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Reproductive Toxicology 24 (2007) 131-138. doi:10.1016/j.reprotox.2007.07.005Received by publisher: 2007-06-08Harvest Date: 2016-01-04 12:22:51DOI: 10.1016/j.reprotox.2007.07.005Page Range: 131-13

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.

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.

BackgroundIn 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.ObjectivesWe 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.DiscussionAlthough 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.ConclusionsPublic 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.