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Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways

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Bisphenol-A (BPA) is an estrogenic monomer commonly used in the manufacture of numerous consumer products such as food and beverage containers. Widespread human exposure to significant doses of this compound has been reported. Traditionally, BPA has
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  Review Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways Paloma Alonso-Magdalena ⇑ , Ana Belén Ropero, Sergi Soriano, Marta García-Arévalo, Cristina Ripoll,Esther Fuentes, Iván Quesada, Ángel Nadal ⇑ Instituto de Bioingeniería and CIBERDEM, Universidad Miguel Hernández de Elche, 03202 Elche, Spain a r t i c l e i n f o  Article history: Available online xxxx Keywords: Islets of LangerhansBisphenol-AEstrogen receptors a b s t r a c t Bisphenol-A (BPA) is an estrogenic monomer commonly used in the manufacture of numerous consumerproducts such as food and beverage containers. Widespread human exposure to significant doses of thiscompound has been reported. Traditionally, BPA has been considered a weak estrogen, based on its lowerbinding affinity to the nuclear estrogen receptors (ERs) compared to 17- b  estradiol (E2) as well as its lowtranscriptional activity after ERs activation. However,  in vivo  animal studies have demonstrated that itcan interfere with endocrine signaling pathways at low doses during fetal, neonatal or perinatal periodsas well as in adulthood. In addition, mounting evidence suggests a variety of pathways through whichBPA can elicit cellular responses at very low concentrations with the same or even higher efficiency thanE2. Thus, the purpose of the present review is to analyze with substantiated scientific evidence the strongestrogenic activity of BPA when it acts through alternative mechanisms of action at least in certain celltypes.   2011 Elsevier Ireland Ltd. All rights reserved. Contents 0. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 001. How do natural estrogens signal on their target tissues? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. The discovery of xenoestrogens, the need for assessing estrogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. The case of bisphenol-A (BPA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. Estrogenic activity of BPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005. Mechanistic evidence that reveals a potent estrogenic action of BPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 0. Introduction Natural estrogens have been defined as one of the most impor-tant female reproductive hormones since they play a key role inthe development of female secondary sex characteristics and pro-mote the growth and the maintenance of the female reproductivesystem. They are formed from androgen precursors and synthe-sized mainly in the ovaries, and to a lesser extent in the adrenalglands, adipose tissue, brain and testis. After menopause, the ovar-ian production of estrogen declines, and the adrenal cortex andovaries secrete mostly androgens, which are converted to estro-gens in the peripheral tissues, such as adipose tissue and muscle(Simpson et al., 2002; Hillier et al., 1994; Nelson and Bulun,2001). Although several estrogens are synthesized throughout life,17 b -estradiol (E2) is normally considered the most potent andimportant estrogen. Besides this sexual and reproductive role, weknowthatE2exertsalargenumberofactionsinothersystemssuchasthebone,liver,brain,endocrinepancreas,adiposetissue,skeletalmuscle and cardiovascular systems (Gustafsson, 2003; Heldringetal.,2007).Inaddition,anysyntheticorsemisyntheticsteroidthatmimicstheeffectsofnaturalestrogens(http://www.merriam-web-ster.com/medlineplus/estrogen, M.D.o.t.U.N.L.M.) is considered an estrogen. 1. How do natural estrogens signal on their target tissues? From the classical mechanism point of view, E2 binds to theestrogen receptors (ER  a  or ER  b ) that act as transcriptional factors 0303-7207/$ - see front matter    2011 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.mce.2011.12.012 ⇑ Corresponding authors. Tel.: +34 965222165; fax: +34 965222033 (P. Alonso-Magdalena), tel.: +34 965222002; fax: +34 965222033 (A. Nadal). E-mail addresses:  palonso@umh.es (P. Alonso-Magdalena), nadal@umh.es (Á. Nadal).Molecular and Cellular Endocrinology xxx (2012) xxx–xxx Contents lists available at SciVerse ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce Please cite this article in press as: Alonso-Magdalena, P., et al. Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways. Mo-lecular and Cellular Endocrinology (2012), doi:10.1016/j.mce.2011.12.012  after being activated. In their non-liganded state, both estrogenreceptors (ERs) are associated with inhibitory protein complexescontaining several chaperone proteins in the cytosolic or nuclearcompartment, that repress their function (Pratt and Toft, 1997). Upon ligand activation, the receptors dissociate from the inhibitorycomplex, change their conformation, trigger functional dimers incertain DNA-elements and recruit coregulators. Finally, the recep-tor complex binds to the promoter regions of target genes (Held-ring et al., 2007; Hall et al., 2001).Several mechanisms for ER transcriptional regulation have beendescribed; the classical one involves the binding of ER-dimers di-rectly to estrogen response elements (EREs) through the DNA-binding domain. Alternatively, ER may also activate non-ERE targetgenes by binding to other transcription factor complexes such asFos/Jun, also named AP-1 responsive elements or SP-1 (Safe andKim, 2004; Kushner et al., 2000; Saville et al., 2000).In addition, non-nuclear initiation mechanisms have beenshown to exist. Both ER  a  and ER  b  have been identified outsidethe nucleus: in the cytosol, mitochondria and associated to or nearthe plasma membrane (Hammes and Levin, 2007). Interestingly, it has been shown that from these locations they are able to rapidlyactivate other signaling cascades. To make this signaling scenarioeven more complex, other novel membrane estrogen receptorshave also been described. Some of them have been molecularlycharacterized; such is the case of the orphan protein-coupledreceptor (GPR30) (Kelly and Levin, 2001; Losel et al., 2003; Marinet al., 2003; Nadal et al., 2001; Toran-Allerand, 2004). Additionally,E2 can act by binding directly to other neurotransmitter receptorsand to ion channels, triggering rapid cellular responses (Nadalet al., 2001; Valverde et al., 1999).These effects triggered outside the nucleus, normally elicit a ra-pid activationof different signaling moleculessuch as those involv-ingSrc, PI3kinaseand receptortyrosine kinases(EGFR,IGF-1R),andof second messengers cAMP, cGMP, intracellular Ca 2+ , etc. which inturn can also regulate gene expression (Revankar et al., 2005; Filar-do et al., 2007; Song et al., 2002; Migliaccio et al., 1996). 2. The discovery of xenoestrogens, the need for assessing estrogenicity  Xenoestrogens encompass a variety of chemicals that haveestrogen-like effects. Most frequently, xenoestrogens are agricul-ture chemicals such as pesticides and industrial by-products (cer-tain plastics or detergents) widely spread in the environment,compounds from plants (phytoestrogens) such as isoflavones fromsoy (geniestein, daidzein), or coumesterol from red clover. In addi-tion, there are synthetic drugs like DES, a potent synthetic estrogenthat was widely prescribed to pregnant women from the 1940sthrough the 1970s in the mistaken belief that it could preventthreatened miscarriages.Over the 1990s, the appearance of adverse reproductive effectsin aquatic and wildlife species living within or near contaminatedareas was reported (Colborn et al., 1993; Sonnenschein and Soto,1998; Guillette et al., 1994; Sumpter and Jobling, 1993). In parallel,the estrogenic activity of some of these compounds such as octyl-phenol and bisphenol-A was accidentally discovered in the labora-tory, because they disrupted experiments that studied the effectsof natural estrogens (Soto et al., 1991; Krishnan et al., 1993). Throughout the years, substantial evidence has pointed to thefact that these chemicals can mimic the action of the natural hor-mone E2, although they do not exhibit a similar structure to estro-gens. Thus it became necessary to develop assays accurately whichcould evaluate the risk of suspected xenoestrogens.Competitive receptor-binding assays, yeast-based reporter geneassays and in particularthe inductionof cell proliferation, are someof the most commonlyused in vitro screeningmethods. All of themare based on the classical concept of estrogenicity, that is the prop-erty of a compound to bind to ER  a or ER  b  and act subsequently as atranscription factor when binding directly to DNA through ERE.However, as described above, the action of E2 can be mediatedby many other signaling pathways and not all of them are evalu-ated in these assays.In addition, we have to consider thatan  in vitro  assay cannot de-tect pro-estrogen which is metabolized  in vivo  into estrogen. Fur-thermore, the assessment of theoretically weak estrogen activitycan be underestimated if it acts as a source of more potent estro-genic metabolites.To solve these difficulties, one can deduce that  in vivo  methodswould be more convenient. In fact, although the  in vivo  uterotroph-ic assay which measures the increase in wet weight of the uterushas been considered a ‘‘gold standard technique’’, it is not free fromdiscrepancies. Many authors consider it is not as sensitive in theendpoints as it initially was thought to be and it often proves