Combinations of physiologic estrogens with xenoestrogens alter ERK phosphorylation profiles in rat pituitary cells

Abstract

Background: Estrogens are potent nongenomic phospho-activators of extracellular-signal-regulated kinases (ERKs). A major concern about the toxicity of xenoestrogens (XEs) is potential alteration of responses to physiologic estrogens when XEs are present simultaneously.

Objectives: We examined estrogen-induced ERK activation, comparing the abilities of structurally related XEs (alkylphenols and bisphenol A) to alter ERK responses induced by physiologic concentrations (1 nM) of estradiol (E2), estrone (E1), and estriol (E3).

Methods: We quantified hormone/mimetic-induced ERK phosphorylations in the GH3/B6/F10 rat pituitary cell line using a plate immunoassay, comparing effects with those on cell proliferation and by estrogen receptor subtype-selective ligands.

Results: Alone, these structurally related XEs activate ERKs in an oscillating temporal pattern similar (but not identical) to that with physiologic estrogens. The potency of all estrogens was similar (active between femtomolar and nanomolar concentrations). XEs potently disrupted physiologic estrogen signaling at low, environmentally relevant concentrations. Generally, XEs potentiated (at the lowest, subpicomolar concentrations) and attenuated (at the highest, picomolar to 100 nM concentrations) the actions of the physiologic estrogens. Some XEs showed pronounced nonmonotonic responses/inhibitions. The phosphorylated ERK and proliferative responses to receptor-selective ligands were only partially correlated.

Conclusions: XEs are both imperfect potent estrogens and endocrine disruptors; the more efficacious an XE, the more it disrupts actions of physiologic estrogens. This ability to disrupt physiologic estrogen signaling suggests that XEs may disturb normal functioning at life stages where actions of particular estrogens are important (e.g., development, reproductive cycling, pregnancy, menopause).

Figure 1

Time-dependent changes in pERK elicited by combinations of short-chain alkylphenols with E₂, E₁, or E₃. GH₃/B6/F10 cells were cotreated with 1 nM EP (A–C) or PP (D–F) and 1 nM E₂ (A,D), E₁ (B,E), or E₃ (C,F). The pERK levels were measured by plate immunoassay after different times of cotreatment. *p < 0.05 compared with vehicle-treated control cells, shaded to match each set of data symbols (*, , ✫). ^#p < 0.05 compared with cells treated with 1 nM E₂, E₁, or E₃ alone.

Figure 2

Time-dependent changes in pERK elicited by combinations of long-chain alkylphenols with E₂, E₁, or E₃. GH₃/B6/F10 cells were cotreated with 1 nM OP (A–C) or NP (D–F) and 1 nM E₂ (A,D), E₁ (B,E), or E₃ (C,F). The pERK levels were measured by plate immunoassay after different times of cotreatment. *p < 0.05 compared with vehicle-treated control cells, shaded to match each set of data symbols (*, ). ^#p < 0.05 compared with cells treated with 1 nM E₂, E₁, or E₃ alone.

Figure 3

Time-dependent changes in pERK elicited by combinations of BPA at two different environmentally relevant concentrations. GH₃/B6/F10 cells were cotreated with 10⁻¹⁴ M BPA (A–C) or 10⁻⁹ M BPA (D–F) plus 1 nM E₂ (A,D), E₁ (B,E), or E₃ (C,F). The pERK levels were measured after different times of cotreatment. *p < 0.05 compared with vehicle-treated control cells, shaded to match each set of data symbols (*, , ✫). #p < 0.05 compared with cells treated with 1 nM E₂, E₁, or E₃ alone.

Figure 4

Concentration-dependent changes in pERK caused by short-chain alkylphenols. Cells were treated for 5 min with a combination of 1 nM E₂ (A,D), E₁ (B,E), or E₃ (C,F) plus different concentrations of EP (A–C) or PP (D–F), and pERK was assayed. The blue horizontal bar indicates the pERK level and error range in vehicle-treated cells (V); the crosshatched horizontal bar indicates the pERK value in cells treated with nanomolar concentrations of E₂, E₁, or E₃ alone. *p < 0.05 compared with vehicle-treated cells. ^#p < 0.05 compared with cells treated with E₂, E₁, or E₃ alone.

Figure 5

Concentration-dependent changes in pERK caused by the long-chain alkylphenols. Cells were treated for 5 min with a combination of 1 nM E₂ (A,D), E₁ (B,E), or E₃ (C,F) plus different concentrations of OP (A–D) or NP (D–F), and pERK was assayed. The blue horizontal bar indicates the pERK level and error range in vehicle-treated cells (V); the crosshatched horizontal bar indicates the pERK value in cells treated with nanomolar concentrations of E₂, E₁, or E₃ alone. *p < 0.05 compared with vehicle-treated cells. #p < 0.05 compared with cells treated with E₂, E₁, or E₃ alone.

Figure 6

Concentration-dependent alteration of physiologic estrogen-induced pERK by BPA. Cells were treated for 5 min with different concentrations of BPA with or without 1 nM E₂ (A), E₁ (B), or E₃ (C). The blue horizontal bar indicates the pERK level and error range in vehicle-treated cells (V); the crosshatched horizontal bar indicates the pERK value in cells treated with nanomolar concentrations of E₂, E₁, or E₃ alone. *p < 0.05 compared with vehicle-treated cells. ^#p < 0.05 compared with cells treated with E₂, E₁, or E₃ alone.

Figure 7

Concentration dependence of pERK (A–D) and proliferation (E–H) on selective ER agonists. pERK was measured in cells after 5 min of treatment with different concentrations of E₂ (A), the ERα agonist PPT (B), the ERβ agonist DPN (C), or the GPER agonist G1 (D). Proliferation was measured (via the crystal violet assay) in cells treated for 3 days with matching concentrations of E₂ (E), PPT (F), DPN (G), or G1 (H). V, vehicle-treated cells. *p < 0.05 compared with vehicle-treated cells.