Cheryl S. Watson

Xenoestrogens at Picomolar to Nanomolar Concentrations Trigger Membrane Estrogen Receptor-α–Mediated Ca2+ Fluxes and Prolactin Release in GH3/B6 Pituitary Tumor Cells

Xenoestrogens (XEs) are widespread in our environment and are known to have deleterious effects in animal (and perhaps human) populations. Acting as inappropriate estrogens, XEs are thought to interfere with endogenous estrogens such as estradiol (E2) to disrupt normal estrogenic signaling. We investigated the effects of E2 versus several XEs representing organochlorine pesticides (dieldrin, endosulfan, o′p′-dichlorodiphenylethylene), plastics manufacturing by-products/detergents (nonylphenol, bisphenol A), a phytoestrogen (coumestrol), and a synthetic estrogen (diethylstilbestrol) on the pituitary tumor cell subline GH3/B6/F10, previously selected for expression of high levels of membrane estrogen receptor-α. Picomolar to nanomolar concentrations of both E2 and XEs caused intracellular Ca2+ changes within 30 sec of administration. Each XE produced a unique temporal pattern of Ca2+ elevation. Removing Ca2+ from the extracellular solution abolished both spontaneous and XE-induced intracellular Ca2+ changes, as did 10 μM nifedipine. This suggests that XEs mediate their actions via voltage-dependent L-type Ca2+ channels in the plasma membrane. None of the Ca2+ fluxes came from intracellular Ca2+ stores. E2 and each XE also caused unique time- and concentration-dependent patterns of prolactin (PRL) secretion that were largely complete within 3 min of administration. PRL secretion was also blocked by nifedipine, demonstrating a correlation between Ca2+ influx and PRL secretion. These data indicate that at very low concentrations, XEs mediate membrane-initiated intracellular Ca2+ increases resulting in PRL secretion via a mechanism similar to that for E2, but with distinct patterns and potencies that could explain their abilities to disrupt endocrine functions.

Differential regulation of dopamine transporter function and location by low concentrations of environmental estrogens and 17β-estradiol

Background The effects of 17β-estradiol (E2) and xenoestrogens (XEs) on dopamine transport may have important implications for the increased incidence of neurologic disorders, especially in women during life stages characterized by frequent hormonal fluctuations. Objective We examined low concentrations of XEs [dieldrin, endosulfan, o′, p′-dichlorodiphenyl-ethylene (DDE), nonylphenol (NP), and bisphenol A (BPA)] for nongenomic actions via action of membrane estrogen receptors (ERs). Methods We measured activity of the dopamine transporter (DAT) by the efflux of 3H-dopamine in nontransfected nerve growth factor–differentiated PC12 rat pheochromocytoma cells expressing membrane DAT, ER-α, ER-β, and G-protein–coupled receptor 30. We used a plate immunoassay to monitor trafficking of these proteins. Results All compounds at 1 nM either caused efflux or inhibited efflux, or both; each compound evoked a distinct oscillatory pattern. At optimal times for each effect, we examined different concentrations of XEs. All XEs were active at some concentration < 10 nM, and dose responses were all nonmonotonic. For example, 10−14 to 10−11 M DDE caused significant efflux inhibition, whereas NP and BPA enhanced or inhibited efflux at several concentrations. We also measured the effects of E2/XE combinations; DDE potentiated E2-mediated dopamine efflux, whereas BPA inhibited it. In E2-induced efflux, 15% more ER-α trafficked to the membrane, whereas ER-β waned; during BPA-induced efflux, 20% more DAT was trafficked to the plasma membrane. Conclusions Low levels of environmental estrogen contaminants acting as endocrine disruptors via membrane ERs can alter dopamine efflux temporal patterning and the trafficking of DAT and membrane ERs, providing a cellular mechanism that could explain the disruption of physiologic neurotransmitter function.

Combinations of physiologic estrogens with xenoestrogens alter ERK phosphorylation profiles in rat pituitary cells.