tobe expensive and time-consuming (Nagel et al., 2001; Markeyet al., 2001). In addition, this kind of tests would not detect thosecompounds with estrogenic properties in some tissues, but withno uterotrophic activity, as it is the case for STX and the estro-gen-dendrimer conjugate (Chambliss et al., 2010; Qiu et al.,2003). STX is a non-steroidal diphenylacrylamide compound withsome estrogenic properties acting through a new Gq-coupledmembrane estrogen receptor molecularly unidentified (Qiu et al.,2003; Roepke et al., 2009). STX has been shown to prevent theovariectomy-induced body weight gain and increase in body coretemperature in guinea pigs. It modulates the activity of hypotha-lamic POMC and dopamine neurons (Qiu et al., 2003, 2006, 2008;Zhang et al., 2010), increases K ATP  channel activity in hypothalamicGnRH neurons (Zhang et al., 2010) and protects against ischemia-induced hippocampal neuron loss (Lebesgue et al., 2010). Estrogen dendrimer conjugate is 17 a -ethynylestradiol linked to apoly(amido)amine dendrimer (PAMAM) . This conjugate binds toER  a  and it remains outside the nucleus, because of its size andcharge and therefore, it does not activate nuclear ER  a  function(Harrington et al., 2006). 3. The case of bisphenol-A (BPA) Bisphenol-A (BPA) was first synthesized by Dianin in 1891 andreported to be a synthetic estrogen in the 1930s (Dodds et al.,1936). By that time, the estrogenic properties of diethylstilbestrol(DES) were also tested and because of its strong estrogenic activity,BPA essentially took the backseat. In the 1950s, BPA was rediscov-ered as a compound that could be polymerized to make polycar-bonate plastic, and from that moment on until now, it has beencommonly used in the plastic industry. BPA is one of the highestvolume chemicals produced worldwide, with over 6 billion poundsproducedeachyearandover100 tonsreleasedintotheatmosphereby the yearly production (Vandenberg et al., 2009). It is used as the base compound in the manufacture of polycarbonate plastic andthe resin lining of food and beverage cans, and as an additive inother widely used plastics such as polyvinyl chloride and polyeth-yleneterephthalate.Itispresentnotonlyinfoodandbeveragecon-tainers, but also in some dental material (Olea et al., 1996). Numerous studies have found that BPA can leach from polycarbon-ate containers; heat and either acidic or basic conditions acceleratethehydrolysisoftheesterbondlinkingBPAmonomers,leadingtoarelease of BPA with the concomitant potential human exposure(Kang et al., 2006; Richter et al., 2007). Indeed, the potential for BPA exposure has already been demonstrated since BPA was de-tected in 95% of the urine samples in the USA (Calafat et al.,2005). Its concentration in human serum ranges from 0.2 to 2  P. Alonso-Magdalena et al./Molecular and Cellular Endocrinology xxx (2012) xxx–xxx Please cite this article in press as: Alonso-Magdalena, P., et al. Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways. Mo-lecular and Cellular Endocrinology (2012), doi:10.1016/j.mce.2011.12.012  1.6 ng/ml (0.88–7.0 nM) Sajiki et al., 1999; Takeuchi and Tsutsumi,2002. Moreover, it has been detected in amniotic fluid, neonatalblood, placenta, cord blood and human breast milk (Richter et al.,2007). Concerning the potential risk of this compound, in the1980s the lowest-observable-adverse effect-level (LOAEL) for BPAwas determined at 50 mg/kgbw/day, and the Environmental Pro-tection Agency (EPA) calculated a ‘‘reference dose’’ or safe dose of 50 l g/kgbw/day in a series of studies in which the changes of bodyweight in animals fed diets containing BPA were analyzed (http://www.epa.gov/iris/subst/0356.htm, U.E.P.A.). However, since that time, numerous scientific evidence supports that BPA can interferewith the endocrine signaling pathways at doses below the calcu-lated safe dose, particularly after fetal, neonatal or perinatal expo-sure, but also after adult exposure. Examples of low doses  in vivo effects of BPA have been reported in a wide variety of tissues andcell types. A review by Richter and colleagues provides a compre-hensive account of these findings (Richter et al., 2007). There are several reasons why both natural hormones and EDCsare active at low doses. Relevant one is based on the relationshipbetween the receptor occupancy and the elicited response. Thefinding that the maximum biological response can be achieved atconcentrations of hormones lower than required to occupy all thereceptors on the cell suggested the there are ‘‘spare receptors’’. Inpractice, the determination is usually made by comparing the con-centration for 50% of maximal effect (EC 50 ) with the concentrationfor50%of maximalbinding( K  d ).If theEC 50  islessthanthe K  d , sparereceptors are said to exist. This is because the duration of the acti-vation of the effector may be greater