Bisphenol A Disrupts HNF4α-Regulated Gene Networks Linking to Prostate Preneoplasia and Immune Disruption in Noble Rats

Abstract

Exposure of humans to bisphenol A (BPA) is widespread and continuous. The effects of protracted exposure to BPA on the adult prostate have not been studied. We subjected Noble rats to 32 weeks of BPA (low or high dose) or 17β-estradiol (E2) in conjunction with T replenishment. T treatment alone or untreated groups were used as controls. Circulating T levels were maintained within the physiological range in all treatment groups, whereas the levels of free BPA were elevated in the groups treated with T+low BPA (1.06 ± 0.05 ng/mL, P < .05) and T+high BPA (10.37 ± 0.43 ng/mL, P < .01) when compared with those in both controls (0.1 ± 0.05 ng/mL). Prostatic hyperplasia, low-grade prostatic intraepithelial neoplasia (PIN), and marked infiltration of CD4+ and CD8+ T cells into the PIN epithelium (P < .05) were observed in the lateral prostates (LPs) of T+low/high BPA-treated rats. In contrast, only hyperplasia and high-grade PIN, but no aberrant immune responses, were found in the T+E2-treated LPs. Genome-wide transcriptome analysis in LPs identified differential changes between T+BPA vs T+E2 treatment. Expression of multiple genes in the regulatory network controlled by hepatocyte nuclear factor 4α was perturbed by the T+BPA but not by the T+E2 exposure. Collectively these findings suggest that the adult rat prostate, under a physiologically relevant T environment, is susceptible to BPA-induced transcriptomic reprogramming, immune disruption, and aberrant growth dysregulation in a manner distinct from those caused by E2. They are more relevant to our recent report of higher urinary levels BPA found in patients with prostate cancer than those with benign disease.

Figure 1.

A, Representative hematoxylin/eosin-stained sections from LPs of the five treatment groups. Untreated or T-treated control LPs show normal glandular architecture with little or no infiltration by inflammatory cells. T+E2-treated LPs exhibit an HG-PIN lesion with a typical cribriform pattern. T+low/high BPA exposure induced the development of LG-PIN in LPs, as evidenced by the presence of pleomorphic nuclei in the dysplastic epithelium (blue arrows). B, Mean PIN scores across treatment groups. Bar, 100 μm. *, P < .01 vs control.

Figure 2.

A, Representative Ki-67 staining (green arrows) of specific regions of LPs across treatment groups. B, Ki-67 index of normal or specific histological lesions (hyperplasia, LG-PINs, or HG-PINs) of LPs across treatment groups. Bar, 100 μm. a, P < .05 vs control: normal; T+E2: normal; T+E2: HG-PIN; T+low BPA: hyperplasia. b, P < .05 vs control: normal; T+E2: normal; T+low BPA: LG-PIN; T+high BPA: LG-PIN. c, P < .05 vs control: normal; T+E2: normal; T+E2: hyperplasia. d, P < .05 vs control: normal; control: hyperplasia; T+low BPA: normal; T+E2: hyperplasia; T+high BPA: hyperplasia; T: hyperplasia. e, P < .05 vs control: normal; T+low BPA: normal; T+E2: LG-PIN. f, P < .05 vs control: normal; control: hyperplasia; T+low BPA: hyperplasia; T+high BPA: normal. g, P < .05 vs control: normal; T+E2: LG-PIN; T+high BPA: normal. h, P < .05 vs control: normal; T: normal; T+low BPA: hyperplasia

Figure 3.

