Effects of chronic exposure to arsenic and estrogen on epigenetic regulatory genes expression and epigenetic code in human prostate epithelial cells.

Chronic exposures to arsenic and estrogen are known risk factors for prostate cancer. Though the evidence suggests that exposure to arsenic or estrogens can disrupt normal DNA methylation patterns and histone modifications, the mechanisms by which these chemicals induce epigenetic changes are not fully understood. Moreover, the epigenetic effects of co-exposure to these two chemicals are not known. Therefore, the objective of this study was to evaluate the effects of chronic exposure to arsenic and estrogen, both alone and in combination, on the expression of epigenetic regulatory genes, their consequences on DNA methylation, and histone modifications. Human prostate epithelial cells, RWPE-1, chronically exposed to arsenic and estrogen alone and in combination were used for analysis of epigenetic regulatory genes expression, global DNA methylation changes, and histone modifications at protein level. The result of this study revealed that exposure to arsenic, estrogen, and their combination alters the expression of epigenetic regulatory genes and changes global DNA methylation and histone modification patterns in RWPE-1 cells. These changes were significantly greater in arsenic and estrogen combination treated group than individually treated group. The findings of this study will help explain the epigenetic mechanism of arsenic- and/or estrogen-induced prostate carcinogenesis

Representative RAPD fingerprints showing DNA methylation changes.

<p>Fingerprints generated by primers OPC07 (5′-GTCCCGACGA-3′) [upper panel], OPC17 (5′-TTCCCCCCAG-3′) [middle panel], OPC20 (5′-ACTTCGCCAC-3′) [lower panel] showing the effects of As and E2 treatment alone and in combination on the methylation status in RWPE-1 cells as revealed by loss/gain of amplification products. Genomic DNA isolated from chronically treated RWPE-1 cells was digested with methylation sensitive restriction enzymes (<i>MspI</i> and <i>HpaII</i>) and then used for RAPD amplifications as described in Materials and Methods section. The loss/gain of RAPD amplification products in the fingerprints from treated RWPE-1 cells are indicated by arrows. Primers used are indicated next to each fingerprint. PCR amplification failed in lane 7 of middle panel and lane 3 of lower panel.</p

Representative Western blots (3a) and their relative band intensity histograms (3b).

<p>Western blots showing the effect of As, E2 and their combination on the expression of MBD4 at protein level as well as the levels of phosphorylated and acetylated histone H3 [p-Ac-histone H3 (S 11/K 15)] and methylated histone H3 [H3K4me3]. The whole cell lysates from As and E2 treated cells were prepared and protein expression/modifications were determined by Western blot analysis as described in Materials and Methods. The histograms represent the signal intensity of protein bands in arbitrary units after normalization with the signal intensity of GAPDH internal control for each sample. The graph represents means of duplicate values. An * indicates significant (<i>p</i><0.05) difference between treatment groups and control. Similarly, † indicates a significant difference (<i>p<0.05</i>) between As alone and As in combination with E2. ‡ indicates a significant difference (<i>p<0.05</i>) between E2 alone and E2 in combination with As.</p

A list of forward and reverse primers sequence of the genes used in gene expression analysis by quantitative real time PCR.

<p>A list of forward and reverse primers sequence of the genes used in gene expression analysis by quantitative real time PCR.</p

Real-time quantitative reverse transcription PCR analysis of gene expression of DNA methylation related genes.

<p>Total RNA isolated from RWPE-1 cells with chronic exposure to arsenic and/or estrogen was used to perform one-step real-time quantitative reverse transcription PCR as described in materials and methods. Cycle threshold value (Ct value) of each gene was normalized to the Ct value of housekeeping gene GAPDH obtained from the same sample. The gene expression in fold- change was calculated and histogram was plotted using the means of triplicate values. Statistically significant change in gene expression in treated groups as compared to the vehicle treated control is indicated by an *. Similarly, † indicates a significant difference (<i>p<0.05</i>) between As alone and As in combination with E2. ‡ indicates a significant difference (<i>p<0.05</i>) between E2 alone and E2 in combination with As.</p

Real-time quantitative reverse transcription PCR analysis of gene expression of histone modifications- related genes.

<p>Total RNA isolated from RWPE-1 cells with chronic exposure to arsenic and/or estrogen was used to perform one-step real-time quantitative reverse transcription PCR as described in materials and methods. Cycle threshold value (Ct value) of each gene was normalized to the Ct value of housekeeping gene GAPDH obtained from the same sample. The gene expression in fold- change was calculated and histogram was plotted using the means of triplicate values. Statistically significant change in gene expression in treated groups as compared to the vehicle treated control is indicated by an *. Similarly, † indicates a significant difference (<i>p<0.05</i>) between As alone and As in combination with E2. ‡ indicates a significant difference (<i>p<0.05</i>) between E2 alone and E2 in combination with As.</p