Evaluation of Bisphenol A (BPA) Exposures on Prostate Stem Cell Homeostasis and Prostate Cancer Risk in the NCTR-Sprague-Dawley Rat: An NIEHS/FDA CLARITY-BPA Consortium Study.

BACKGROUND: Previous work determined that early life exposure to low-dose Bisphenol A (BPA) increased rat prostate cancer risk with aging. Herein, we report on prostate-specific results from CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity), which aims to resolve uncertainties regarding BPA toxicity. OBJECTIVES: We sought to a) reassess whether a range of BPA exposures drives prostate pathology and/or alters prostatic susceptibility to hormonal carcinogenesis, and b) test whether chronic low-dose BPA targets prostate epithelial stem and progenitor cells. METHODS: Sprague-Dawley rats were gavaged daily with vehicle, ethinyl estradiol (EE) or [Formula: see text] BPA/kg-BW during development or chronically, and prostate pathology was assessed at one year. One developmentally exposed cohort was given testosterone plus estradiol ([Formula: see text]) implants at day 90 to promote carcinogenesis with aging. Epithelial stem and progenitor cells were isolated by prostasphere (PS) culture from dorsolateral prostates (DLP) of rats continuously exposed for six months to [Formula: see text] BPA/kg-BW. Gene expression was analyzed by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR). RESULTS: Exposure to BPA alone at any dose did not drive prostate pathology. However, rats treated with EE, 2.5, 250, or [Formula: see text] BPA/kg-BW plus [Formula: see text] showed greater severity of lateral prostate intraepithelial neoplasia (PIN), and DLP ductal adenocarcinoma multiplicity was markedly elevated in tumor-bearing rats exposed to [Formula: see text]-BW. DLP stem cells, assessed by PS number, doubled with chronic EE and [Formula: see text] exposures. PS size, reflecting progenitor cell proliferation, was greater at 25 and [Formula: see text] BPA doses, which also shifted lineage commitment toward basal progenitors while reducing luminal progenitor cells. CONCLUSIONS: Together, these results confirm and extend previous evidence using a rat model and human prostate epithelial cells that low-dose BPA augments prostate cancer susceptibility and alters adult prostate stem cell homeostasis. Therefore, we propose that BPA exposures may contribute to the increased carcinogenic risk in humans that occurs with aging. https://doi.org/10.1289/EHP3953.

Exposure of Human Prostaspheres to Bisphenol A Epigenetically Regulates SNORD Family Noncoding RNAs via Histone Modification.

Bisphenol A (BPA) is a ubiquitous endocrine disruptor exerting lifelong effects on gene expression in rodent prostate cancer (PCa) models. Here, we aimed to determine whether epigenetic events mediating the action of BPA on human prostaspheres enriched in epithelial stem-like/progenitor cells is linked to PCa. We performed genome-wide transcriptome and methylome analyses to identify changes in prostaspheres treated with BPA (10 nM, 200 nM, and 1000 nM) or estradiol-17ß (E2) (0.1 nM) for 7 days and validated changes in expression, methylation, and histone marks in parallel-treated prostaspheres. BPA/E2-treatment altered expression of 91 genes but not the methylation status of 485,000 CpG sites in BPA/E2-treated prostaspheres. A panel of 26 genes was found repressed in all treatment groups. Fifteen of them were small nucleolar RNAs with C/D motif (SNORDs), which are noncoding, small nucleolar RNAs known to regulate ribosomal RNA assembly and function. Ten of the most down-regulated SNORDs were further studied. All 10 were confirmed repressed by BPA, but only 3 ratified as E2-repressed. SNORD suppression showed no correlation with methylation status changes in CpG sites in gene regulatory regions. Instead, BPA-induced gene silencing was found to associate with altered recruitments of H3K9me3, H3K4me3, and H3K27me3 to 5'-regulatory/exonic sequences of 5 SNORDs. Expression of 4 out of these 5 SNORDs (SNORD59A, SNORD82, SNORD116, and SNORD117) was shown to be reduced in PCa compared with adjacent normal tissue. This study reveals a novel and unique action of BPA in disrupting expression of PCa-associated SNORDs and a putative mechanism for reprogramming the prostasphere epigenome via histone modification.

