No abstract

Environmental estrogens are suspected of being involved in the current increase in the incidence of human reproductive malfunctions, such as a decrease in male reproductive capacity and an increased incidence of breast cancer in women. The influences of these compounds have been proposed to be mediated through binding to macromolecules, such as estrogen receptor alpha or beta. In this study we examined whether the low-affinity Type II estrogen binding site (Type II EBS), originally identified in the rat uterus, is a possible mediator of environmental estrogens such as bisphenol A (BPA). Analysis of BPA's binding to an enriched fraction of Type II EBS, using a competition assay, indicated that BPA was able to compete with estradiol in binding to this site. At a concentration of 10-15 microM (comparable to that required to induce uterine proliferation), BPA inhibited the binding of estradiol to Type II EBS by greater than 50%. The binding affinity of BPA for the Type II EBS was only 8-10-fold lower than that of the synthetic estrogen diethylstilbestrol. The binding of BPA to Type II EBS appeared specific to BPA, in that endosulfan, another environmental estrogen, failed to displace estradiol from the site. A comparison of the relative binding affinities of BPA for rat uterine estrogen receptor alpha to that of the Type II EBS implies that BPA preferentially binds to the Type II EBS.

No abstract

The low incidence of prostate cancer in Asians has been attributed to chemopreventative properties of certain chemicals found in their diet. This study characterized the androgenic and chemopreventative properties of the Jamaican bush tea “Bizzy,” using androgen receptor positive and negative cell lines. Exposure of prostate cells to Biz-2 resulted in a growth inhibition (GI50) of 15 ppm in LNCaP cells and 3.6 ppm in DU145 cells. Biz-2 elicited a 2-fold increase in the mRNA of the anti-apoptotic gene Bcl2, with a 10-fold increase in that of the proapoptotic gene Bax. We observed a 2.4- to 7.5-fold change in apoptotic cells in both cell lines. Biz-2 at 10 ppm elicited a time- and dose-dependent stimulation of both the protein and mRNA levels of several androgen-regulated genes. Biz-2 caused a 36% decrease in PSA secretion and a significant increase in PSA mRNA. The relative binding affinity (IC50) of Biz-2 for AR was 2- to 5-fold lower than that of the synthetic androgen R1881. Biz-2 was found to be a specific ligand for the AR in that the natural ligand, DHT, and the anti-androgen, flutamide, displaced Biz-2 bound to AR and inhibited Biz-2-induced transcription and PSA secretion. This study provided evidence that Biz-2 extract possesses the ability to modulate prostate cancer cell biology in an AR-dependent manner.

Estrogen receptor (ER) alpha is commonly thought to bind to a consensus estrogen response element (ERE) as a homodimer, but previous experiments have not ruled out the presence of other proteins in the ERalpha/ERE complex. To characterize this interaction in more detail, we overexpressed mouse (m) ERalpha in a baculovirus system, using the selective advantage of the apoptosis inhibitor p35. Recombinant mERalpha possesses the predicted molecular weight and binds 17beta-estradiol and an oligonucleotide containing a consensus vitellogenin ERE with high affinity. Over a wide concentration range of mERalpha protein (0.1-50 nM), only one complex was detected between mERalpha and vitellogenin ERE in gel shift assays. The ratio of E2:vitellogenin ERE bound by mERalpha was close to 2:1, and each complex contained only one ERE. The molecular weight of the complex was determined to be 160 000, very close to that predicted for two mERalpha proteins and one ERE oligonucleotide, therefore providing strong evidence that no other proteins were present. Recombinant mERalpha was purified such that it was the only protein observable by silver stain. Purified mERalpha and mERalpha in a nuclear extract behaved identically in Ferguson analysis, providing more evidence that only mERalpha was binding to the ERE. Purified mERalpha bound vitellogenin ERE with high affinity (Kd = 0. 92 +/- 0.20 nM), indicating that no other proteins are necessary for high-affinity mERalpha interaction with a consensus ERE. To determine whether ERalpha in an estrogen-responsive mammalian tissue behaves the same as the overexpressed mERalpha, we tested rat uterine cytosol by Ferguson analysis. ERalpha in rat uterine cytosol behaved identically to overexpressed mERalpha, suggesting that ERalpha in the uterine extract also binds to DNA predominantly as a homodimer with no additional proteins.