Developmental exposure to estradiol and bisphenol A increases susceptibility to prostate carcinogenesis and epigenetically regulates phosphodiesterase type 4 variant 4

Abstract

Early developmental perturbations have been linked to adult-onset prostate pathology, including excessive exposure to estrogenic compounds; however, the molecular basis for this imprinting event is not known. An important and controversial health concern is whether low-dose exposures to hormonally active environmental estrogens, such as bisphenol A, can promote human diseases, including prostate cancer. Here, we show that transient developmental exposure of rats to low, environmentally relevant doses of bisphenol A or estradiol increases prostate gland susceptibility to adult-onset precancerous lesions and hormonal carcinogenesis. We found permanent alterations in the DNA methylation patterns of multiple cell signaling genes, suggesting an epigenetic basis for estrogen imprinting. For phosphodiesterase type 4 variant 4 (PDE4D4), an enzyme responsible for cyclic AMP breakdown, a specific methylation cluster was identified in the 5'-flanking CpG island that was gradually hypermethylated with aging in normal prostates, resulting in loss of gene expression. Early and prolonged hypomethylation at this site following neonatal estradiol or bisphenol A exposure resulted in continued, elevated PDE4D4 expression. Cell line studies confirmed that site-specific methylation is involved in transcriptional silencing of the PDE4D4 gene and showed hypomethylation of this gene in prostate cancer cells. Importantly, the PDE4D4 alterations in the estrogen-exposed prostates were distinguishable before histopathologic changes of the gland, making PDE4D4 a candidate molecular marker for prostate cancer risk assessment as a result of endocrine disruptors. In total, these findings indicate that low-dose exposures to ubiquitous environmental estrogens affect the prostate epigenome during development and, in so doing, promote prostate disease with aging.

Figure 1

Effects of neonatal estrogens on adult prostate. Representative data for the dorsal prostate at 6 months. A, dorsal prostate weights at day 200. *, P < 0.05 versus oil/testosterone + estradiol (T + E). B, columns, mean PIN scores; bars, SE. *, P < 0.05 versus oil alone; **, P < 0.05 versus oil/testosterone + estradiol; †, P < 0.01 versus bisphenol A (BPA) alone. C, incidence of PIN lesions across treatment groups at day 200. LGPIN, low-grade PIN; HGPIN, high-grade PIN. D, representative H&E sections from dorsal prostates of the eight treatment groups. Bar, 50 µm.

Figure 2

Proliferation and apoptosis rates following estrogenic exposures. A, proliferation index for dorsal prostate epithelial cells as determined by Ki-67 immunostaining. Black and red-hatched columns, counts in histologically normal regions; red-stripped columns, areas of high-grade PIN. Basal proliferation was elevated in high-dose EB prostates. Proliferation rates were further elevated in high-grade PIN lesions of high-dose EB and bisphenol A/testosterone + estradiol prostates. *, P < 0.05 versus oil and low-dose EB; **, P < 0.01 versus oil and high-dose EB/testosterone + estradiol/PIN region; †, P < 0.05 versus bisphenol A/testosterone + estradiol/normal region; † †, P < 0.001 versus normal regions of all testosterone + estradiol treatment groups. B, representative regions of histologically normal dorsal prostates immunostained for Ki-67. Inset, regions of high-grade PIN within each group. C, apoptotic index for dorsal prostate epithelial cells as determined by TUNEL. Areas of high-grade PIN in high-dose EB and bisphenol A/testosterone + estradiol tissues showed increased apoptosis. *, P < 0.05 versus bisphenol A/testosterone + estradiol; **, P < 0.01 versus normal regions of all testosterone + estradiol treatment groups; ***, P < 0.001 versus normal regions in all treatments. D, representative TUNEL-labeled dorsal prostates. Normal region for oil and high-grade PIN region for all others. Bar, 50 µm.

Figure 3

Bisulfite genomic sequencing of prostatic PDE4D4 gene methylation. A, schematic of CpG content (%) in the 5′-flanking region of the rat PDE4D4 gene identifies a CpG island (blue) between −310 to +390 bp. Vertical lines, individual CpG sites and the translation start site (ATG+1) and transcription start site (TSS). Blanket, 700-bp nested PCR-amplified region used for bisulfite sequencing. B, bisulfite genomic sequencing data from 4 to 6 clones each of three individual DNA samples taken from day 200 oil/testosterone + estradiol and bisphenol A/testosterone + estradiol dorsal prostates. Methylation status of specific CpG sites. ○, unmethylated; ●, methylated. Boxed region, potential CpG sites epigenetically altered by bisphenol A. C, percentage methylation at each CpG site within the 5′-CpG island of PDE4D4 in the separate treatment groups at days 10, 90, and 200 with or without adult testosterone + estradiol. The percentage methylation at each site was averaged from three individual sample sets. ◆, oil control; □, high-dose estradiol; △, low-dose estradiol; ×, bisphenol A.

Figure 4

Comparison of PDE4D4 CpG methylation and mRNA transcript levels. A, methylation-specific PCR analysis: dorsal prostate genomic DNA was bisulfite treated followed by methylation-specific PCR using methylated specific (M) or unmethylated-specific (U) primer sets. The amplified region is indicated in Fig. 3. The PCR products are representative data from three individual sets of samples. B, PDE4D4 mRNA transcript levels as determined by real-time RT-PCR. Relative expression of day 10 oil samples was set to 1. Columns, mean; bars, SD. All EB/bisphenol A groups at days 90, 200, and 200/testosterone + estradiol were significantly different (P < 0.05) from respective groups at day 10. *, P < 0.05; **, P < 0.01 versus oil controls at the same time interval; †, P < 0.05 versus day 200 oil controls; † †, P < 0.05 versus day 10 oil controls.

Figure 5

Alterations in PDE4D4 CpG methylation and gene expression in NbE-1 and AIT cell lines by a demethylating agent. A, percentage CpG methylation for PDE4D4 CpGs 49 to 56 in normal NbE-1 cells without (◆) or with 5-Aza-dC treatment (□, 0.5 µmol/L 5-Aza-dC; △, 1 µmol/L 5-Aza-dC). B, percentage CpG methylation for PDE4D4 CpGs 49 to 56 in tumorigenic AIT cells without (◆) or with 5-Aza-dC treatment. C, relative PDE4D4 mRNA levels in NbE-1 and AIT cells. White columns, control cells (A0); bars, SD. Black columns, cells treated with 5-Aza-dC. Expression of control NbE-1 cells was set to 1. **, P < 0.01 versus controls; ‡, P < 0.05 versus control NbE-1 cells.