Alkylphenol xenoestrogens with varying carbon chain lengths differentially and potently activate signaling and functional responses in GH₃/B₆/F10 somatomammotropes

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.