CHEMICAL MIXTURES OF XENOESTROGENS AND THEIR METABOLITES ALTER ESTRADIOL-INDUCED NON-GENOMIC SIGNALING

BACKGROUND: Xenoestrogens (XEs) pose a threat to human health by disrupting natural responses of physiologic estrogens. Bisphenol-A (BPA), a plastics monomer, is potently estrogenic via non-genomic signaling; however, bisphenol-S (BPS), a BPA substitute, has unknown endocrine actions. Nonylphenol is a surfactant and a ubiquitous contaminant. Our goals were to use a well-characterized estrogen-responsive cell model, the GH3/B6/F10 pituitary line, to characterize the potentially potent nongenomic signaling and functional responses to these compounds alone, as mixtures, as metabolites and congeners, and in combination with estradiol (E2), while also automating these assays to facilitate future screening of a potentially large number of XEs. METHODOLOGY: Environmentally relevant concentrations of XEs (10-15-10-7M) were assessed individually and as mixtures by challenging 1nM E2-induced responses. We quantified phospho-activation of extracellular signal-regulated kinases (ERKs) and c-Jun-N-terminal kinases (JNKs) by multi-well plate immunoassays. Cell proliferation was assessed by crystal violet assay, while apoptosis (caspase-8, -9) was assessed via the release of 7-amino-4-trifluoromethylcoumarin. Prolactin release was measured by radio-immunoassay after 1 min XE exposures. The BIOMEK FXP workstation was used to develop an automated screening system for changes in MAPKs activities due to XE exposures. RESULTS: XEs often activated MAPKs in a non-monotonic dose- and oscillating time-dependent (2.5-60 min) manner and attenuated 1nM E2 responses. While individual bisphenols did not activate JNK as NP did, the combination of all XEs with E2 generated an enhanced non-monotonic JNK dose-response. E2 and all XE compounds induced cell proliferation, while mixtures of these compounds with E2 suppressed proliferation. Caspase 8 activity was suppressed by E2, and elevated by BPS, while caspase 9 activity was inhibited by E2 and some XE combinations at later times. Mono- and di-chlorinated BPA activated, while tri-chlorinated BPA dephosphorylated ERK. Di- and tri-chlorinated BPAs caused JNK dephosphorylation. Phase II metabolites (sulfated and glucuronidated) were mostly unable to activate either kinase and in some cases severely inactivated them. CONCLUSIONS: Novel chemical analogues and conjugated forms of BPA individually or as mixtures with other known XEs had dramatic disrupting effects on physiologic estrogens, disrupting mechanisms of cell regulation and their downstream functional responses.