Background Estrogens are potent nongenomic phospho-activators of extracellular-signal–regulated kinases (ERKs). A major concern about the toxicity of xenoestrogens (XEs) is potential alteration of responses to physiologic estrogens when XEs are present simultaneously. Objectives We examined estrogen-induced ERK activation, comparing the abilities of structurally related XEs (alkylphenols and bisphenol A) to alter ERK responses induced by physiologic concentrations (1 nM) of estradiol (E2), estrone (E1), and estriol (E3). Methods We quantified hormone/mimetic-induced ERK phosphorylations in the GH3/B6/F10 rat pituitary cell line using a plate immunoassay, comparing effects with those on cell proliferation and by estrogen receptor subtype-selective ligands. Results Alone, these structurally related XEs activate ERKs in an oscillating temporal pattern similar (but not identical) to that with physiologic estrogens. The potency of all estrogens was similar (active between femtomolar and nanomolar concentrations). XEs potently disrupted physiologic estrogen signaling at low, environmentally relevant concentrations. Generally, XEs potentiated (at the lowest, subpicomolar concentrations) and attenuated (at the highest, picomolar to 100 nM concentrations) the actions of the physiologic estrogens. Some XEs showed pronounced nonmonotonic responses/inhibitions. The phosphorylated ERK and proliferative responses to receptor-selective ligands were only partially correlated. Conclusions XEs are both imperfect potent estrogens and endocrine disruptors; the more efficacious an XE, the more it disrupts actions of physiologic estrogens. This ability to disrupt physiologic estrogen signaling suggests that XEs may disturb normal functioning at life stages where actions of particular estrogens are important (e.g., development, reproductive cycling, pregnancy, menopause).

Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding.

GH3/B6 rat pituitary tumor cells exhibit rapid prolactin release (within 5 min) when treated with nanomolar amounts of estrogen. However, the putative protein mediator of this nongenomic action has not been described. Using antibodies directed against a peptide representing the hinge region of the intracellular estrogen receptor (iER), we have demonstrated that these cells contain a membrane ER (mER). We now report that confocal scanning laser microscopy of cells labeled live with the anti‐peptide antibody further supports a membrane localization of ER. The monoclonal antibodies H226 and H222 and a polyclonal antibody, ER21, each recognizing a unique epitope on iER (NH2 terminal to the DNA‐binding region, within the steroid binding region, and the NH2‐terminal end, respectively), also immunohistochemically label membrane proteins of immuno‐selected GH3/B6 cells. These cells also specifically bind a fluorescent estrogen‐BSA conjugate. Coincubation of cells with anti‐ER antibody and the fluorescent estrogen‐BSA conjugate reveals that these labels colocalize on cells. These results suggest that mER may be structurally similar to iER.—Pappas, T. C., Gametchu, B., Watson, C. S. Membrane estrogen receptors identified by multiple antibody labeling and impeded‐ligand binding. FASEB J. 9, 404–410 (1995)

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Rapid actions of estrogens in GH3/B6 pituitary tumor cells via a plasma membrane version of estrogen receptor-α

The focus of our work on rapid actions of estrogens has been on the immuno-identification of a membrane version of the estrogen receptor-alpha (mERalpha) and the correlation of the presence of this receptor to the rapid secretion of prolactin in pituitary tumor cells. We demonstrated the mERalpha by both fluorescence and immuno-enzyme-cytochemistry and with both conventional and confocal microscopy in the cell line GH3/B6 and its sublines. Its presence on cells (including recently subcloned ones) is very heterogenous, unlike the nuclear ERalpha, which is present in every cell. An impeded ligand (estradiol covalently linked to BSA) binds to mERalpha and elicits the same response. A total of eight antibodies to ERalpha recognize mERalpha, making it likely that the membrane and nuclear proteins are highly related. Immuno-identification techniques have also been used to identify mERalpha on the MCF-7 human breast cancer cell line. Estradiol at very low concentrations elicits prolactin release from GH3/B6 cells within a few minutes of application. This response is bimodal, with effective concentrations in both the picomolar and nanomolar ranges. Prolactin release is also elicited or inhibited by ERalpha-specific antibodies. The characteristics of mERalpha and the membrane receptor for glucocorticoids have many similarities, suggesting that this mode of subcellular location/function alternative might be used by other members of the gene family.

Estrogen Receptor-α Detected on the Plasma Membrane of Aldehyde-Fixed GH3/B6/F10 Rat Pituitary Tumor Cells by Enzyme-Linked Immunocytochemistry1

A population of estrogen receptor-α (ERα) proteins, located at the plasma membrane, is postulated to mediate the rapid, nongenomic responses of GH3/B6/F10 pituitary cells to estrogen. To demonstrate the presence of ERα at the plasma membrane and to distinguish this receptor population from that in the nucleus, GH3/B6/F10 cells were first prepared in 2% paraformaldehyde/0.1% glutaraldehyde in PBS (P/G) without detergent, then exposed to one of several antibodies (Abs) raised against nuclear ERα. Ab binding was visualized as a fluorescent/chromagenic reaction product catalyzed by avidin-biotin-complexed alkaline phosphatase. With P/G fixation, Abs could only access antigens at the cell surface, as evidenced by the inability of 70K mol wt dextrans to permeate cells and the absence of intracellular staining by Abs to cytoplasmic or nuclear antigens. ERα Abs generated membrane, but not nuclear, staining in P/G-fixed cells; nuclear receptor labeling could only be detected in detergent-treated cells. Specificity...