than the duration of the hor-mone-receptor activation or because the actual number of receptors may exceed the number of molecules available. In anycase, the presence of spare receptors provides a mechanism forobtaininga responseatverylowconcentration ofthehormonethatnonethelesshasarelativelowaffinityforthereceptor.AsWelshonsand colleagues describe, the relationship between receptor occu-pancy and hormone concentration, as well as between receptoroccupancy and response, are approximately linear up to 10% recep-tor occupancy. At concentrations above the  K  d , saturation of re-sponse occurs first, and then at higher concentrations, saturationof receptor is observed (Welshons et al., 2003). This should helps us to understand how some EDCs like BPA can have biological ef-fects at doses extremely low without the need of non classical acti-vated pathways. Nevertheless, we describe below how novelestrogen activated pathways may also explain low dose estrogenicactions of BPA.BPA was srcinally thought to exert actions primarily by dis-rupting the activity of the classic estrogen triggered pathways,using ERs as transcription factors binding to the ERE site in theDNA (Kuiper et al., 1998; Sheeler et al., 2000). Nowadays, increas- ing basic scientific research shows that BPA triggers non-classicalestrogen activated pathways via binding to extra-nuclear ERs(Alonso-Magdalena et al., 2008; Wozniak et al., 2005) and perhaps other estrogen binding proteins in the membrane which are stillmolecularly unidentified. In addition, the actions of BPA can in-volve many other signaling systems such as thyroid function (Zoel-ler et al., 2005; Zoeller, 2005) and androgen signaling (Lee et al.,2003; Sohoni and Sumpter, 1998).There is a notable amount of information available regardingsources of exposure to BPA, biomonitoring studies, mechanismsof action as well as exposure effects both  in vivo  and  in vitro  (Van-denberg et al., 2009, 2010; Richter et al., 2007). Hence, we do notaim to review exhaustively the low dose effects of BPA, or all theBPA elicited molecular mechanisms (vom Saal and Hughes, 2005;Wetherill et al., 2007), but to attempt to call the reader’s attentionto the strong estrogenic activity of BPA when acting through novelestrogen activated pathways. Thus, we will focus on the ability of BPA to mimic the activity of 17 b -estradiol and review the evidenceusing estrogen receptor knock out models to demonstrate that, atleast in some cell types, BPA is not a weak but a strongxenoestrogen. 4. Estrogenic activity of BPA In 1993 the estrogenic activity of BPA was rediscovered. Whilelooking for an estrogen-binding protein in yeast, a group of scien-tists found that BPA leached from polycarbonate (PC) flasks whenautoclaving, and that this estrogenicity did not come from theyeastbut fromBPA.This was confirmedby performingdifferent as-says such as: competitive binding to ER, proliferation of MCF-7breast cancer cells, induction of progesterone receptors, and rever-sal estrogen action by tamoxifen with the lowest effective dosebeing 10–20 nM (Krishnan et al., 1993). Some years later, an exten- sive uterotrophic analysis of BPA was performed confirming that ithad the capacity to induce proliferative and stimulatory changes inestrogen targets, analogous to those induced by E2 in concentra-tions ranging from 0.1 to 100 mg/kg (Markey et al., 2001). More re- cently, a new study has been published that increases the extent towhich BPA, among other EDCs, can initiate ER  a gene regulationin amouse uterine model compared to E2. The authors confirmed thatBPA at a dose of 750 l g/mouse stimulates uterine proliferation inan ER  a  dependent manner and found that the BPA-activated geneprofile in the early response phase was highly correlated to that of E2 (Hewitt and Korach, 2011). Although the estrogenicity of BPA was accepted from the earlybeginning, it was considered a weak estrogen since its bindingaffinity to the estrogen receptors alfa (ER  a ) and beta (ER  b ) wasestimated to be over 1000–10,000-fold lower than the natural hor-mone E2 (EC 50  = 2–7  10  7 M compared to 1–6  10  13 M for E2)(Kuiper et al., 1998; Andersen et al., 1999; Fang et al., 2000). BPA selectively binds to ER  a  and ER  b  although it has a higher affinityfor ER  b  (Kuiper et al., 1997; Matthews et al., 2001; Routledgeet al., 2000). In some cell types, it has been proposed that BPAexhibits estradiol-like agonist activity via ER  b  and a mixed agonistand antagonist activity via ER  a  (Kurosawa et al., 2002).In contrast, recent studies have revealed that BPA can promoteestrogen-like activities that are similar or stronger than E2 (Alon-so-Magdalena et al., 2005, 2006; Hugo et al., 2008; Zsarnovszkyet al., 2005). These low dose effects can be explained at least par-tiallybecause BPA elicits rapidresponses via