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Annales dEndocrinologie 74 (2013) 211220

Review

Bisphenol A: An endocrine and metabolic disruptorBisphnol A : un perturbateur endocrinien et mtabolique

Patrick Fenichel , Nicolas Chevalier , Francoise Brucker-DavisInserm U1065/C3M, service dendocrinologie, diabtologie et reproduction, hpital de lArchet 2, CHU de NICE, 151,

route Saint-Antoine-de-Ginestire, 06202 Nice cedex 3, France

Abstract

Bisphenol A (BPA), initially designed, like diethylstilbestrol, as a synthetic estrogen, has been rapidly and widely used for its cross-linkingproperties in the manufacture of polycarbonate plastics and epoxy resins. Because of incomplete polymerization and degradation of the polymersby exposure to higher than usual temperatures, BPA leaches out from food and beverage containers, as well as from dental sealants. In humans,free active unconjugated BPA is metabolized by rapid glucurono- or sulfo-conjugation and eliminated via renal clearance. However, exposure toenvironmental nanomolar concentrations of BPA is ubiquitous and continuous via different routes: oral, air, skin. In rodents, fetal and perinatalexposure to such environmentally relevant doses of BPA has been shown to affect the brain, liver, gut, adipose tissue, endocrine pancreas, mammarygland and reproductive tract and function. Similar concentrations are also able in vitro to impact human malignant breast, prostate, male germor adipocyte cell lines (with a promoting effect and by interfering with chemotherapy drugs), or to stimulate pancreatic ! cell insulin secretion.High levels of BPA have recently been correlated with obesity, diabetes, cardiovascular diseases, polycystic ovarian disease or low sperm count.However, before the real impact of BPA on human health can be clearly assessed, prospective longitudinal epidemiological studies are needed aswell as characterization of selective biomarkers to verify long-term exposure and selective imprinting. 2013 Published by Elsevier Masson SAS.

Rsum

Le bisphnol A (BPA) est une substance chimique trs ubiquitaire prsente dans la plupart des plastiques polycarbons et les rsines poxy, qui estrelargu sous leffet de la chaleur et considre comme prsentant un effet de perturbateur endocrinien estrogno-mimtique. Il est essentiellementabsorb dans lespce humaine par voie alimentaire et est retrouv sous forme libre active dans le sang et sous forme conjugue dans les urinesen vue de son limination, chez la majorit des individus. La mise en vidence de troubles du dveloppement, de la reproduction, du mtabolismechez les rongeurs exposs des taux quivalents, pendant des priodes critiques dexposition ftale et/ou prinatale, a conduit sinterrogersur sa responsabilit dans les pathologies humaines correspondantes. Dautant quil est capable dinduire in vitro sur des cellules humaines, viades rcepteurs membranaires des estrognes, la prolifration de cellules malignes mammaires ou testiculaires ou dadipocytes et de rguler lascrtion insulinique par les lots !-pancratiques. Bien que des tudes pidmiologiques transversales, rtrospectives mettent en vidence unecorrlation entre obsit, insulinorsistance, diabte, hypofertilit et les taux de BPA, elles ne permettent pas elles seules dattester dune relationcausale et dvaluer exactement la part de ce potentiel facteur de risque. Seules des tudes prospectives longitudinales pourraient conforter cettehypothse ainsi que la caractrisation de biomarqueurs permettant la fois dvaluer lexposition prolonge et continue de faibles doses de BPAet en mme temps de mettre en vidence des modifications molculaires conduisant faire la part de lexposition chronique et de la susceptibilitindividuelle. 2013 Publi par Elsevier Masson SAS.

1. Introduction

The concept of endocrine disruptor was proposed 20 yearsago [1] when various observations of deleterious effects on

Corresponding author.E-mail address: fenichel.p@chu-nice.fr (P. Fenichel).

wildlife or humans were put together, in association with theintensive use of pesticides in agriculture. It refers to natural orsynthetic chemical products, which are able to mimic and/orinterfere with hormone receptors generating as unexpected lig-ands deleterious effects on animal or human health [2]. Many ofthese products, such as dichlorodiphenyltrichloroethane (DDT)or other organochlorides, demonstrated estrogenic or anti-androgenic effects and could interact during development and/or

0003-4266/$ see front matter 2013 Published by Elsevier Masson SAS.http://dx.doi.org/10.1016/j.ando.2013.04.002

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reproduction [3]. These observations have been compared withthe human unwilling experiment of fetal exposure to diethyl-stilbestrol (DES) used in prevention of miscarriages from 1950to 1975. An increased risk of different pathologies, includinggenital malformations, infertility, or hormone dependent can-cers, have been reported [4] for these exposed children, some-times several decades later [5], underlining the critical windowof fetal period for exposure to environmental exposure disruptors(EED). This concept is very similar to the one proposed by DavidBarker, based on the fetal nutritional environment influencingthe developmental origin of chronic adult diseases [6].