Bisphenol A promotes human prostate stem-progenitor cell self-renewal and increases in vivo carcinogenesis in human prostate epithelium.

Previous studies in rodent models have shown that early-life exposure to bisphenol A (BPA) reprograms the prostate and enhances its susceptibility to hormonal carcinogenesis with aging. To determine whether the human prostate is similarly sensitive to BPA, the current study used human prostate epithelial stem-like cells cultured from prostates of young, disease-free donors. Similar to estradiol-17ß (E2), BPA increased stem-progenitor cell self-renewal and expression of stem-related genes in a dose-dependent manner. Further, 10 nM BPA and E2 possessed equimolar membrane-initiated signaling with robust induction of p-Akt and p-Erk at 15 minutes. To assess in vivo carcinogenicity, human prostate stem-progenitor cells combined with rat mesenchyme were grown as renal grafts in nude mice, forming normal human prostate epithelium at 1 month. Developmental BPA exposure was achieved through oral administration of 100 or 250 µg BPA/kg body weight to hosts for 2 weeks after grafting, producing free BPA levels of 0.39 and 1.35 ng/mL serum, respectively. Carcinogenesis was driven by testosterone plus E2 treatment for 2 to 4 months to model rising E2 levels in aging men. The incidence of high-grade prostate intraepithelial neoplasia and adenocarcinoma markedly increased from 13% in oil-fed controls to 33% to 36% in grafts exposed in vivo to BPA (P < .05). Continuous developmental BPA exposure through in vitro (200 nM) plus in vivo (250 µg/kg body weight) treatments increased high-grade prostate intraepithelial neoplasia/cancer incidence to 45% (P < .01). Together, the present findings demonstrate that human prostate stem-progenitor cells are direct BPA targets and that developmental exposure to BPA at low doses increases hormone-dependent cancer risk in the human prostate epithelium.

Actions of estrogens and endocrine disrupting chemicals on human prostate stem/progenitor cells and prostate cancer risk.

Estrogen reprogramming of the prostate gland as a function of developmental exposures (aka developmental estrogenization) results in permanent alterations in structure and gene expression that lead to an increased incidence of prostatic lesions with aging. Endocrine disrupting chemicals (EDCs) with estrogenic activity have been similarly linked to an increased prostate cancer risk. Since it has been suggested that stem cells and cancer stem cells are potential targets of cancer initiation and disease management, it is highly possible that estrogens and EDCs influence the development and progression of prostate cancer through reprogramming and transforming the prostate stem and early stage progenitor cells. In this article, we review recent literature highlighting the effects of estrogens and EDCs on prostate cancer risk and discuss recent advances in prostate stem/progenitor cell research. Our laboratory has recently developed a novel prostasphere model using normal human prostate stem/progenitor cells and established that these cells express estrogen receptors (ERs) and are direct targets of estrogen action. Further, using a chimeric in vivo prostate model derived from these normal human prostate progenitor cells, we demonstrated for the first time that estrogens initiate and promote prostatic carcinogenesis in an androgen-supported environment. We herein discuss these findings and highlight new evidence using our in vitro human prostasphere assay for perturbations in human prostate stem cell self-renewal and differentiation by natural steroids as well as EDCs. These findings support the hypothesis that tissue stem cells may be direct EDC targets which may underlie life-long reprogramming as a consequence of developmental and/or transient adult exposures.

Estrogen-initiated transformation of prostate epithelium derived from normal human prostate stem-progenitor cells.