non-classicalestrogentriggered pathways (Nadal et al., 2000; Quesada et al., 2002; Wat-son et al., 2005). In addition, BPA may bind differently than E2within the ligand domain of estrogen receptors (ERs) Gould et al.,1998 .There are also differences in the BPA co-activator recruit-ment, as is indicated by the fact that the BPA/ER  a complex showedover a 500-fold greater potency than BPA/ER  a  in recruiting the co-activator TIF2. This is a reflection of the more efficient capacity thatER  b  has to potentiate receptor gene activity in some cell types(Routledge et al., 2000; Safe et al., 2002).It has become increasingly apparent that BPA can activatetransduction signaling pathways which can vary from cell type tocell type and that the total disruption effect arises from the combi-nation of rapid mechanisms and longer signaling effects in estro-gen-responsive gene expression. 5. Mechanistic evidence that reveals a potent estrogenic actionof BPA As previously described, E2 can evoke rapid signaling effects viathe induction of second messengers such as Ca 2+ , cAMP, cGMP, NOas well as stimulate different types of kinases ERKs, PI3K, etc. Someof these responses are thought to be initiated at the plasma mem-brane, although the distinction between an estrogen-triggered P. Alonso-Magdalena et al./Molecular and Cellular Endocrinology xxx (2012) xxx–xxx  3 Please cite this article in press as: Alonso-Magdalena, P., et al. Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways. Mo-lecular and Cellular Endocrinology (2012), doi:10.1016/j.mce.2011.12.012  membrane effect and estrogen-triggered cytoplasmic effect is notclear. In 1995, studies performed in the prolactinoma cell lineGH 3 /B6/F10 proposed that this subclone of pituitary clonal cellswas characterized by the high expression of ER  a  in the plasmamembrane (mER  a ) and the rapid capacity that E2 has to induceprolactin release (Pappas et al., 1995). The authors also anticipated that environmental estrogens could initiate their actions from theplasma membrane (Watson et al., 1995). The first time it was dem- onstrated that xenoestrogens may act via non-classical estrogenactivated pathways was in 1998, when Ruehlman et al reportedthat 4-octylphenol,  p -nonylphenol,  o ,  p 0 -DDT rapidly blocked L-typecalcium channels and provoked vascular relaxation (Ruehlmannet al., 1998). In the year 2000, it was reported that E2, and thexenoestrogens DES and bisphenol-A produced a rapid regulationof Ca 2+ signals in pancreatic beta cells; the three chemicals wereequally potent at the dose of 1 nM (Nadal et al., 2000). BPA and E2-induced calcium signals were initiated at the plasma mem-brane and activated the transcription factor CREB in the nucleuswithin 15 min after exposure. Therefore, a membrane BPA-initi-ated action regulated gene expression at the same doses as E2(Quesada et al., 2002). The molecular pathway activated by E2, BPA and DES was insensitive to the antiestrogen ICI182, 780, sug-gesting that they may activate a different pathway from those ini-tiated after binding at the classical ER  a and ER  b . Moreover, bindingexperiments using E2 conjugated to peroxidase indicated that theplasma membrane binding site was the same for E2, BPA and DES(Alonso-Magdalena et al., 2005; Nadal et al., 2000, 2004; Quesadaet al., 2002). DDT and its metabolites also induced gene expressionin human uterine cell lines independently of ERs (Frigo et al.,2002). In any case, more molecular evidence is needed to charac-terize which ERs independent pathways are involved.Extranuclear ER  a , however, mediates rapid xenoestrogen ac-tions at low doses. Further research leads to the conclusion thatcertain xenoestrogens can induce ERK-1 and ERK-2 activation inthe nanomolar range.It has been proposed that this responseis ini-tiated by the binding of nonylphenol and coumestrol, among otherendocrine disruptor chemicals (EDCs), to mER  a  and occurs withinthe first 30 min after application (Bulayeva et al., 2004; Bulayevaand Watson, 2004) Although this was not the case for BPA, itwas demonstrated that as with E2, it induces a rapid Ca 2+ influx(within 30 s) which is necessary for rapidly induced prolactin re-lease. This effect occurs with extremely low doses of BPA, at thepicomolar range and apparently takes place by binding to the samemER  a  as indicated by the performed E2 linked to horseradish per-oxidase (E2-HRP) binding assays (Wozniak et al., 2005). In a recentstudy carried out by the same group, the effect of BPA in a range of low concentrations in combination with E2 was studied and re-vealed that in the range of femtomolar or nanomolar concentra-tions, BPA had a marked inhibitory effect on estrogen-inducedERK activity ( Jeng and Watson, 2011). Despite the main drawbacksof these studies: the failure to use estrogen receptor knock out ani-mals and thus provide