However, an objective causative role for EED exposure inhuman chronic disease remains very difficult to assess, exceptfor this unique experimental story of distilbene, becauseof methodological limitations. These difficulties concern epi-demiological designs as well as toxicological and mechanisticaspects. These concerns represent a real challenge for the scien-tists and the physicians who have to assess the possible toxicityor the harmlessness of a given molecule, and for the politicianswho will have to establish regulation for allowed thresholds ordecide the authorization for common use of a new molecule(REACH program).

The case of bisphenol A represents a perfect example forthis emerging concept and should be of particular interest forFrench endocrinologists and diabetologists, considering that itslinks with this compound has been pointed out for reproduction,diabetes, obesity and endocrine related cancers in the very recentANSES document (Agence nationale de scurit sanitaire delalimentation, de lenvironnement et du travail) published inDecember 2011 by the French Ministry of Environment [7].

2. Historical record

BPA is a chemical product, first synthesized at the begin-ning of the 20th century from two phenol molecules (bisphenol)linked by one acetone molecule (A) (Fig. 1). BPA was firstdesigned as a synthetic estrogen because of its action on rodentsuterus [8]. In fact, diethylstilbestrol, a structural analog, wasfound to be much more potent. Later, BPA has been widely usedfor its cross-linking properties in the manufacture of polycarbo-nate plastics and epoxy resins. Due to real advantages in termsof heat resistance and elasticity, the use of BPA has progres-sively increased all over the world with an annual production,higher than 10 millions of tons. It has become ubiquitous in a lotof domestic products such as plastic bags, bottles, coated tins,dental sealants, paintings, CD-Rom, etc. Because of incompletepolymerization and degradation of the polymers by exposure tohigher than usual temperatures such as microwaves use withplastic film or containers [9], BPA has been found to leachout from food and beverage, as well as from dental sealants.Exposure to environmental nanomolar concentrations of BPA isubiquitous and continuous, via different routes: oral absorption,air, skin. In humans, free active unconjugated BPA is metabo-lized by rapid glucurono- or sulfono-conjugation and eliminatedvia renal clearance. It is present in the urine of 90% of cases inan American reference population (0.21.6 ng/mL) [10] and inmost bloods, maternal milk or amniotic fluid [11].

Its structural analogy with DES (Fig. 1), with its supposedxenoestrogenic effect, as well as its increasing underspreadbackground exposure have led to numerous toxicological tests inrodents. After fetal or perinatal exposure, these tests have shown,at low, environmentally relevant concentrations, to affect thebrain, liver, gut, adipose tissue, endocrine pancreas, mammarygland and reproductive tract and function. Similar concentra-tions are also able in vitro to impact human malignant breast,prostate, male germ or adipocyte cell lines with a promotingeffect [12,13], or by interfering with chemotherapy drugs, orto stimulate pancreatic ! cell insulin secretion. High levels offree active BPA have recently been correlated with obesity, dia-betes, cardiovascular diseases, polycystic ovarian disease or lowsperm count. All these experimental and epidemiological datahave led to increasing concerns about the real impact of chronicBPA exposure on human health and what should be an objec-tive and realistic regulatory policy concerning the determinationof maximum thresholds or even its prohibition, as was recentlydecided [14] in France concerning baby bottles in 2011 leadingto complete prohibition after 2014.

3. Obesity and metabolic syndrome

A recent working document of ANSES proposed the rec-ommendation of a total BPA prohibition because of indirectevidence supporting its involvement in obesity, metabolic syn-drome and diabetes [7]. In mice, fetal or perinatal exposureto low, environmentally relevant doses of BPA induces in theoffspring an increase of adult weight, depending on sex, doseand window of exposure [1519], associated to metabolic syn-drome [20]. Similar effects have been described after neonatalexposure to DES [21]. BPA stimulates in vitro differentiation ofmurine fibroblasts into adipocytes and increases lipid storage ina dose-dependent manner [2224]. Low doses of BPA inhibitadiponectin secretion by human adipocytes cultures in vitroand stimulate the secretion of interleulin 6 and TNF", twoinflammatory adipokines, suggesting its possible involvementin obesity, metabolic syndrome and insulin resistance [2527].It also results in deleterious effects observed both on the ener-getic inbalance and glucose homeostasis via its effects on liver,adipose tissue and pancreatic islets [27]. In humans, althoughrapidly metabolized in the liver into inactivated glucurono- orsulfo-conjugates and theoretically immediately eliminated byrenal excretion, BPA was shown to accumulate into the adiposetissue. This could explain its blood persistence during fasting[28]. A recent French epidemiological study reported a non-monotonous, inversed U shape dose-response relation betweenmaternal urinary BPA and newborn weight [29] quite concordantwith what was observed in rodents [15]. Several recent epidemi-ological studies in adults have tried to correlate urinary BPAand obesity. First an American study concerning 2700 adultsreported a positive correlation with general obesity, central obe-sity, and insulin resistance [30]. A Chinese study involving 3400middle age adults found quite similar results [31]. However,both studies were retrospective, cross-sectional with a singleurinary sample, traducing only the recent exposure. Both won-dered with honesty whether this correlation was causal or linked

P. Fenichel et al. / Annales dEndocrinologie 74 (2013) 211220 213

Fig. 1. Xenoestrogens: notice the bisphenol A structural analogy with various xenoestrogens like diethylstilbestrol, un estrogne de synthse, DDT, an organochloridepesticide, PCB an industrial chemical and resveratrol, a phytoestrogen.

to an excessive intake of sweet beverages from canned tins wellappreciated by over-weight people. In women, blood BPA levelshave been correlated with PCO, an endocrine disease associatedwith obesity, insulin resistance and hyperandrogenia [32,33].

