title: Mixtures of xenoestrogens disrupt estradiol-induced non-genomic signaling and downstream functions in pituitary cells.
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.
authors.forename: René
authors.initials: R
authors.lastname: Viñas
authors.forename: Cheryl S
authors.initials: CS
authors.lastname: Watson
mesh_terms: D000818:Animals; D001559:Benzhydryl Compounds / Q000633:toxicity; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004785:Environmental Pollutants / Q000633:toxicity*; D004958:Estradiol / Q000378:metabolism; D004967:Estrogens / Q000378:metabolism* / Q000633:toxicity*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D010636:Phenols / Q000633:toxicity; D010766:Phosphorylation; D010902:Pituitary Gland / Q000187:drug effects*; D051381:Rats; D015398:Signal Transduction; D013450:Sulfones / Q000633:toxicity; D000098831:Bisphenol S Compounds; D000098826:Bisphenol A Compounds
chemical_list: D001559:Benzhydryl Compounds; D004785:Environmental Pollutants; D004958:Estradiol; D004967:Estrogens; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D010636:Phenols; D013450:Sulfones; C025256:nonylphenol; C543008:bisphenol S; D000098831:Bisphenol S Compounds; C006780:bisphenol A; D000098826:Bisphenol A Compounds