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Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells.

Viñas R, Watson CS - Environ Health (2013)

Bottom Line: Our study examines the effects of xenoestrogen mixtures on estradiol-induced non-genomic signaling and associated functional responses.Individual bisphenol compounds did not activate JNK, while nonylphenol did; however, the combination of two or three xenoestrogens with estradiol generated an enhanced non-monotonic JNK dose-response.In mixtures expected to be found in contaminated environments, xenoestrogens can have dramatic disrupting effects on hormonal mechanisms of cell regulation and their downstream functional responses, altering cellular responses to physiologic estrogens.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Our study examines the effects of xenoestrogen mixtures on estradiol-induced non-genomic signaling and associated functional responses. Bisphenol-A, used to manufacture plastic consumer products, and nonylphenol, a surfactant, are estrogenic by a variety of assays, including altering many intracellular signaling pathways; bisphenol-S is now used as a bisphenol-A substitute. All three compounds contaminate the environment globally. We previously showed that bisphenol-S, bisphenol-A, and nonylphenol alone rapidly activated several kinases at very low concentrations in the GH3/B6/F10 rat pituitary cell line.

Methods: For each assay we compared the response of individual xenoestrogens at environmentally relevant concentrations (10-15 -10-7 M), to their mixture effects on 10-9 M estradiol-induced responses. We used a medium-throughput plate immunoassay to quantify phosphorylations of extracellular signal-regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs). Cell numbers were assessed by crystal violet assay to compare the proliferative effects. Apoptosis was assessed by measuring caspase 8 and 9 activities via the release of the fluorescent product 7-amino-4-trifluoromethylcoumarin. Prolactin release was measured by radio-immunoassay after a 1 min exposure to all individual and combinations of estrogens.

Results: Individual xenoestrogens elicited phospho-activation of ERK in a non-monotonic dose- (fM-nM) and mostly oscillating time-dependent (2.5-60 min) manner. When multiple xenoestrogens were combined with nM estradiol, the physiologic estrogen's response was attenuated. Individual bisphenol compounds did not activate JNK, while nonylphenol did; however, the combination of two or three xenoestrogens with estradiol generated an enhanced non-monotonic JNK dose-response. Estradiol and all xenoestrogen compounds induced cell proliferation individually, while the mixtures of these compounds with estradiol suppressed proliferation below that of the vehicle control, suggesting a possible apoptotic response. Extrinsic caspase 8 activity was suppressed by estradiol, elevated by bisphenol S, and unaffected by mixtures. Intrinsic caspase 9 activity was inhibited by estradiol, and by xenoestrogen combinations (at 10-14 and 10-8 M). Mixtures of xenoestrogens impeded the estradiol-induced release of prolactin.

Conclusions: In mixtures expected to be found in contaminated environments, xenoestrogens can have dramatic disrupting effects on hormonal mechanisms of cell regulation and their downstream functional responses, altering cellular responses to physiologic estrogens.

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XEs cause PRL release, and XE mixtures alter these responses. We measured PRL release into the culture medium by RIA after a 1-min exposure to (A) individual XEs (10-15 M – 10-7 M) and also as (B) XE mixtures (10-15 M – 10-7 M) with a constant physiologic E2 concentration (10-9 M) throughout the dose-response range of the XEs. The amount of PRL secreted for each well was normalized to the CV value for cell number, and expressed as a percentage of vehicle (V)-treated controls. Error bars are means ± S.E. For positive (E2) and negative (V) controls, the width of the bars indicate error ranges (V ±1.5; 10-9 M E2 ± 1.6). n = 24 over 3 experiments. * = p < 0.05 compared to vehicle; in B, # = p < 0.05 compared to 10-9 M E2. The E2 (10-9 M) response is significantly different compared to the vehicle control.
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Figure 7: XEs cause PRL release, and XE mixtures alter these responses. We measured PRL release into the culture medium by RIA after a 1-min exposure to (A) individual XEs (10-15 M – 10-7 M) and also as (B) XE mixtures (10-15 M – 10-7 M) with a constant physiologic E2 concentration (10-9 M) throughout the dose-response range of the XEs. The amount of PRL secreted for each well was normalized to the CV value for cell number, and expressed as a percentage of vehicle (V)-treated controls. Error bars are means ± S.E. For positive (E2) and negative (V) controls, the width of the bars indicate error ranges (V ±1.5; 10-9 M E2 ± 1.6). n = 24 over 3 experiments. * = p < 0.05 compared to vehicle; in B, # = p < 0.05 compared to 10-9 M E2. The E2 (10-9 M) response is significantly different compared to the vehicle control.

Mentions: The rapid non-genomic secretion response for PRL caused by estrogenic exposure in GH3/B6/F10 cells has become a standard tool in our lab for evaluating functional endpoints [30-32,38,42,43]. After a 1-min exposure, BPS could not increase PRL secretion as did E2 (Figure 7A [28]). At certain concentrations, BPA and NP were able to significantly increase PRL release, even above that caused by nM E2 (Figure 7A); the shape of these dose–response curves are non-monotonic (as confirmed by determining that values at the peaks of activation were statistically different than those at other, usually higher, concentrations). As XE mixtures with 10-9 M E2, the 3-compound mixture inhibited E2-induced PRL release at low concentrations (significantly at 10-11 M). The 4-compound mixture caused more extreme inhibitions, even below the vehicle level at the lower concentrations (Figure 7B). Though the 4-compound mixture at 10-8 M appears to have resulted in PRL release, the errors in these mixture measurements did not allow this response to be distinguished as statistically different from vehicle, and the mixed signaling patterns caused by the multiple ligands may contribute to this variability.


Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells.

