Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.
Environ Health. 2010 Oct 15;9:61. doi: 10.1186/1476-069X-9-61.
Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens.
We studied the effects of low concentrations of endogenous estrogens (estradiol, estriol, and estrone) at 10 pM (representing pre-development levels), and 1 nM (representing higher cycle-dependent and pregnancy levels) in combinations with the same levels of xenoestrogens in GH3/B6/F10 pituitary cells. These levels of xenoestrogens represent extremely low contamination levels. We monitored calcium entry into cells using Fura-2 fluorescence imaging of single cells. Prolactin release was measured by radio-immunoassay. Extracellular-regulated kinase (1 and 2) phospho-activations and the levels of three estrogen receptors in the cell membrane (ERα, ERβ, and GPER) were measured using a quantitative plate immunoassay of fixed cells either permeabilized or nonpermeabilized (respectively).
All xenoestrogens caused responses at these concentrations, and had disruptive effects on the actions of physiologic estrogens. Xenoestrogens reduced the % of cells that responded to estradiol via calcium channel opening. They also inhibited the activation (phosphorylation) of extracellular-regulated kinases at some concentrations. They either inhibited or enhanced rapid prolactin release, depending upon concentration. These latter two dose-responses were nonmonotonic, a characteristic of nongenomic estrogenic responses.
Responses mediated by endogenous estrogens representing different life stages are vulnerable to very low concentrations of these structurally related xenoestrogens. Because of their non-classical dose-responses, they must be studied in detail to pinpoint effective concentrations and the directions of response changes.
烷基酚等异雌激素以及结构相关的塑料副产物双酚 A 最近被证明可以通过非基因组信号通路和雌激素受体-α 的膜形式发挥强大作用。尽管这些化合物的反应通常是单独测量的，但它们通常会污染已经存在内源性雌激素的生物体。因此，我们使用定量中通量筛选测定法来测量生理雌激素与这些异雌激素结合的影响。
我们研究了低浓度内源性雌激素（雌二醇、雌三醇和雌酮）在 10 pM（代表发育前水平）和 1 nM（代表更高的周期依赖性和妊娠水平）与 GH3/B6/F10 垂体细胞中相同水平的异雌激素组合时的影响。这些异雌激素水平代表极低的污染水平。我们使用 Fura-2 荧光成像单细胞监测细胞内钙离子进入。通过放射免疫测定法测量催乳素释放。使用固定细胞的定量板免疫测定法测量细胞外调节激酶 (1 和 2) 磷酸化和细胞膜上的三种雌激素受体（ERα、ERβ 和 GPER）的水平，该方法可对透化或非透化的细胞（分别）进行测量。
所有异雌激素在这些浓度下都能引起反应，并对生理雌激素的作用具有破坏作用。异雌激素通过钙通道打开减少了对雌二醇有反应的细胞的百分比。它们还在某些浓度下抑制细胞外调节激酶的激活（磷酸化）。它们依赖于浓度抑制或增强快速催乳素释放。后两种剂量反应是非单调的，这是一种非基因组雌激素反应的特征。
代表不同生命阶段的内源性雌激素介导的反应容易受到这些结构相关异雌激素的极低浓度的影响。由于它们的非经典剂量反应，必须详细研究它们以确定有效浓度和反应变化的方向。
