3 in drinking water for 52 and 100 wk. Kidneys were removed, frozen, and stored at -80°C, then used for Affymetrix microarray analysis. Gene expression patterns were examined using a non-carcinogenic (20 ppm) and carcinogenic dose (400 ppm) at 52 wk, and compared to 100 wk high dose (400 ppm) and adenoma gene expression.ResultsStatistical analysis revealed 144, 224, 43, and 994 genes out of 15866 from the 52 wk low, 52 wk high, 100 wk high, and adenomas respectively, were differentially expressed when compared to control kidneys. Gene ontology classification of the 52 wk high dose showed alterations of gene transcripts involved in oxidative stress, lipid metabolism, kidney function/ion transport, and cellular function. In a comparison of kidney development gene expression, alterations were seen in the adenomas but not in the 52 wk bromate-treated kidneys. However, the normal kidney from the high dose group resembled the adenoma expression pattern with early kidney development genes being up-regulated and adult phase genes being down-regulated. Moreover, eight genes were identified which could serve as biomarkers of carcinogenic exposure to bromate. The most promising of these was Pendrin, or Slc26a4, a solute carrier of chloride and iodide active in the kidney, thyroid, and inner ear. All these tissues are targets of KBrO3 toxicity. Expression array results were verified with quantitative real-time rtPCR.ConclusionsThese data demonstrate that the 400 ppm carcinogenic dose of KBrO3 showed marked gene expression differences from the 20 ppm non-carcinogenic dose. Comparison of kidney development gene expression showed that the adenoma patterns were more characteristic of embryonic than adult kidneys, and that the normal kidney from the high dose group resembled the adenoma-like gene expression pattern. Taken together, the analysis from this study identifies potential biomarkers of exposure and illuminates a possible carcinogenic mode of action for KBrO3.

Project description:BackgroundDiabetic nephropathy is the leading cause of end stage renal disease. All three cell types of the glomerulus, podocytes, endothelial cells and mesangial cells, play important roles in diabetic nephropathy. In this report we used Meis1-GFP transgenic mice to purify mesangial cells from normal mice and from db/db mice, which suffer diabetic nephropathy. The purpose of the study is to better define the unique character of normal mesangial cells, and to characterize their pathogenic and protective responses during diabetic nephropathy.MethodsComprehensive gene expression states of the normal and diseased mesangial cells were defined with microarrays. By comparing the gene expression profiles of mesangial cells with those of multiple other renal cell types, including podocytes, endothelial cells and renal vesicles, it was possible to better define their exceptional nature, which includes smooth muscle, phagocytic and neuronal traits.ResultsThe complete set of mesangial cell expressed transcription factors, growth factors and receptors were identified. In addition, the analysis of the mesangial cells from diabetic nephropathy mice characterized their changes in gene expression. Molecular functions and biological processes specific to diseased mesangial cells were characterized, identifying genes involved in extracellular matrix, cell division, vasculogenesis, and growth factor modulation. Selected gene changes considered of particular importance to the disease process were validated and localized within the glomuerulus by immunostaining. For example, thrombospondin, a key mediator of TGF? signaling, was upregulated in the diabetic nephropathy mesangial cells, likely contributing to fibrosis. On the other hand the decorin gene was also upregulated, and expression of this gene has been strongly implicated in the reduction of TGF? induced fibrosis.ConclusionsThe results provide an important complement to previous studies examining mesangial cells grown in culture. The remarkable qualities of the mesangial cell are more fully defined in both the normal and diabetic nephropathy diseased state. New gene expression changes and biological pathways are discovered, yielding a deeper understanding of the diabetic nephropathy pathogenic process, and identifying candidate targets for the development of novel therapies.

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Epigenetic changes in p21 expression in renal cells after exposure to bromate.

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