Because many xenoestrogenic compounds bioaccumulate in fat tissues, resulting in prolonged and escalating human exposures, the exposure levels causing possible deleterious health effects are somewhat difficult to determine and are actively debated (Myers et al., 2009; Whitten and Patisaul, 2001). Although physiological estrogens can normally influence the growth and functioning of both female and male reproductive, skeletal, and cardiovascular systems (Cornwell et al., 2004), prolonged estrogen exposures have also been linked to the development of cancer in tissues, such as breast, colon, and pituitary (Brownson et al., 2002; Fritz et al., 1998; Mueller and Gooren, 2008). It is thus important to determine which of these estrogenic attributes are shared by xenoestrogens, and via which cellular mechanism(s) they operate, so that their suspected effects on health can be predicted, prevented, perhaps remediated, or even used therapeutically, such as in the case of phytoestrogens (Adlercreutz, 1995).

In the past, xenoestrogenic compounds have undergone extensive testing for actions via nuclear estrogen receptor–mediated gene transcription but have largely been found to act very weakly, if at all, via these genomic pathways (generally at least 1000-fold more weakly than estradiol [E₂]). We have instead probed the ability of different classes and structurally variant estrogens to trigger signal cascades initiated at the plasma membrane via membrane estrogen receptors (α, β, and GPR30). To link these effects to the presence of specific receptor subtypes, our laboratory has demonstrated the importance of a membrane form of the estrogen receptor-α (mERα) in clonal rat pituitary cell lines that naturally express high or low receptor levels (Pappas et al., 1994); mERα was also the predominant receptor through which effects were mediated in neuronal cells, although the other estrogen receptors mERβ and GPR30 (Filardo and Thomas, 2005; Revankar et al., 2005) had inhibitory roles (Alyea and Watson, 2009a; Alyea et al., 2008). We also linked E₂’s and xenoestrogens’ abilities to mediate membrane-initiated signaling via Ca⁺⁺ elevation, prolactin (PRL) release, and various kinase activations to complex cellular events, such as cell proliferation and apoptosis (Jeng and Watson, 2009; Zivadinovic et al., 2005). Others have likewise found that specific estrogen receptor subtypes can mediate effects of xenoestrogens (Kuiper et al., 1998; Nadal et al., 2009). Xenoestrogen-altered responses could explain a variety of exposed tissue malfunctions, including both short-term functional deficits and long-term changes in cell and tissue activities (such as teratogenesis and cancer).

Estrogen-induced cell proliferation is part of the normal response of the pituitary but can also produce pituitary tumors (Gorski et al., 1997). PRL is believed to be a growth factor for many target tissues, including breast and prostate (Adams, 1992; Nevalainen et al., 1997), so its overproduction may lead to pathologies or tumors of these tissues (Clevenger et al., 2003; Gutzman et al., 2004; Rose-Hellekant et al., 2003). If any xenoestrogens act differently than the normal timing and levels of endogenous estrogens, then imbalances of PRL secretion and stimulation could occur; these could be developmental stage specific or gender specific and might cause unanticipated harmful responses. Our studies demonstrated how this could occur mechanistically for different estrogens and xenoestrogens via membrane-initiated signaling pathways (Bulayeva and Watson, 2004; Jeng and Watson, 2009; Jeng et al., 2009; Kochukov et al., 2009; Watson et al., 2008).

While E₂ is the physiological estrogen most often studied and associated with reproductive function during the reproductive years, other endogenous estrogenic compounds can be more prevalent during other life phases. These other estrogens may have significant effects on tissue development, function, and disease states (such as the development of cancers in reproductive tissues). Estrone (E₁) is a significant estrogenic hormone contributor in both reproductive (~0.5–1nM) and postmenopausal (150–200pM) women and in men (~100pM); estriol (E₃) levels are significantly higher in pregnant women (~10–100nM) than in nonpregnant women (< 7nM) (Greenspan and Gardner, 2004). Lowered E₃ levels in pregnancy have been associated with complications of eclampsia (Shenhav et al., 2003) and the incidence of Down’s syndrome in offspring (Chard and Macintosh, 1995). These estrogenic metabolites are also produced by aromatases in a number of nonreproductive tissues where their effects may extend beyond reproductive functions (Meinhardt and Mullis, 2002). One example is that E₃ has protective effects against the development of arthritis in certain experimental models (Jansson and Holmdahl, 2001), as has been known previously for E₂. Effects in brain, bone, cardiovascular system, and many other tissues may be affected differentially by these three endogenous estrogenic compounds during different life stages; therefore, loss or enhancement of these effects due to interference by xenoestrogenic compounds could affect human health in a large number of tissues. These metabolites also present an interesting structure-activity study group as their modifications are simple variations at only two positions on their D-rings (see Fig. 1). Scant previous information about the actions of physiological concentrations of E₁ and E₃ via nongenomic steroid signaling mechanisms (Morley et al., 1992; Selles et al., 2005) have now been augmented by our recent studies (Alyea and Watson, 2009b; Watson et al., 2008). We saw that E₁ and E₃ were similarly potent with E₂ in some responses for both pituitary cells (increasing the number of Ca⁺⁺-responding cells and evoking extracellular-regulated kinase [ERK] phosphorylation) and neuronal cells (evoking dopamine efflux). However, in neuronal cells, E₁ and E₃ (inhibitory) had opposite effects from E₂ (stimulatory) on the activation of dopamine efflux by the dopamine transporter (DAT), and these physiological hormones achieved this by differentially causing rapid trafficking of the estrogen receptors (α, β, and GPR30) and DAT to and from the plasma membrane. Further exploration of these potent and differential effects of physiological estrogen metabolites, and interference with their activities by xenoestrogens, could illuminate other life stage–specific changes in estrogen-related disease vulnerabilities.

