| Alkylphenol Xenoestrogens with Varying Carbon Chain Lengths
Differentially and Potently Activate Signaling and Functional Responses
in GH3/B6/F10 Somatomammotropes Mikhail Y. Kochukov, Yow-Jiun Jeng, and Cheryl S. Watson Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas, USA Abstract
Background: Alkylphenols varying in their side-chain lengths [ethyl-, propyl-, octyl-, and nonylphenol (EP, PP, OP, and NP, respectively) ] and bisphenol A (BPA) represent a large group of structurally related xenoestrogens that have endocrine-disruptive effects. Their rapid nongenomic effects that depend on structure for cell signaling and resulting functions are unknown. Objectives: We compared nongenomic estrogenic activities of alkylphenols with BPA and 17β–estradiol (E2) in membrane estrogen receptor-α–enriched GH3/B6/F10 pituitary tumor cells. These actions included calcium (Ca) signaling, prolactin (PRL) release, extracellular-regulated kinase (ERK) phosphorylation, and cell proliferation. Methods: We imaged Ca using fura-2, measured PRL release via radioimmunoassay, detected ERK phosphorylation by fixed cell immunoassay, and estimated cell number using the crystal violet assay. Results: All compounds caused increases in Ca oscillation frequency and intracellular Ca volume at 100 fM to 1 nM concentrations, although long-chain alkylphenols were most effective. All estrogens caused rapid PRL release at concentrations as low as 1 fM to 10 pM ; the potency of EP, PP, and NP exceeded that of E2. All compounds at 1 nM produced similar increases in ERK phosphorylation, causing rapid peaks at 2.5–5 min, followed by inactivation and additional 60-min peaks (except for BPA) . Dose–response patterns of ERK activation at 5 min were similar for E2, BPA, and PP, whereas EP caused larger effects. Only E2 and NP increased cell number. Some rapid estrogenic responses showed correlations with the hydrophobicity of estrogenic molecules ; the more hydrophobic OP and NP were superior at Ca and cell proliferation responses, whereas the less hydrophobic EP and PP were better at ERK activations. Conclusions: Alkylphenols are potent estrogens in evoking these nongenomic responses contributing to complex functions ; their hydrophobicity can largely predict these behaviors. Key words: bisphenol A, calcium oscillation, ERK activation, estradiol, hydrophobicity, nongenomic response, prolactin release. Environ Health Perspect 117:723–730 (2009) . doi:10.1289/ehp.0800182 available via http://dx.doi.org/ [Online 31 December 2008]
Address correspondence to C.S. Watson, Biochemistry and Molecular Biology Dept., University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0645 USA. Telephone or fax: (409) 772-2382. E-mail: cswatson@utmb.edu We thank D. Konkel for expert editorial assistance, L. Vergara for core lab assistance with the calcium imaging studies, and W. Bolen and V.M.S. Ramanujam for helpful discussions about the chemistry of the compounds. This study was supported by the National Institutes of Health grant R01 ES015292. The authors declare they have no competing financial interests. Received 11 September 2008 ; accepted 31 December 2008.
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