Differential regulation of dopamine transporter function and location by low concentrations of environmental estrogens and 17beta-estradiol

Abstract

Background: The effects of 17beta-estradiol (E2) and xenoestrogens (XEs) on dopamine transport may have important implications for the increased incidence of neurologic disorders, especially in women during life stages characterized by frequent hormonal fluctuations.

Objective: We examined low concentrations of XEs [dieldrin, endosulfan, o', p'-dichlorodiphenyl-ethylene (DDE), nonylphenol (NP), and bisphenol A (BPA)] for nongenomic actions via action of membrane estrogen receptors (ERs).

Methods: We measured activity of the dopamine transporter (DAT) by the efflux of 3H-dopamine in nontransfected nerve growth factor-differentiated PC12 rat pheochromocytoma cells expressing membrane DAT, ER-alpha, ER-beta, and G-protein-coupled receptor 30. We used a plate immunoassay to monitor trafficking of these proteins.

Results: All compounds at 1 nM either caused efflux or inhibited efflux, or both; each compound evoked a distinct oscillatory pattern. At optimal times for each effect, we examined different concentrations of XEs. All XEs were active at some concentration < 10 nM, and dose responses were all nonmonotonic. For example, 10(-14) to 10(-11) M DDE caused significant efflux inhibition, whereas NP and BPA enhanced or inhibited efflux at several concentrations. We also measured the effects of E2/XE combinations; DDE potentiated E(2)-mediated dopamine efflux, whereas BPA inhibited it. In E2-induced efflux, 15% more ER-alpha trafficked to the membrane, whereas ER-beta waned; during BPA-induced efflux, 20% more DAT was trafficked to the plasma membrane.

Conclusions: Low levels of environmental estrogen contaminants acting as endocrine disruptors via membrane ERs can alter dopamine efflux temporal patterning and the trafficking of DAT and membrane ERs, providing a cellular mechanism that could explain the disruption of physiologic neurotransmitter function.

Keywords: dopamine efflux; low concentrations; nongenomic; xenoestrogens.

Figure 1

Effects of 10⁻⁹ M E₂ (n = 18), DES (n = 23), DDE (n = 14), NP (n = 18), dieldrin (n = 14), BPA (n = 14), and endosulfan (n = 18) on the dopamine efflux time course. The 10⁻⁹ M E₂ time course is reproduced from Alyea et al. (2008), with permission of Journal of Neurochemistry, and is included here for purposes of comparison. Values are means and SEs. Points above the zero point line indicate a positive efflux of dopamine from the cells. *p < 0.05 compared with control. ^#p < 0.05 compared with E₂ treatment.

Figure 2

Concentration-dependent dopamine efflux patterns for E₂ and XEs at 9 (A) and 5 min (B), using optimal time points for each compound chosen from the 10⁻⁹ M time course (Figure 1). (A) A 9-min dopamine efflux for E₂, DES, endosulfan, DDE, NP, and BPA at concentrations ranging from 10⁻¹⁴ to 10⁻⁹ M. (B) A 5-min dopamine efflux for E₂, dieldrin, NP, and BPA at concentrations ranging from 10⁻¹⁴ to 10⁻⁹ M. Values are means and SEs; numbers per treatment are as follows: E₂, n = 18; dieldrin, n= 12; DES, n= 18; endosulfan, n= 12; DDE, n= 12; BPA, n= 23; NP, n= 15. Points above the zero point line indicate a positive efflux of dopamine from the cells. *p < 0.05 compared with control. #p < 0.05 compared with E₂ treatment.

Figure 3

Quantitative plate assay measuring immunoreactive protein levels for plasma membrane and total ER-α, ER-β, GPR30, and DAT after treatment with E₂ for 9 min (A), or BPA for 5 (B) or 9 min (C). (A) 10⁻⁹ M E₂ for 9 min (dopamine efflux was significantly increased at this time point and concentration). (B) 10⁻¹⁴ M BPA for 5 min (DA efflux was significantly increased at this time point and concentration). (C) 10⁻¹² M BPA for 9 min (DA efflux was significantly inhibited at this time point and concentration). (A) is modified from Alyea et al. (2008) with permission of Journal of Neurochemistry. Values are means and SEs; n = 24 for all compounds. Points above the zero point line indicate a positive efflux of dopamine from the cells. *p < 0.05 compared with control. ^#p < 0.05 compared with membrane, shown here for comparison with changes caused by BPA alone.

Figure 4

Effects of physiologic levels of E₂ (10⁻⁹ M) combined with multiple concentrations of DDE or BPA measured by dopamine efflux assay. (A) E₂ plus increasing concentrations of DDE during a 9-min dopamine efflux assay compared with E₂-mediated dopamine efflux and DDE-mediated dopamine efflux. (B) E₂ plus increasing concentrations of BPA during a 5-min dopamine efflux assay compared with E₂-mediated dopamine efflux and BPA-mediated dopamine efflux. Values are means and SEs; numbers per treatment are as follows: E₂, n = 18; BPA, n = 14; DDE, n = 14; DDE + E₂, n = 15; BPA + E₂, n = 15. Points above the zero point line indicate a positive efflux of dopamine from the cells. *p < 0.05 compared with control for combinations. ^#p < 0.05 compared with E₂ treatment. ^##p < 0.05 compared with control for E₂.