Viñas R, Watson CS - Environ Health (2013)

XEs cause PRL release, and XE mixtures alter these responses. We measured PRL release into the culture medium by RIA after a 1-min exposure to (A) individual XEs (10-15 M – 10-7 M) and also as (B) XE mixtures (10-15 M – 10-7 M) with a constant physiologic E2 concentration (10-9 M) throughout the dose-response range of the XEs. The amount of PRL secreted for each well was normalized to the CV value for cell number, and expressed as a percentage of vehicle (V)-treated controls. Error bars are means ± S.E. For positive (E2) and negative (V) controls, the width of the bars indicate error ranges (V ±1.5; 10-9 M E2 ± 1.6). n = 24 over 3 experiments. * = p < 0.05 compared to vehicle; in B, # = p < 0.05 compared to 10-9 M E2. The E2 (10-9 M) response is significantly different compared to the vehicle control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643824&req=5

Figure 7: XEs cause PRL release, and XE mixtures alter these responses. We measured PRL release into the culture medium by RIA after a 1-min exposure to (A) individual XEs (10-15 M – 10-7 M) and also as (B) XE mixtures (10-15 M – 10-7 M) with a constant physiologic E2 concentration (10-9 M) throughout the dose-response range of the XEs. The amount of PRL secreted for each well was normalized to the CV value for cell number, and expressed as a percentage of vehicle (V)-treated controls. Error bars are means ± S.E. For positive (E2) and negative (V) controls, the width of the bars indicate error ranges (V ±1.5; 10-9 M E2 ± 1.6). n = 24 over 3 experiments. * = p < 0.05 compared to vehicle; in B, # = p < 0.05 compared to 10-9 M E2. The E2 (10-9 M) response is significantly different compared to the vehicle control.
Mentions: The rapid non-genomic secretion response for PRL caused by estrogenic exposure in GH3/B6/F10 cells has become a standard tool in our lab for evaluating functional endpoints [30-32,38,42,43]. After a 1-min exposure, BPS could not increase PRL secretion as did E2 (Figure 7A [28]). At certain concentrations, BPA and NP were able to significantly increase PRL release, even above that caused by nM E2 (Figure 7A); the shape of these dose–response curves are non-monotonic (as confirmed by determining that values at the peaks of activation were statistically different than those at other, usually higher, concentrations). As XE mixtures with 10-9 M E2, the 3-compound mixture inhibited E2-induced PRL release at low concentrations (significantly at 10-11 M). The 4-compound mixture caused more extreme inhibitions, even below the vehicle level at the lower concentrations (Figure 7B). Though the 4-compound mixture at 10-8 M appears to have resulted in PRL release, the errors in these mixture measurements did not allow this response to be distinguished as statistically different from vehicle, and the mixed signaling patterns caused by the multiple ligands may contribute to this variability.

Bottom Line: Our study examines the effects of xenoestrogen mixtures on estradiol-induced non-genomic signaling and associated functional responses.Individual bisphenol compounds did not activate JNK, while nonylphenol did; however, the combination of two or three xenoestrogens with estradiol generated an enhanced non-monotonic JNK dose-response.In mixtures expected to be found in contaminated environments, xenoestrogens can have dramatic disrupting effects on hormonal mechanisms of cell regulation and their downstream functional responses, altering cellular responses to physiologic estrogens.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: Our study examines the effects of xenoestrogen mixtures on estradiol-induced non-genomic signaling and associated functional responses. Bisphenol-A, used to manufacture plastic consumer products, and nonylphenol, a surfactant, are estrogenic by a variety of assays, including altering many intracellular signaling pathways; bisphenol-S is now used as a bisphenol-A substitute. All three compounds contaminate the environment globally. We previously showed that bisphenol-S, bisphenol-A, and nonylphenol alone rapidly activated several kinases at very low concentrations in the GH3/B6/F10 rat pituitary cell line.

Methods: For each assay we compared the response of individual xenoestrogens at environmentally relevant concentrations (10-15 -10-7 M), to their mixture effects on 10-9 M estradiol-induced responses. We used a medium-throughput plate immunoassay to quantify phosphorylations of extracellular signal-regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs). Cell numbers were assessed by crystal violet assay to compare the proliferative effects. Apoptosis was assessed by measuring caspase 8 and 9 activities via the release of the fluorescent product 7-amino-4-trifluoromethylcoumarin. Prolactin release was measured by radio-immunoassay after a 1 min exposure to all individual and combinations of estrogens.

Results: Individual xenoestrogens elicited phospho-activation of ERK in a non-monotonic dose- (fM-nM) and mostly oscillating time-dependent (2.5-60 min) manner. When multiple xenoestrogens were combined with nM estradiol, the physiologic estrogen's response was attenuated. Individual bisphenol compounds did not activate JNK, while nonylphenol did; however, the combination of two or three xenoestrogens with estradiol generated an enhanced non-monotonic JNK dose-response. Estradiol and all xenoestrogen compounds induced cell proliferation individually, while the mixtures of these compounds with estradiol suppressed proliferation below that of the vehicle control, suggesting a possible apoptotic response. Extrinsic caspase 8 activity was suppressed by estradiol, elevated by bisphenol S, and unaffected by mixtures. Intrinsic caspase 9 activity was inhibited by estradiol, and by xenoestrogen combinations (at 10-14 and 10-8 M). Mixtures of xenoestrogens impeded the estradiol-induced release of prolactin.

Conclusions: In mixtures expected to be found in contaminated environments, xenoestrogens can have dramatic disrupting effects on hormonal mechanisms of cell regulation and their downstream functional responses, altering cellular responses to physiologic estrogens.

Show MeSH
Related in: MedlinePlus