Alkylphenols represent a group of ubiquitous environmental estrogens that are highly related in structure, although somewhat different from E₂. These compounds are surfactants or monomer byproducts of plastic manufacturing or product breakdown. They have been found at surprisingly high concentrations in human fluids (Lakind and Naiman, 2008; Stahlhut et al., 2009) and at environmental sites (Kolpin et al., 2002; Talsness et al., 2009; Thomas and Doughty, 2004). Our laboratory compared several members of this class with either different lengths of carbon side-chain modifications at Position 4 on the phenol ring (nonyl-, octyl-, propyl-, and ethylphenol [NP, OP, PP, EP]; see Fig. 1), or instead an added phenolic group (bisphenol A [BPA]). These compounds were active at very low doses in our studies (Kochukov et al., 2009), a potency confirmed by others studying other endocrine tissues (Alonso-Magdalena et al., 2008; Nadal et al., 2009). These comparisons represent low environmentally common concentrations (femtomoles to nanomoles). Overall, the alkylphenols are quite potent in several of our assays for nongenomic responses, including PRL release, cell proliferation, calcium (Ca⁺⁺) influx, and in the activation of mitogen-activated protein kinases (MAP kinases) (Bulayeva and Watson, 2004; Kochukov et al., 2009; Wozniak et al., 2005). These activities are summarized for all our publications to date in Figure 2.

By comparison, BPA and nonylphenol have shown very low potency in nuclear transcription assays for estrogen-responsive genes (Gaido et al., 1997; Gutendorf and Westendorf, 2001; Kloas et al., 1999; Sheeler et al., 2000; Singleton et al., 2004; Steinmetz et al., 1997). The long carbon side-chain alkylphenols were previously shown to have weak estrogenic activity in genomic assays, and the shorter side-chain versions were even less active (Kwack et al., 2002; Routledge and Sumpter, 1997; Tabira et al., 1999). In contrast, their nongenomic activities are quite robust, and short or long carbon chain variants are more effective in different responses (Kochukov et al., 2009). Therefore, inactivity in genomic assays does not predict inactivity in nongenomic mechanisms. In addition, this class of xenoestrogens is becoming increasingly important to consider for further modification by chlorination in manufacturing and waste water treatment plants (Fukazawa et al., 2001; Gallard et al., 2004; Gross et al., 2004; Hu et al., 2002; Petrovic et al., 2003), so structure-activity knowledge about their estrogenic effects will become increasingly important.

Our laboratory has also performed nongenomic signaling studies with several chlorinated pesticides known to be estrogenic—dieldrin, dichlorodiphenyldichloroethylene, and endosulfan. These compounds break down slowly, and so persist in the soil even though their use has largely been banned (U.S. Department of Health and Human Services, 2005). Plants and animals that are part of the food supply become exposed, subsequently passing on these exposures to humans. Because many xenoestrogens bioaccumulate in fat tissues, resulting in prolonged and escalating human exposures, the exposure levels causing deleterious health effects are actively debated (Myers et al., 2009). In our studies, these chlorinated xenoestrogen pesticides were quite effective in eliciting all the responses examined, including ERK activation (Bulayeva and Watson, 2004), PRL release, and Ca⁺⁺ influx (Wozniak et al., 2005). The studies of others have also demonstrated rapid signaling actions of these compounds on endocrine cells (Wu et al., 2006).

