Nongenomic effects of estradiol vs. the birth control estrogen ethinyl estradiol on signaling and cell proliferation in pituitary tumor cells, and differences in the ability of R-equol to neutralize or enhance these effects

Abstract

Ethinyl estradiol (EE₂, the active component of many birth control formulations) persists in treated waste waters and it has become a concerning endocrine-disrupting contaminant throughout the world. Previous studies have not examined the behavior of EE₂ in nongenomic signaling pathways and the subsequent functional responses (either alone or in mixtures) or conducted comparisons with the physiological estrogen estradiol (E₂). In this study, mitogen-activated protein kinases (MAPKs), ERK, and JNK were activated in pituitary tumor cells by fM EE₂, but p38 activation was insensitive to <nM doses. Both E₂ and EE₂ (10 fM to 10 nM) caused cell proliferation as well as triggering intracellular calcium increases and GTP charging of Gαi. E₂ was more effective at causing prolactin release. Previously, we reported that the soy-based diadzein synthetic metabolite R-equol (R-eq) activated nongenomic responses in pituitary cells and impaired the actions of E₂, thereby affecting both prolactin release and cell proliferation. In the present study, as expected, R-eq activated all MAPKs in a dose-dependent manner at concentrations ranging from fM to 100 nm, and it also modified the effects of environmentally and therapeutically relevant levels of EE₂. The physiological/therapeutic doses of E₂/EE₂ that activated p38 were most effectively challenged by R-eq at ≥fM concentrations. R-eq did not alter the proliferative response to E₂ but it elevated the cell numbers induced by EE₂ at all concentrations of added R-eq. The more pronounced ability of R-eq to inhibit the cell-killing mechanisms associated with p38-induced inflammatory responses may explain its capacity to increase the numbers of EE₂-stimulated pituitary tumor cells. Thus, widespread exposure to persistent pharmaceutical estrogens that imperfectly mimic endogenous estrogens may exacerbate cell proliferation in these responsive cells.

Keywords: Calcium; ERK JNK p38 activation; Ethinyl estradiol; G(α)i; Kinase; Membrane ERα; Non-genomic effect; Phytoestrogen; Prolactin; R-equol.