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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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Xenoestrogens alter the amount of prolactin released by physiologic estrogens. The levels of prolactin (PRL) were measured in cells treated with (A-C) 10 pM of the physiologic estrogens (E2, E1, or E3 ) alone or in combinations with 10 pM of different alkylphenols (EP, PP, OP, NP, or BPA), row labeled lo PE - lo XE; (D-F) 1 nM of the physiologic estrogens alone or in combinations with 10 pM of different alkylphenols, row labeled hi PE - lo XE; (G-I) 10 pM of the physiologic estrogens alone or in combinations with 1 nM of different alkylphenols, row labeled lo PE - hi XE; (J-L) 1 nM of the physiologic estrogens alone or in combinations with 1 nM of different alkylphenols, row labeled hi PE - hi XE. * = p < 0.05 compared to vehicle (V) control cells. # = p < 0.05 compared to E2, E1, or E3 alone.
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Figure 5: Xenoestrogens alter the amount of prolactin released by physiologic estrogens. The levels of prolactin (PRL) were measured in cells treated with (A-C) 10 pM of the physiologic estrogens (E2, E1, or E3 ) alone or in combinations with 10 pM of different alkylphenols (EP, PP, OP, NP, or BPA), row labeled lo PE - lo XE; (D-F) 1 nM of the physiologic estrogens alone or in combinations with 10 pM of different alkylphenols, row labeled hi PE - lo XE; (G-I) 10 pM of the physiologic estrogens alone or in combinations with 1 nM of different alkylphenols, row labeled lo PE - hi XE; (J-L) 1 nM of the physiologic estrogens alone or in combinations with 1 nM of different alkylphenols, row labeled hi PE - hi XE. * = p < 0.05 compared to vehicle (V) control cells. # = p < 0.05 compared to E2, E1, or E3 alone.

Mentions: Using higher through-put assays (in multi-well plates for the ERK and PRL studies), we tested multiple estrogen concentrations. Figures 4 and 5 show how combinations of two different endogenous estrogen concentrations with two different concentrations of each xenoestrogen (10 pM and 1 nM for all estrogens) can affect ERK activation and PRL release. These low concentrations of xenoestrogens correspond to ~20 ppb for the 1 nM concentration, and 200 ppt for the 10 pM concentration. They were chosen to represent high and low values of serum and urine levels reported in Americans [34]. For comparison, physiologic estrogens are present in these assays at ~27 ppb and 270 ppt concentrations.


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)

Xenoestrogens alter the amount of prolactin released by physiologic estrogens. The levels of prolactin (PRL) were measured in cells treated with (A-C) 10 pM of the physiologic estrogens (E2, E1, or E3 ) alone or in combinations with 10 pM of different alkylphenols (EP, PP, OP, NP, or BPA), row labeled lo PE - lo XE; (D-F) 1 nM of the physiologic estrogens alone or in combinations with 10 pM of different alkylphenols, row labeled hi PE - lo XE; (G-I) 10 pM of the physiologic estrogens alone or in combinations with 1 nM of different alkylphenols, row labeled lo PE - hi XE; (J-L) 1 nM of the physiologic estrogens alone or in combinations with 1 nM of different alkylphenols, row labeled hi PE - hi XE. * = 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
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Figure 5: Xenoestrogens alter the amount of prolactin released by physiologic estrogens. The levels of prolactin (PRL) were measured in cells treated with (A-C) 10 pM of the physiologic estrogens (E2, E1, or E3 ) alone or in combinations with 10 pM of different alkylphenols (EP, PP, OP, NP, or BPA), row labeled lo PE - lo XE; (D-F) 1 nM of the physiologic estrogens alone or in combinations with 10 pM of different alkylphenols, row labeled hi PE - lo XE; (G-I) 10 pM of the physiologic estrogens alone or in combinations with 1 nM of different alkylphenols, row labeled lo PE - hi XE; (J-L) 1 nM of the physiologic estrogens alone or in combinations with 1 nM of different alkylphenols, row labeled hi PE - hi XE. * = p < 0.05 compared to vehicle (V) control cells. # = p < 0.05 compared to E2, E1, or E3 alone.
Mentions: Using higher through-put assays (in multi-well plates for the ERK and PRL studies), we tested multiple estrogen concentrations. Figures 4 and 5 show how combinations of two different endogenous estrogen concentrations with two different concentrations of each xenoestrogen (10 pM and 1 nM for all estrogens) can affect ERK activation and PRL release. These low concentrations of xenoestrogens correspond to ~20 ppb for the 1 nM concentration, and 200 ppt for the 10 pM concentration. They were chosen to represent high and low values of serum and urine levels reported in Americans [34]. For comparison, physiologic estrogens are present in these assays at ~27 ppb and 270 ppt concentrations.

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