a definitive demonstration of ER participa-tion, along with the obvious limited applicability of in vitroexperimental approaches to whole-animal physiology, they dem-onstrate that BPA can influence intracellular signaling via rapidmechanisms with the same efficiency as E2.The spatial and temporal influence of E2 on ERK signaling hasbeen proposed to be directly involved in the development andmaturation of cerebellar neurons (Wong et al., 2003). Curiously, long-term exposure to E2 (10  10 M) in primary rat cerebellar gran-ule cells induces neuroprotective effects with a concomitant de-crease in LDH levels whereas short exposure to E2 (10  10 M)increases necrotic programmed cell death. Mechanistically, thereare clear differences between both phenomena; the first one ismediated by classical ER-mediated transactivation while the sec-ond one is mediated by membrane receptor-initiated ERK1/2signaling. What is even more interesting is that a brief exposureto BPA (up to 15 min) at exactly the same doses as E2 is able to mi-mic the cytotoxic effect in granule cell precursors and results in aclear increment of LDH secretion. Actually BPA exposure increasesLDH release at all concentrations examined within the range10  10 –10  6 M. The rapid intracellular signaling cascade involves,in both cases, E2 and BPA, the participation of a G protein-depen-dent mechanism as well as a PKA and PTX sensitive pathway. Inaddition, it has been demonstrated that this rapid effect is initiatedby an ER  b -like receptor system since the effect has been shown tobe mimicked by the ER  b  agonist DPN, but not by the ER  a  agonistPPT (Zsarnovszky et al., 2005; Le and Belcher, 2010; Belcher,2008; Belcher et al., 2005). As regards in vivo effects, the intracer-ebellar injection of BPA induced ERK activation in a non-monotonicdose dependent fashion, with a positive effect at low doses (10  12 –10  10 M) and at higher ones (10  7 –10  6 M). Remarkably, BPA ac-tion was as potent as E2. In addition, the injection of a mix com-posed of BPA (10  12 –10  10 M) plus E2 (10  10 M) completelyblocked the estrogenic-induced ERK signaling. This paradoxicalphenomenon suggests that they may act as mutual inhibitors of each other’s rapid actions (Zsarnovszky et al., 2005). Although we are conscious about the fact that intracerebellar injections do notreflect the normal route of BPA administration, the experimentsabove described demonstrate a direct adverse effect of BPA indeveloping cerebellar neurons. In summary, we consider that thiscompendium of scientific reports constitutes an elegant demon-stration of the effect the disruptor BPA has on normal neurodevel-opment, as well as a clear demonstration of the equal potency of BPA and E2 at least in the cell type used in the study. Nevertheless,the use of KO mice or a silencing system will be the best tools tounequivocally demonstrate that BPA uses ER  a  and ER  b  to produceactions at low doses.Knockout mouse models have been developed to define thephysiological action of E2 through their cognate receptors and theyprovide a stable genetic system for evaluating estrogen signaling.In addition, they constitute an excellent tool for the discriminationof estrogen versus non-estrogen actions of EDCs. If we combinethese animal models with the use of synthetic agonists of ERs,we will probably obtain advanced knowledge of the estrogenreceptor role in mediating EDCs effects. This is exactly what hasbeen pursued when looking at the effects of low doses of BPA onthe endocrine pancreas.The endocrine pancreas was not considered a classic estrogentarget; however, estrogen receptors ER  a  and ER  b  are present inthe islet of Langerhans and E2 has been demonstrated to modulateinsulin secretion and to be an important player in the maintenanceof normal insulin sensitivity (Nadal et al., 2004, 2009; Sutter-Dub,2002). This was the basis that led us to hypothesize that exposureto an exogenous chemical acting as the natural hormone but in aninappropriate concentration and during an improper time windowmight affect energy balance and glucose homeostasis. We focusedour attention on BPA effects and found that both pancreatic alphaand beta cells were extremely sensitive to this compound (Roperoet al., 2008). In vivo  experiments showed that exposure to BPA at a dose of 100 l g/kg induced hyperinsulinemia, mild insulin resistance andglucose intolerance in adult male mice with exactly the same po-tency as E2. We proposed that these alterations resulted, at leastin part, from a direct effect of BPA on pancreatic beta cells. Whenbeta cells from BPA-treated mice were isolated they showed a clearincrease in insulin content compared to cells from control mice. Inaddition,theyrespondedmorevigorouslytoglucose,mostprobablybecause their insulin content was higher. Therefore, we suggestedthatthiswasresponsibleforthehyperinsulinemiathatBPA-treatedmice presented in a fed state (Alonso-Magdalena et al., 2006). We should keep in mind that chronic hyperinsulinemia can lead to the 4  P. Alonso-Magdalena et al./Molecular and Cellular Endocrinology xxx (2012) xxx–xxx Please cite this article in press as: Alonso-Magdalena, P., et al. Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways. Mo-lecular and Cellular Endocrinology (2012), doi:10.1016/j.mce.2011.12.012  development of insulin resistance (Del Prato et al., 1994) such as it canhappeninthisexperimentalmodel.Thiswasthefirstreportthatconnected BPA exposure with altered insulin sensitivity. But wewanted to move further on and gain insight into the mechanismresponsible for this estrogenic effect. So we performed a series of  in vitro  experiments that confirmed the direct effect of low dosesof E2 (100 pM–10 nM) on pancreatic insulin content (Alonso-Mag-dalena et al., 2008, 2006). We demonstrated it was related to an in-creaseininsulingeneexpressionandanenhancedinsulinreleaseinresponse to stimulatory glucose concentrations. Remarkably, 1nMBPA was equally effective as E2. By using the pure antiestrogen ICI182, 780, we determined that nuclear estrogen receptors (ER  a , ER  b or both) mediate E2 and BPA effects since they completely blockE2andBPAaction.An invivo injectionofICI182,780ledtothesameconclusion (Alonso-Magdalena et al., 2008, 2006). The ER  a  agonist4,4 0 ,4 00 -(4-Propyl-[1H]-pyrazole-1,3,5-triyl) tris phenol (PPT) hadthe same effect on insulin content as E2 but the ER  b  agonist, 2,3- bis (4-Hydroxyphenyl)-propionitrile (DPN) did not, indicating thatthe E2 and BPA effect was mediated by ER  a . In order to provideunequivocal evidence of the indispensable role of ER  a in mediatingBPAandE2effectsonpancreaticbetacells,weusedisletsfromER  b-  KO and ER  a KO mice. Thus, we confirmed that ER  a was the main ER involved in the regulation of insulin content by both E2 and BPA. Inaddition we demonstrated that the activation of ER  a  involvesERK1/2.As a final remark we concluded that E2 and BPA have the sameeffects at exactly the same doses and proposed that this paradigmcould be explained based on the fact that ER  a  activation by lowdoses of BPA occurs outside the nucleus which in turn activatesalternative signaling pathways (Alonso-Magdalena et al., 2008). Another study in which KO animal models were used describedhow BPA at low doses interferes with ER  b  mediated cellular re-sponses. The authors demonstrated that C57BL/6 female pups pre-natally exposed to BPA at a dose of 20 l g/kg/day underwent adisruption of early oogenesis. By analyzing meiotic prophase ova-ries from female fetuses at day 18.5 of gestation they demon-strated that BPA provoked synaptic abnormalities andaberrations in recombination in those oocytes from BPA-exposedfemales. In addition they observed that the altered synaptic andrecombination profiles found at the onset of female meiosis werecorrelated with increased aneuploidy in eggs and embryos frommature females. Interestingly similar abnormalities were observedin ER  b -null females which suggests that BPA acts as an antagonistof E2 since it mimics the effect that the lack of ER  b  triggers on theoocyte development (Susiarjo et al., 2007). Such as the temporal influence that E2 has on cerebellar neu-rons, E2 also exerts different time-dependent effects in pancreaticbeta cells which are explained by different mechanistic pathways.Unlike the E2 long-term effects described above, E2 rapidly pro-vokes the closure of the K ATP  channel of beta cells, a key moleculeinvolved in glucose-stimulated insulin secretion. Interestingly, themaximum inhibition of the channel is reached 3–7 min after E2application and the effect is transient, returning to normal levels30 min later. This provokes an increased frequency of [Ca 2+ ] oscil-lations, which finally results in a rapid increased insulin secretion.By using ER  b KO mice as well as the selective agonist PPT and DPN,we demonstrated that the fast modulation of insulin secretion isnot a genomic effect and that this effect is mediated by ER  b  andnot by ER  a . The data indicate that when E2 binds to ER  b , the guan-ylate cyclase A receptor is activated through a yet unknown mech-anism. As a consequence, K ATP  channel activity decreases in acGMP/PKG-dependent manner, which finally potentiates and en-hances insulin secretion (Soriano et al., 2009). In vivo, the injectionof 10 l g/kg of E2 provokes a rapid increase in plasma insulin levels(Alonso-Magdalena et al., 2006). Once again, BPA is able to mimicE2 action at exactly the same doses both  in vivo and in vitro  andalthough the mechanism that mediates the BPA effect is still un-solved, unpublished experiments by our group indicate that itmost probably is the same as the one described for E2. 