Perimembrane Localization of the Estrogen Receptor α Protein in Neuronal Processes of Cultured Hippocampal Neurons

There is clear evidence of rapid, nongenomic responses to estrogen in a variety of neuronal model systems. To address the question of whether some of these rapid estrogen signals might be transduced by the classical estrogen receptor (ER) α or a closely related protein in nontransformed neurons, we undertook the present study using isolated fetal rat hippocampal neurons. Several antibodies developed to detect ERα were tested in this system and showed positive membrane staining in nonpermeabilized neurons. MC-20, an affinity purified anti-ERα, rabbit polyclonal IgG antibody which does not recognize ERβ was selected to carry out the majority of the experiments. When permeabilized, the hippocampal neurons exhibited low levels of nuclear staining for ERα, but abundant labeling for ERα throughout the entire cell including the neurites. In addition to traditional immunocytochemistry controls, incubation of neurons for 24 h in the presence of 10 µM antisense oligonucleotide directed against the translation start site of ERα reduced ERα immunoreactivity throughout the neurons providing further evidence that the immunostaining was specific for ERα. Confocal and conventional microscopy demonstrated that the antigen was predominately extranuclear and localization of ERα in the neurites suggests that the receptor is in close proximity to the plasma membrane. This localization is consistent with a role for ERα as a transducer of rapid, nongenomic estrogen responses in hippocampal neurons.

Antibodies to the estrogen receptor-α modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors

Antibodies (Abs) raised against the estrogen receptor‐α (ERα) were used to investigate the role of ERα proteins located at the plasma membrane in mediating the rapid, estrogen‐stimulated secretion of prolactin (PRL) from rat pituitary GH3/B6/F10 cells. Exposure of the cells to 1 nM 17 β‐estradiol (E2) significantly increased PRL release after 3 or 6 min. When ERα Abs that bind specifically to ERα but are too large to diffuse into cells were tested for activity at the cell membrane, Ab R4, targeted to an ERα hinge region sequence, increased PRL release in a time‐ and concentration‐dependent fashion. Ab H151, directed against a different hinge region epitope, decreased PRL release and blocked the stimulatory action of E2. Abs raised against the DNA binding domain (H226) or the carboxyl terminus (C542) were not biologically active. When each Ab was examined for recognition of ERα on the cell surface by immunocytochemistry, all except H151 generated immunostaining in aldehyde‐fixed cells. In live cells, however, Ab H151 but not Ab R4 blocked the membrane binding of fluorescently tagged E2‐BSA. Overall, the data indicate that plasma membrane ERα proteins mediate estrogen‐stimulated PRL release from GH3/B6/F10 cells. These results may also convey information about conformationally sensitive areas of the membrane form of ERα involved in rapid, nongenomic responses to estrogens.—Norfleet, A. M., Clarke, C. H., Gametchu, B., Watson, C. S. Antibodies to the estrogen receptor‐α modulate rapid prolactin release from rat pituitary tumor cells through plasma membrane estrogen receptors. FASEB J. 14, 157–165 (2000)

Xenoestrogens are potent activators of nongenomic estrogenic responses

Studies of the nuclear transcriptional regulatory activities of non-physiological estrogens have not explained their actions in mediating endocrine disruption in animals and humans at the low concentrations widespread in the environment. However, xenoestrogens have rarely been tested for their ability to participate in the plethora of nongenomic steroid signaling pathways elucidated over the last several years. Here we review what is known about such responses in comparison to our recent evidence that xenoestrogens can rapidly and potently elicit signaling through nongenomic pathways culminating in functional endpoints. Both estradiol (E(2)) and compounds representing various classes of xenoestrogens (diethylstilbestrol, coumestrol, bisphenol A, DDE, nonylphenol, endosulfan, and dieldrin) act via a membrane version of the estrogen receptor-alpha on pituitary cells, and can provoke Ca(2+) influx via L-type channels, leading to prolactin (PRL) secretion. These hormones and mimetics can also cause the oscillating activation of extracellular regulated kinases (ERKs). However, individual estrogen mimetics differ in their potency and temporal phasing of these activations compared to each other and to E(2). It is perhaps in these ways that they disrupt some endocrine functions when acting in combination with physiological estrogens. Our quantitative assays allow comparison of these outcomes for each mimetic, and let us build a detailed picture of alternative signaling pathway usage. Such an understanding should allow us to determine the estrogenic or antiestrogenic potential of different types of xenoestrogens, and help us to develop strategies for preventing xenoestrogenic disruption of estrogen action in many tissues.