T+BPA treatment induced infiltration of CD4+ and CDa8+ T cells into PIN epithelium of LPs. A, Immunohistochemical localization of CD4+ T cells (red arrows: epithelium; red open arrows: stroma) in LPs across treatments: in normal and T-treated control LPs, CD4+ T cells were occasionally found in the epithelium and stroma; in T+E2-treated LP, CD4+ T cells were found in the epithelium and stroma of LG- and HG-PIN lesions; in T+low BPA-treated LP, numerous CD4+ T cells were infiltrated to the LG-PIN gland. An adjacent normal gland is free of any CD4+ cell infiltration (blue arrow). In T+high BPA-treated LP, massive infiltration of CD4+ T cells was observed in the PIN epithelium. B, Quantitation of CD4+ T-cell infiltration into the normal and PIN epithelium across treatments. The number of CD4+ T cells was significantly increased in the PIN epithelium of T+low/high BPA-treated LPs when compared with untreated or T-treated controls or LG-PIN glands in T+E2-treated LPs. a, P < .05 vs control: normal; T+E2: LG-PIN; T: normal. b, P < .05 vs T+E2: HG-PIN. C, Immunohistochemical localization of CD8a+ T cells (black arrows) in LPs across treatments: in untreated or T-treated control LPs, CD8+ T cells were sporadically detected in normal glandular epithelium; in T+E2-treated LP, a few CD8a+ T cells were localized in the LG-/HG-PIN epithelium; in T+low BPA-treated LP, clusters of CD8a+ T cells were often detected in PIN epithelium; in T+high BPA-treated LP, numerous CD8a+ cells were infiltrated into PIN epithelium. Bar, 100 μm. D, Quantitation of CD8a+ T-cell infiltration into the normal and PIN epithelium across treatments. The number of epithelium-infiltrating CD8a+ T cells was significantly higher in the PIN glands of T+low/high BPA-treated LPs than in the normal/PIN glands of untreated, T+E2-treated, and T-treated LPs. a, P < .05 vs control: normal; T+E2: LG-PIN; T+E2: HG-PIN; T: normal. b, P < .05 vs T+high BPA: LG-PIN.

Figure 4.

Two-way hierarchical clustering of differential genes expressed across treatment groups. A, Heat map showing genes differentially expressed in the LP of T+E2-treated, T+low BPA-treated, and T+high BPA-treated groups as compared with the untreated control group. Red, Up-regulated; green, down-regulated. Specific blocks (A–I) of gene clusters are denoted as high and/or low BPA-sensitive clusters. Pink boxes, Clusters of genes showing the same response to T+low BPA and T+E2 but displaying a distinctive pattern of expression in response to T+high BPA. Blue boxes, Clusters of genes revealing the same pattern of response to T+low BPA and T+high BPA but showing a different pattern of response to T+E2. Yellow boxes, Clusters of genes showing similar responses in all treatment groups. B, A schematic diagram demonstrating approaches for gene categorization, shaving with the filter 1 and filter 2 (two-way clustering, Venn diagram representation, network mapping, and post hoc confirmation). C, Venn diagram represents the population of significantly differentiated genes in the treatment groups after shaving with the filter 2 (P < .01 vs control, fold change > 1.2). Numbers of genes that are significant for a specific treatment were shown.

Figure 5.

HNF4α network and post hoc real-time qPCR analyses of the T+low BPA-responsive genes. Molecular interaction network by IPA demonstrated that HNF4α as a central node of genes was regulated by T+low BPA (A) and T+high BPA (B) treatment in the rat LPs. The intensity of genes indicates the degree of up-regulation (red) or down-regulation (green) of a specific gene. Different shapes represented the functional classes of the gene, and the direct or indirect relationship between genes is linked by lines (see legend). Molecules in white were not directly involved in treatment response but were related to the imported molecules. C, Genes involved in the HNF4α-related pathway were validated in the LPs of control-, T+E2-, T+low BPA-, T+high BPA-, and T-treated rats. Values are normalized to Rpl19 and are expressed relative to the LPs of control-treated rats for a specific transcript. Results are analyzed by a one-way ANOVA with a Bonferroni test. Data are expressed as mean ± SEM. *, P < .05,**, P < .01, and ***, P < .001 compared with control unless otherwise specified.