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Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells.

Jeng YJ, Kochukov M, Watson CS - Environ Health (2010)

Bottom Line: 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.Because of their non-classical dose-responses, they must be studied in detail to pinpoint effective concentrations and the directions of response changes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.

ABSTRACT

Background: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

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Movement of mERs in or out of the membrane in response to different physiologic (E2, E1, or E3 ) or environmental (EP, PP, OP, NP, or BPA) estrogens. Levels were assessed by a quantitative plate assay measuring immunoreactive protein levels in the plasma membrane for ERα, ERβ, and GPER, after treatment with (A) 10 pM of estrogenic compounds or (B) 1 nM of estrogenic compounds for 5 min. *p < 0.05 compared to vehicle (V) control cells. #:p < 0.05 compared to E2, E1, or E3 alone.
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Figure 6: Movement of mERs in or out of the membrane in response to different physiologic (E2, E1, or E3 ) or environmental (EP, PP, OP, NP, or BPA) estrogens. Levels were assessed by a quantitative plate assay measuring immunoreactive protein levels in the plasma membrane for ERα, ERβ, and GPER, after treatment with (A) 10 pM of estrogenic compounds or (B) 1 nM of estrogenic compounds for 5 min. *p < 0.05 compared to vehicle (V) control cells. #:p < 0.05 compared to E2, E1, or E3 alone.

Mentions: Finally, we studied the effects of all of these estrogens on mER trafficking five minutes after hormone administration (Figure 6), choosing this time because it is unambiguously nongenomic, and because five-min responses were universally present for both ERK and Ca signaling pathways, and for the PRL secretion endpoint, for all classes of estrogens [1,15]. We had previously reported the lack of ERβ in our cell line [37], as values were not significantly different from controls (but did have wide errors of measurement). Perhaps due to assay improvements, cell line evolution, or change of Ab, we now see measurable amounts of ERβ in the membrane of these GH3/B6/F10 cells; whole cell ERβ had been reported by others in the parent GH3 cell line previously [77]. We detect a 57 kD band for ERβon an immunoblot at the same migration distance as ERβ isolated from LNCAP human prostate cancer cells (data not shown). Some xenoestrogen effects were seen at the 10 pM concentrations for most compounds; OP, NP and BPA all decreased mERα, and PP decreased mERβ. However, these effects were more prominent at the higher (1 nM) concentrations for all compounds. EP, PP, and BPA all decreased mERα in the membrane; all alkylphenols and BPA decreased mERβ in the membrane. It is interesting that physiologic estrogens also had some effects on these mERs. E1, ࿠increased levels of mERβ at the lower concentration and E3 decreased levels of mERα at the higher concentration. None of these estrogens significantly changed GPER levels.


Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells.

Jeng YJ, Kochukov M, Watson CS - Environ Health (2010)

Movement of mERs in or out of the membrane in response to different physiologic (E2, E1, or E3 ) or environmental (EP, PP, OP, NP, or BPA) estrogens. Levels were assessed by a quantitative plate assay measuring immunoreactive protein levels in the plasma membrane for ERα, ERβ, and GPER, after treatment with (A) 10 pM of estrogenic compounds or (B) 1 nM of estrogenic compounds for 5 min. *p < 0.05 compared to vehicle (V) control cells. #:p < 0.05 compared to E2, E1, or E3 alone.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2967504&req=5

Figure 6: Movement of mERs in or out of the membrane in response to different physiologic (E2, E1, or E3 ) or environmental (EP, PP, OP, NP, or BPA) estrogens. Levels were assessed by a quantitative plate assay measuring immunoreactive protein levels in the plasma membrane for ERα, ERβ, and GPER, after treatment with (A) 10 pM of estrogenic compounds or (B) 1 nM of estrogenic compounds for 5 min. *p < 0.05 compared to vehicle (V) control cells. #:p < 0.05 compared to E2, E1, or E3 alone.
Mentions: Finally, we studied the effects of all of these estrogens on mER trafficking five minutes after hormone administration (Figure 6), choosing this time because it is unambiguously nongenomic, and because five-min responses were universally present for both ERK and Ca signaling pathways, and for the PRL secretion endpoint, for all classes of estrogens [1,15]. We had previously reported the lack of ERβ in our cell line [37], as values were not significantly different from controls (but did have wide errors of measurement). Perhaps due to assay improvements, cell line evolution, or change of Ab, we now see measurable amounts of ERβ in the membrane of these GH3/B6/F10 cells; whole cell ERβ had been reported by others in the parent GH3 cell line previously [77]. We detect a 57 kD band for ERβon an immunoblot at the same migration distance as ERβ isolated from LNCAP human prostate cancer cells (data not shown). Some xenoestrogen effects were seen at the 10 pM concentrations for most compounds; OP, NP and BPA all decreased mERα, and PP decreased mERβ. However, these effects were more prominent at the higher (1 nM) concentrations for all compounds. EP, PP, and BPA all decreased mERα in the membrane; all alkylphenols and BPA decreased mERβ in the membrane. It is interesting that physiologic estrogens also had some effects on these mERs. E1, ࿠increased levels of mERβ at the lower concentration and E3 decreased levels of mERα at the higher concentration. None of these estrogens significantly changed GPER levels.

Bottom Line: 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.Because of their non-classical dose-responses, they must be studied in detail to pinpoint effective concentrations and the directions of response changes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA.

ABSTRACT

Background: 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.

Methods: 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).

Results: 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.

Conclusions: 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.

Show MeSH