Xenoestrogens are potent activators of nongenomic estrogenic responses

Abstract

Studies of the nuclear transcriptional regulatory activities of non-physiological estrogens have not explained their actions in mediating endocrine disruption in animals and humans at the low concentrations widespread in the environment. However, xenoestrogens have rarely been tested for their ability to participate in the plethora of nongenomic steroid signaling pathways elucidated over the last several years. Here we review what is known about such responses in comparison to our recent evidence that xenoestrogens can rapidly and potently elicit signaling through nongenomic pathways culminating in functional endpoints. Both estradiol (E₂) and compounds representing various classes of xenoestrogens (diethylstilbestrol, coumestrol, bisphenol A, DDE, nonylphenol, endosulfan, and dieldrin) act via a membrane version of the estrogen receptor-α on pituitary cells, and can provoke Ca²⁺ influx via L-type channels, leading to prolactin (PRL) secretion. These hormones and mimetics can also cause the oscillating activation of extracellular regulated kinases (ERKs). However, individual estrogen mimetics differ in their potency and temporal phasing of these activations compared to each other and to E₂. It is perhaps in these ways that they disrupt some endocrine functions when acting in combination with physiological estrogens. Our quantitative assays allow comparison of these outcomes for each mimetic, and let us build a detailed picture of alternative signaling pathway usage. Such an understanding should allow us to determine the estrogenic or antiestrogenic potential of different types of xenoestrogens, and help us to develop strategies for preventing xenoestrogenic disruption of estrogen action in many tissues.

Introduction

Xenoestrogens are compounds other than physiological estrogens that can nonetheless evoke estrogenic responses. Xenoestrogens are known to contaminate our environment and alter the reproductive health of wildlife, and probably humans [1]. Such estrogen mimetics were noted for their effects on wildlife in the 1960s when naturalists such as Rachel Carson drew attention to the endocrine-disrupting effects of some pesticides (notably DDT, [2]). These compounds may act as inappropriate estrogens, and/or could interfere with the actions of endogenous estrogens. For many years the mechanisms via which many xenoestrogens act remained a mystery. This lack of a mechanistic explanation existed because while these compounds can affect animal functions and development at relatively low concentrations, experimental systems for testing the classical nuclear transcriptional activities of xenoestrogens showed weak or no activity [3], [4], [5], [6], [7], [8], [9], [10]. Therefore, the question remained, via what cellular mechanisms do xenoestrogens act? Actions mediated through nongenomic pathways and plasma membrane receptors for steroids [11], [12], [13] were largely unstudied until very recently.

Compounds known as xenoestrogens have wide structural diversity, but all have in common lipophillic phenolic rings and other hydrophobic components, a characteristic they share with steroid hormones and related nuclear receptor-activating compounds (see Fig. 1). It has been suggested that the “promiscuity” of estrogen receptors in accepting many diverse ligands may be due to their status as the most evolutionarily primitive versions of ligand-activatable regulatory proteins [14]; as such they probably initially evolved to respond to a diverse set of molecules in the cell's environment. Therefore, many compounds that are byproducts of our modern industrialized life-style (pesticides, herbicides, plastics manufacturing byproducts, fungicides, cosmetics additives, and pharmaceuticals) can serve as estrogenic ligands in an inappropriate way.

We and others have very recently studied compounds representing different functional and structural xenoestrogen classes for actions initiated at the plasma membrane. Our studies, summarized in this review, examined the following diverse xenoestrogenic compound classes displayed in Fig. 1. Dieldrin, endosulfan, and the DDT metabolite o,p′-dichlorodiphenylethylene (DDE) are organochlorine pesticides; because of widespread past usage they still contaminate many agricultural and runoff sites. Detergents used in plastics manufacturing (e.g. p-nonylphenol) and a common precursor monomer that leaches from polycarbonate plastics (bisphenol A) are widespread contaminants in food and water via packaging, and as manufacturing byproducts in the environment [15]. Naturally occurring estrogens from plants and molds can also be abundant; we studied the phytoestrogen coumestrol, which is present in alfalfa sprouts and red clover (entering the food cycle via animals grazing in pastures containing this plant) [16]. Finally, some estrogen mimetics (such as diethylstilbesterol, DES) were designed as pharmaceuticals, but later found to have health-threatening side effects such as vaginal cancer in the neonatally exposed [17]. The potencies of these compounds in nuclear transcription reporter assays range from very weak (dieldrin, DDE, endosulfan), to somewhat weak (bisphenol A and nonylphenol), to quite strong (DES and coumestrol). There is a paucity of data on the ability of environmental estrogens to mediate nongenomic effects at low concentrations [18], [19], [20], [21], [22], [23], [24]. Most published studies examine only very high (μM–mM) concentrations (for example, [25]) in the range required to see any effects on nuclear transcription responses, but which are rarely reached at contamination sites.

Representative examples of the proposed membrane steroid receptor types have recently been reviewed [12]. Such an abundance of credible reports indicates that nongenomic steroid and mimetic actions are likely to result from a very complex sequence of events which can assemble a repertoire of proteins likely to function together. These proteins are probably differentially represented in different cell types and circumstances, and at different response stages. The existence of multiple kinds of steroid-binding proteins (receptors, enzymes, transporters, and blood and cellular binding globulins and their receptors) has long been known, though the exact sequential roles of all of these protein types are still not clear, even in direct genomic response pathways. It is likely that both nuclear receptor-like membrane steroid receptors, and also other unique steroid-binding membrane proteins (such as serpentine receptors and others [19], [26], [27], [28], [29], [30]), play subtly different roles. It is also important to remember that downstream, rapid membrane-initiated steroid effects can ultimately impinge upon nuclear actions via post-translational modifications of transcription factors (including nuclear receptors themselves). Our past studies in both a pituitary tumor cell line selected for robust nongenomic estrogenic responses, and similarly selected MCF-7 breast cancer cells, clearly indicate that a membrane version of ERα is involved. We demonstrated this via antibody (Ab)-elicited responses, increased or decreased receptor expression linked to responses, antisense knockdown of ERα, and the absence of other estrogen receptor types in these cells [31], [32], [33], [34], [35], [36], [37], [38].

In pituitary, estrogens facilitate both genomic (synthesis) and nongenomic (regulated secretion) of PRL [39]. The numerous functional consequences of PRL activity include coordination of the female hormonal cycle with preparation of various tissues for reproduction by inducing protein synthesis and secretion, the growth of new tissue (e.g. mammary gland), and the control of reproductive behavior. In this scenario many different functional endpoints are thus candidates for mis-regulation by xenoestrogens. Our clonal cell line GH3/B6/F10 was selected for its natural (not transfection-driven) expression of high levels of a membrane form of the estrogen receptor-α (mERα). Expression of mERα was correlated with very sensitive responses to E₂, including those for ERK activation [40], Ca²⁺ entry [41], and rapid PRL release [41]. We first observed changes in mERα levels detected in the membrane when cells were treated with low concentrations of xenoestrogens just before fixation for immunocytochemistry. E₂ caused rapid loss (by 3 min) and a slower return (∼15 min) of the mERα epitope. (Whether that be actual exit and return of the protein from the membrane, or a change in epitope recognition, we are not sure.) Xenoestrogens also caused this rapid change in epitope recognition, with a slightly different time course of the slow reversal [42]. This initiated a series of studies comparing physiological versus non-physiological estrogens and their use of membrane-initiated signaling mechanisms. The evidence that we will review here summarizes the arguments for believing that xenoestrogens also effect signaling changes leading to functional endpoints via the same nongenomic pathways as E₂, but with altered pathway kinetics and use preferences.