Limits...
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.

Show MeSH

Related in: MedlinePlus

Activation or deactivation of caspases 8 and 9 by E2, XEs, and mixtures. Over an 8-hr exposure period we measured caspase 8 (A) and 9 (B) activity evoked by two different concentrations of BPS, BPA, and NP (10-14; 10-8 M) separately and together, with each other and with a physiological level of E2 (10-9 M). E2 (10-9 M) is at a constant concentration throughout. Caspase activity was measured by the release of a fluorogenic product (AFC) expressed as the percentage of vehicle (V)-treated controls. Staurosporine (STR, 500nM) was used as a positive control for induction of caspase activities compared to its own DMSO V control (n = 24 over 3 experiments). Error bars are means ± S.E. * = p < 0.05 compared to V.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3643824&req=5

Figure 6: Activation or deactivation of caspases 8 and 9 by E2, XEs, and mixtures. Over an 8-hr exposure period we measured caspase 8 (A) and 9 (B) activity evoked by two different concentrations of BPS, BPA, and NP (10-14; 10-8 M) separately and together, with each other and with a physiological level of E2 (10-9 M). E2 (10-9 M) is at a constant concentration throughout. Caspase activity was measured by the release of a fluorogenic product (AFC) expressed as the percentage of vehicle (V)-treated controls. Staurosporine (STR, 500nM) was used as a positive control for induction of caspase activities compared to its own DMSO V control (n = 24 over 3 experiments). Error bars are means ± S.E. * = p < 0.05 compared to V.

Mentions: Initiation of apoptosis is one of several factors that can influence cell numbers; we therefore assayed caspase 8 and 9 activities to determine if the extrinsic or intrinsic apoptotic pathways were activated over an 8-hr exposure period, the optimum time that was determined previously [28]. Caspase 8 was significantly activated by BPS at both concentrations used (10-14 M and 10-8 M), while BPA, NP, and the mixture solutions at their respective concentrations did not result in significant activations (Figure 6A). Activations of caspase 9 were not detected with either individual XEs or mixtures, indicating that the extrinsic pathway (caspase 8) and not the intrinsic pathway (caspase 9) is the primary apoptotic pathway activated. However, both mixture combinations at the highest concentrations (10-8 M) resulted in a significant deactivation of caspase 9 activities (Figure 6B). Staurosporine, the positive control for activation, was active on both caspases, as expected. E2 by itself suppressed caspase activity below vehicle controls for both apoptotic pathways, as we had seen previously [28,40].


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

Viñas R, Watson CS - Environ Health (2013)

Activation or deactivation of caspases 8 and 9 by E2, XEs, and mixtures. Over an 8-hr exposure period we measured caspase 8 (A) and 9 (B) activity evoked by two different concentrations of BPS, BPA, and NP (10-14; 10-8 M) separately and together, with each other and with a physiological level of E2 (10-9 M). E2 (10-9 M) is at a constant concentration throughout. Caspase activity was measured by the release of a fluorogenic product (AFC) expressed as the percentage of vehicle (V)-treated controls. Staurosporine (STR, 500nM) was used as a positive control for induction of caspase activities compared to its own DMSO V control (n = 24 over 3 experiments). Error bars are means ± S.E. * = p < 0.05 compared to V.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Activation or deactivation of caspases 8 and 9 by E2, XEs, and mixtures. Over an 8-hr exposure period we measured caspase 8 (A) and 9 (B) activity evoked by two different concentrations of BPS, BPA, and NP (10-14; 10-8 M) separately and together, with each other and with a physiological level of E2 (10-9 M). E2 (10-9 M) is at a constant concentration throughout. Caspase activity was measured by the release of a fluorogenic product (AFC) expressed as the percentage of vehicle (V)-treated controls. Staurosporine (STR, 500nM) was used as a positive control for induction of caspase activities compared to its own DMSO V control (n = 24 over 3 experiments). Error bars are means ± S.E. * = p < 0.05 compared to V.
Mentions: Initiation of apoptosis is one of several factors that can influence cell numbers; we therefore assayed caspase 8 and 9 activities to determine if the extrinsic or intrinsic apoptotic pathways were activated over an 8-hr exposure period, the optimum time that was determined previously [28]. Caspase 8 was significantly activated by BPS at both concentrations used (10-14 M and 10-8 M), while BPA, NP, and the mixture solutions at their respective concentrations did not result in significant activations (Figure 6A). Activations of caspase 9 were not detected with either individual XEs or mixtures, indicating that the extrinsic pathway (caspase 8) and not the intrinsic pathway (caspase 9) is the primary apoptotic pathway activated. However, both mixture combinations at the highest concentrations (10-8 M) resulted in a significant deactivation of caspase 9 activities (Figure 6B). Staurosporine, the positive control for activation, was active on both caspases, as expected. E2 by itself suppressed caspase activity below vehicle controls for both apoptotic pathways, as we had seen previously [28,40].

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