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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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JNK activation dose-response analysis by E2, XEs, and mixtures. GH3/B6/F10 rat pituitary cells were exposed to increasing concentrations (10-15 M – 10-7 M) of BPS, BPA, and NP compared to a single physiological level of E2 (10-9 M). E2 (10-9 M) is at a constant concentration throughout the dose-response. Individual XEs (A) and XE mixtures (B) were measured by plate immunoassay at a 5-min exposure time. All error bars represent S.E M. The widths of the vehicle and E2 [10-9 M] bars represent a S.E. of ± 1.3 and ± 1.2 respectively, (n = 24 over 3 experiments). * = p < 0.05 compared to vehicle (V); # = 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 4: JNK activation dose-response analysis by E2, XEs, and mixtures. GH3/B6/F10 rat pituitary cells were exposed to increasing concentrations (10-15 M – 10-7 M) of BPS, BPA, and NP compared to a single physiological level of E2 (10-9 M). E2 (10-9 M) is at a constant concentration throughout the dose-response. Individual XEs (A) and XE mixtures (B) were measured by plate immunoassay at a 5-min exposure time. All error bars represent S.E M. The widths of the vehicle and E2 [10-9 M] bars represent a S.E. of ± 1.3 and ± 1.2 respectively, (n = 24 over 3 experiments). * = p < 0.05 compared to vehicle (V); # = p < 0.05 compared to 10-9 M E2. The E2 (10-9 M) response is significantly different compared to the vehicle control.

Mentions: Individual bisphenol compounds deactivated pJNK below vehicle levels (Figure 4A), unlike E2 and NP that both activated JNK. However, when E2 was administered together with both bisphenol compounds (Figure 4B), JNK was strongly activated, featuring a non-monotonic dose–response curve with the lowest concentrations evoking the largest responses; the 4-compound mixture evoked no activation of JNK and was consistently, though not statistically, below the level of the response to vehicle, thus erasing the response to 1 nM E2.


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

Viñas R, Watson CS - Environ Health (2013)

JNK activation dose-response analysis by E2, XEs, and mixtures. GH3/B6/F10 rat pituitary cells were exposed to increasing concentrations (10-15 M – 10-7 M) of BPS, BPA, and NP compared to a single physiological level of E2 (10-9 M). E2 (10-9 M) is at a constant concentration throughout the dose-response. Individual XEs (A) and XE mixtures (B) were measured by plate immunoassay at a 5-min exposure time. All error bars represent S.E M. The widths of the vehicle and E2 [10-9 M] bars represent a S.E. of ± 1.3 and ± 1.2 respectively, (n = 24 over 3 experiments). * = p < 0.05 compared to vehicle (V); # = 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 4: JNK activation dose-response analysis by E2, XEs, and mixtures. GH3/B6/F10 rat pituitary cells were exposed to increasing concentrations (10-15 M – 10-7 M) of BPS, BPA, and NP compared to a single physiological level of E2 (10-9 M). E2 (10-9 M) is at a constant concentration throughout the dose-response. Individual XEs (A) and XE mixtures (B) were measured by plate immunoassay at a 5-min exposure time. All error bars represent S.E M. The widths of the vehicle and E2 [10-9 M] bars represent a S.E. of ± 1.3 and ± 1.2 respectively, (n = 24 over 3 experiments). * = p < 0.05 compared to vehicle (V); # = p < 0.05 compared to 10-9 M E2. The E2 (10-9 M) response is significantly different compared to the vehicle control.
Mentions: Individual bisphenol compounds deactivated pJNK below vehicle levels (Figure 4A), unlike E2 and NP that both activated JNK. However, when E2 was administered together with both bisphenol compounds (Figure 4B), JNK was strongly activated, featuring a non-monotonic dose–response curve with the lowest concentrations evoking the largest responses; the 4-compound mixture evoked no activation of JNK and was consistently, though not statistically, below the level of the response to vehicle, thus erasing the response to 1 nM E2.

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