Phytoestrogens, another category of nonphysiological estrogens, have diverse estrogenic biological activities due in part to their ability to act as either estrogen agonists or antagonists depending on the dose and the specific tissue involved. These abilities have caused a lot of attention to be focused on these compounds as potential safe, effective, and inexpensive estrogen replacement medications. Coumestrol, first reported to be estrogenic when it was associated with disrupting reproduction in livestock (Bickoff et al., 1957), is found in such dietary sources such as legumes, clover, and sprouts of soybeans and alfalfa. The reported serum concentration resulting from ingesting these foods in humans is approximately 0.01μM (Mustafa et al., 2007). Isoflavones are represented in our studies by daidzein and genistein, and their major source is soy-based foods. In Asia, the intake of soy is high, and plasma concentrations of genistein from 0.1 to 10μM have been measured (Mustafa et al., 2007; Whitten and Patisaul, 2001); Western diets usually contain about 10-fold lower concentrations (Adlercreutz et al., 1993). Some isoflavones, such as genistein, have also been shown to act predominantly via estrogen receptor-β in genomic responses (Kuiper et al., 1998). Trans-resveratrol, a stilbene (Gehm et al., 1997) that has recently attracted significant attention as a potential anti-aging agent, is found in high quantities in foods such as red grapes (or wine) and peanuts and has peak serum concentrations estimated to be close to 2μM in humans (Walle et al., 2004).

We have speculated that such typical bimodal dose responses for these membrane-initiated mechanisms could result from different receptor subpopulations present in unique compartments of the plasma membrane. For example, membrane forms of steroid receptors have also been shown to reside in membrane caveolae by us (Zivadinovic and Watson, 2005) and others (Chambliss and Shaul, 2002; Lu et al., 2001; Norman et al., 2002), where it is well known that lipid content and other signaling molecule and scaffolding protein availability are quite different from non-raft membranes. Basolateral versus apical or endocytosed membrane compartments represent compartments of different accessibility for hormones to their receptors (Cao et al., 1998), although this is less likely to be the case for small lipophilic molecules like steroids or their mimetics than for peptide hormones. Subcellular location–based availabilities could also dictate different physical associations with other proteins by altering hormone-binding and -partnering opportunities. In addition, differences in lipid content simulated in artificial membranes are known to affect the functioning of proteins imbedded therein (Wu and Gorenstein, 1993), likely causing alteration of ligand-binding pockets and protein partner interaction interfaces. Therefore, characteristics of receptors that target to the membrane or membrane subcompartments may affect signaling responses.

We also know from our own work that estrogens, including xenoestrogens, can signal via several different pathways simultaneously although differentially and that these signals traverse their pathways at variable speeds (Bulayeva and Watson, 2004). Different phasing of pathway travel, along with feedback or feedforward regulation or crossing over to parallel paths, can result in complex contributions to dose-response changes, not obvious when examined at a single time point. An example could be the estrogenic activation of a phosphatase, which then inactivates another protein, such as a kinase; if the response being monitored is the kinase activation, then one would see an unexpected decline in the response whenever the phosphatase has been activated. We have some evidence for this effect on response curves in breast cancer cells (Zivadinovic and Watson, 2005 and Banga and Watson, unpublished data). For nuclear receptor actions, it is known that there are different dose-response sensitivities to the same level of nuclear steroid receptor for different genes in the same tissue (Catterall et al., 1985; Simons, 2008), probably also involving receptors partnering with other receptors or coregulators that are target gene specific. Ligand-induced conformations of nuclear receptors can vary due to the structure and level of the hormones bound to them, likely altering their interaction interfaces being presented to other regulatory molecules, thus initiating different responses (Pike et al., 1999). Such mechanisms are also likely to account for associations that regulate membrane-initiated signaling cascades.

The changing circumstances to which a cell must respond are first presented by the ligands that it encounters at its surface, of which there are many that arrive simultaneously. The engagement of surface receptors by these ligands sets in motion coordinated actions, eventually leading to major cellular decision points: proliferation, differentiation, or death. Cells must integrate all these incoming signals and parallel pathways that can eventually contribute to a final common pathway, such as those involving MAP kinases (see Fig. 4). These enzymatic “signal-receiving stations” tally up many inputs from multiple signaling cascades and sum them toward establishing a level of end point of MAP kinase (ERK, JNK, and p38) activities. The final count dictates a decision about how to regulate major cellular responses that require coordination so that the whole cell “is on the same page” during major cellular responses to change. Acting via their membrane receptors, steroids can be one class of input signals to the MAP kinase signal integrator, the cellular rheostat that is dialed up or down according to which pathways feed into it. Not all estrogens elicit identical responses (in level or timing) along these pathways (Bulayeva et al., 2004). Thus, different endogenous metabolites (representing different life-stage challenges) or xenoestrogen mimetic exposures will cause a different tally and resulting cellular response. Estrogens provide a very illustrative example of these cellular strategies because of the availability of many different and medically/environmentally important estrogens that make differential use of the same pathways. This example thus gives new insights into how such signaling webs can lead to variant outcomes depending on which estrogens engage them.