6. Concluding remarks There is no doubt that BPA is an estrogenic compound,however,the matter of debate is to what extent BPA can mimic the action of the natural hormone E2. There are already hundreds of studies onlow-dose BPA published in peer-review journals showing the abil-ity of BPA to imitate E2 action in animal models. In addition, theNational Toxicology Program (NTP) of the US conducted an evalu-ation of the potential for bisphenol A to cause adverse effects onreproduction and development in humans and reached the finalconclusion that ‘‘there is some concern for effects on the brain,behavior, and prostate gland in fetuses, infants, and children atcurrent human exposures to bisphenol A.Remarkably, as stated before, the lowest-observed estrogeniceffect caused by BPA is about 0.1–1 pM. Despite this fact, thereare some authors claiming that the estrogenic effect of BPA doesoccur but that it takes place at high doses or by affecting targetsthat are not estrogen sensitive or ER-dependent (Sharpe, 2010). We have reviewed here evidence that support the extremely po-tent BPA-estrogenic action when acting through non-classicalestrogen activated pathways. Nevertheless, we are aware that, atthe moment, this is applicable only to some cell types and moremechanistic insides in other biological systems are needed to rein-force this concept. References Alonso-Magdalena, P., Laribi, O., Ropero, A.B., Fuentes, E., Ripoll, C., Soria, B., Nadal,A., 2005. Low doses of bisphenol A and diethylstilbestrol impair Ca2+ signals inpancreatic alpha-cells through a nonclassical membrane estrogen receptorwithin intact islets of Langerhans. Environ. Health Perspect. 113, 969–977.Alonso-Magdalena, P., Morimoto, S., Ripoll, C., Fuentes, E., Nadal, A., 2006. Theestrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivoand induces insulin resistance. Environ. Health Perspect. 114, 106–112.Alonso-Magdalena, P., Ropero, A.B., Carrera, M.P., Cederroth, C.R., Baquie, M.,Gauthier, B.R., Nef, S., Stefani, E., Nadal, A., 2008. Pancreatic insulin contentregulation by the estrogen receptor ER alpha. PLoS One 3, e2069.Andersen, H.R., Andersson, A.M., Arnold, S.F., Autrup, H., Barfoed, M., Beresford, N.A.,Bjerregaard, P., Christiansen, L.B., Gissel, B., Hummel, R., Jorgensen, E.B.,Korsgaard, B., Le Guevel, R., Leffers, H., McLachlan, J., Moller, A., Nielsen, J.B.,Olea, N., Oles-Karasko, A., Pakdel, F., Pedersen, K.L., Perez, P., Skakkeboek, N.E.,Sonnenschein, C., Soto, A.M., et al., 1999. Comparison of short-termestrogenicity tests for identification of hormone-disrupting chemicals.Environ. Health Perspect. 107 (Suppl. 1), 89–108.Belcher, S.M., 2008. Rapid signaling mechanisms of estrogens in the developingcerebellum. Brain Res. Rev. 57, 481–492.Belcher, S.M., Le, H.H., Spurling, L., Wong, J.K., 2005. Rapid estrogenic regulation of extracellular signal- regulated kinase 1/2 signaling in cerebellar granule cellsinvolves a G protein- and protein kinase A-dependent mechanism andintracellular activation of protein phosphatase 2A. Endocrinology 146, 5397–5406.Bulayeva, N.N., Watson, C.S., 2004. Xenoestrogen-induced ERK-1 and ERK-2activation via multiple membrane-initiated signaling pathways. Environ.Health Perspect. 112, 1481–1487.Bulayeva, N.N., Gametchu, B., Watson, C.S., 2004. Quantitative measurement of estrogen-induced ERK 1 and 2 activation via multiple membrane-initiatedsignaling pathways. Steroids 69, 181–192.Calafat, A.M., Kuklenyik, Z., Reidy, J.A., Caudill, S.P., Ekong, J., Needham, L.L., 2005.Urinary concentrations of bisphenol A and 4-nonylphenol in a human referencepopulation. Environ. Health Perspect. 113, 391–395.Chambliss, K.L., Wu, Q., Oltmann, S., Konaniah, E.S., Umetani, M., Korach, K.S.,Thomas, G.D., Mineo, C., Yuhanna, I.S., Kim, S.H., Madak-Erdogan, Z., Maggi, A.,Dineen, S.P., Roland, C.L., Hui, D.Y., Brekken, R.A., Katzenellenbogen, J.A.,Katzenellenbogen, B.S., Shaul, P.W., 2010. Non-nuclear estrogen receptoralpha signaling promotes cardiovascular protection but not uterine or breastcancer growth in mice. J. Clin. Invest. 120, 2319–2330.Colborn, T., vom Saal, F.S., Soto, A.M., 1993. Developmental effects of endocrine-disrupting chemicals in wildlife and humans. Environ. Health Perspect. 101,378–384.Del Prato, S., Leonetti, F., Simonson, D.C., Sheehan, P., Matsuda, M., DeFronzo, R.A.,1994. Effect of sustained physiologic hyperinsulinaemia and hyperglycaemia oninsulin secretion and insulin sensitivity in man. Diabetologia 37, 1025–1035. P. Alonso-Magdalena et al./Molecular and Cellular Endocrinology xxx (2012) xxx–xxx  5 Please cite this article in press as: Alonso-Magdalena, P., et al. Bisphenol-A acts as a potent estrogen via non-classical estrogen triggered pathways. Mo-lecular and Cellular Endocrinology (2012), doi:10.1016/j.mce.2011.12.012
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