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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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Timing of JNK activation by E2, XEs, and XE/E2 mixtures. Rat pituitary cells were exposed to BPS (10-14 M), BPA (10-14 M), NP (10-14 M) and/or E2 (10-9 M) over a 60- min time course. Responses to individual XEs (A) and mixtures (B) were measured by plate immunoassay; the pNp signal generated for each well was normalized to cell number (measured by the CV assay). Values are expressed as percentage of vehicle (V)-treated controls. All error bars represent S.E M. The width of the vehicle bar represents a S.E. of ± 1.0 (n = 24 over 3 experiments). * = p < 0.05 compared to vehicle (V); # = p < 0.05 compared to 10-9 M E2.
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Figure 2: Timing of JNK activation by E2, XEs, and XE/E2 mixtures. Rat pituitary cells were exposed to BPS (10-14 M), BPA (10-14 M), NP (10-14 M) and/or E2 (10-9 M) over a 60- min time course. Responses to individual XEs (A) and mixtures (B) were measured by plate immunoassay; the pNp signal generated for each well was normalized to cell number (measured by the CV assay). Values are expressed as percentage of vehicle (V)-treated controls. All error bars represent S.E M. The width of the vehicle bar represents a S.E. of ± 1.0 (n = 24 over 3 experiments). * = p < 0.05 compared to vehicle (V); # = p < 0.05 compared to 10-9 M E2.

Mentions: Neither BPA nor BPS could maintain activation of JNK by themselves during the 60-min time course (Figure 2A); however, their combination with E2 (3-compound mixture) did activate JNK with a significant 60-min response, by which time the response to E2 had declined to control values (Figure 2B). As a 4-compound mixture, E2 plus all XEs inactivated JNK to below vehicle control values early in the time course, but then activated and sustained pJNK after 30 min. Overall, these combinations with XEs markedly attenuated the E2-induced JNK response.


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

Viñas R, Watson CS - Environ Health (2013)

Timing of JNK activation by E2, XEs, and XE/E2 mixtures. Rat pituitary cells were exposed to BPS (10-14 M), BPA (10-14 M), NP (10-14 M) and/or E2 (10-9 M) over a 60- min time course. Responses to individual XEs (A) and mixtures (B) were measured by plate immunoassay; the pNp signal generated for each well was normalized to cell number (measured by the CV assay). Values are expressed as percentage of vehicle (V)-treated controls. All error bars represent S.E M. The width of the vehicle bar represents a S.E. of ± 1.0 (n = 24 over 3 experiments). * = p < 0.05 compared to vehicle (V); # = p < 0.05 compared to 10-9 M E2.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Timing of JNK activation by E2, XEs, and XE/E2 mixtures. Rat pituitary cells were exposed to BPS (10-14 M), BPA (10-14 M), NP (10-14 M) and/or E2 (10-9 M) over a 60- min time course. Responses to individual XEs (A) and mixtures (B) were measured by plate immunoassay; the pNp signal generated for each well was normalized to cell number (measured by the CV assay). Values are expressed as percentage of vehicle (V)-treated controls. All error bars represent S.E M. The width of the vehicle bar represents a S.E. of ± 1.0 (n = 24 over 3 experiments). * = p < 0.05 compared to vehicle (V); # = p < 0.05 compared to 10-9 M E2.
Mentions: Neither BPA nor BPS could maintain activation of JNK by themselves during the 60-min time course (Figure 2A); however, their combination with E2 (3-compound mixture) did activate JNK with a significant 60-min response, by which time the response to E2 had declined to control values (Figure 2B). As a 4-compound mixture, E2 plus all XEs inactivated JNK to below vehicle control values early in the time course, but then activated and sustained pJNK after 30 min. Overall, these combinations with XEs markedly attenuated the E2-induced JNK response.

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