The present study sought to determine whether estrogens with testosterone support are sufficient to transform the normal human prostate epithelium and promote progression to invasive adenocarcinoma using a novel chimeric prostate model. Adult prostate stem/early progenitor cells were isolated from normal human prostates through prostasphere formation in three-dimensional culture. The stem/early progenitor cell status and clonality of prostasphere cells was confirmed by immunocytochemistry and Hoechst staining. Normal prostate progenitor cells were found to express estrogen receptor α, estrogen receptor ß, and G protein-coupled receptor 30 mRNA and protein and were responsive to 1 nm estradiol-17ß with increased numbers and prostasphere size, implicating them as direct estrogen targets. Recombinants of human prostate progenitor cells with rat urogenital sinus mesenchyme formed chimeric prostate tissue in vivo under the renal capsule of nude mice. Cytodifferentiation of human prostate progenitor cells in chimeric tissues was confirmed by immunohistochemistry using epithelial cell markers (p63, cytokeratin 8/18, and androgen receptor), whereas human origin and functional differentiation were confirmed by expression of human nuclear antigen and prostate-specific antigen, respectively. Once mature tissues formed, the hosts were exposed to elevated testosterone and estradiol-17ß for 1-4 months, and prostate pathology was longitudinally monitored. Induction of prostate cancer in the human stem/progenitor cell-generated prostatic tissue was observed over time, progressing from normal histology to epithelial hyperplasia, prostate intraepithelial neoplasia, and prostate cancer with local renal invasion. These findings provide the first direct evidence that human prostate progenitor cells are estrogen targets and that estradiol in an androgen-supported milieu is a carcinogen for human prostate epithelium.

[Effect of ginsenoside Rg1 on expression of p21, cyclin E and CDK2 in the process of cell senescence].

AIM: To explore the possible role of p21, cyclin E and cyclin-dependent kinase 2 (CDK2) in the protection of ginsenoside Rg1 against tert-butylhydroperoxide (t-BHP)-induced senescence in WI-38 cells. METHODS: The cellular ultrastructure, cytometric assay and beta-galactosidase (beta-gal) cytochemistry staining were used to evaluate cell senescence. The levels of of p21, cyclin E and CDK2 protein were detected by Western blot. RESULTS: Pretreatment with Rg1 significantly attenuated t-BHP-induced senescence in WI-38 cells. Simultaneously, compared with cells treated with t-BHP alone, Rg1 pretreatment markedly decreased the level of p21 protein and increased the levels of CDK2 and cyclin E. CONCLUSION: p21, cyclin E and CDK2 may be involved in the process of ginsenoside Rg1 protection against t-BHP-induced senescence in WI-38 cells.

Curcumin protects mitochondria from oxidative damage and attenuates apoptosis in cortical neurons.

AIM: To investigate the effect of curcumin on tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in rat cortical neurons and to explore the possible mechanism. METHODS: Primary cultured rat cortical neurons were performed in vitro and cell viability was measured by MTT assay. DNA fragmentation was used to evaluate cell apoptosis. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (Deltapsim) was determined by flow cytometric assay. Cellular glutathione (GSH) content was measured by spectrophotometer. Bcl-2 family proteins, cytochrome c, cleaved caspase-3, and poly (ADP-ribose) polymerase (PARP) were detected by Western blot. RESULTS: Exposure of tBHP 100 micromol/L to neurons for 60 min resulted in DYm loss and cytochrome c release from mitochondria and subsequent activation of caspase-3 and PARP cleavation, and cell apoptosis. After removal of tBHP and then further treatment with curcumin (2.5-20 micromol/L) for 18 h, curcumin abrogated Deltapsim loss and cytochrome c release, blocked activation of caspase 3, and altered the expression of Bcl-2 family. Further curcumin treatment also prevented cellular GSH and decreased intracellular ROS generation markedly. Curcumin eventually attenuated tBHP-induced apoptosis in cortical neurons. CONCLUSION: Curcumin may attenuate oxidative damages in cortical neurons by reducing intracellular production of ROS and protecting mitochondria from oxidative damage.