Indeed, nowadays exposure to BPA has emerged as a possiblerisk factor able to accelerate, the development of obesity andmetabolic syndrome [7,27].

4. Diabetes

Animal experimental models, cell cultures using murine orhuman pancreatic ! cells and epidemiologiocal studies, suggestalso that exposure to BPA might participate to the increasingincidence of type 2 diabetes. Adult male mice exposed to BPAdevelop both hyperinsulinemia and mild insulin resistance [20].This impairment occurs through a direct effect on pancreaticcells, as described later, but also by impairment of insulin signaltransduction at the level of peripheral tissues (muscle, liver, adi-pose tissue). BPA induces an oxidative stress in rodent liver bydecreasing expression of anti-oxydative enzymes [34]. Saku-rai et al. [35] have shown that in adipocytes, BPA increases,basal and insulin-activated glucose transport, by stimulatingGLUT4 expression. At the level of pancreatic islets, BPA isable to suppress in vitro, glucose-induced calcium oscillations,

which control glucagon secretion [36] in " cells. On ! cells,very low doses of BPA (0.1 nM) increase glucose-induced cal-cium oscillations frequency leading to a higher insulin secretion[37]. These rapid effects observed in vitro are confirmed onentire murine pancreatic islets [37]. These effects mimic therapid effects induced by estrogens, which are mediated bynon-classical membrane estrogen receptors as described later[20]. In vivo BPA is able as estrogens to decrease very rapidly(30 minutes) glucose blood level by stimulating insulin secre-tion [20] and by stimulating AMP dependent transcription factorCREB which regulates both insulin gene expression and ! cellssurvival [38]. A longer exposure to BPA in rats, induces after 4days, chronic hyperinsulinemia and insulin resistance [20]. Twokinds of arguments allow a possible extrapolation to humans.First, identical rapid effects of BPA have been reported on humanpancreatic ! cells [39]. Secondly, several epidemiological, ret-rospective cross-sectional studies found a correlation. All thesestudies belong to the American study of the National Healthand Nutrition Examination Survey (NHANES). The first onewas performed during the period of 20032004 with adultsaged 18 to 74 years from the general. After normalization forage, sex, ethnic background, education level, smoking, BMI,waist circumference, and urinary creatinine level, it found apositive correlation between urinary BPA and the presence of

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a self-reported diabetes [13]. The highest levels of BPA wereassociated with the highest BMI values and one may wonderone more time whether it represents the recent exposure to BPAleached out from soda cans, which are more frequently usedby the obese people or if it could be a marker of exposure tomultiple EED including BPA [13]. Several similar studies per-formed from 2005 to 2008 found discordant results [40,41].However, Shankar et al. [42], selecting from these differentstudies of NHNAES the patients for whom biological resultswere provided, could find after similar normalization, a positivecorrelation with diabetes diagnosed either by fasting glycaemiagreater than 1.26 g/L; fasting or not fasting glycaemia greaterthan 2.00 g/L or HBA1c greater than 6.5%. This possible riskfactor independent from classical risk factors of type 2 diabeteshas to be confirmed by prospective longitudinal studies.

5. Reproductive studies

5.1. Polycystic ovaries and BPA exposure

Polycystic ovaries (PCO) is considered to be the result of bothgenetic and environmental factors. Neonatal exposure to BPAin rats induces an adult syndrome very similar to human PCO[43]. In vitro BPA stimulates testosterone secretion by ovariantheca cells in rats [44]. In adult women with PCO, blood BPAlevel is higher than in control women with normal ovulation andwithout hyperandrogenia, independently of the presence or notof obesity [33], and BPA correlates with androgens.

5.2. Cryptorchidism

Cryptorchidism or undescended testis (UDT) is the most fre-quent male congenital malformation and its incidence at birth(>2%) may have increased over the last decades. UDT is a riskfactor for hypofertility and testicular cancer. It remains idio-pathic in most cases but genetic and environmental factors havebeen suggested [45,46]. We and others have found in mothermilk higher levels of some EED in UDT [46,47]. While cordblood BPA was not found significantly increased in UDT [48](Fig. 2), we could identify in the whole population (UDT andcontrols) a negative correlation between cord blood BPA andinsulin-like peptide 3 (iNSL3) a Leydigian hormone involved intesticular descent, with INSL3 significantly lower in the UDTgroup versus control [49].

5.3. Male hypofertility

Estrogenic and anti-androgenic effects of BPA have beendemonstrated in vivo and in vitro in rodents [50]. For this reason,testicular function has been particularly studied. In rodents, BPAreduces Leydigian secretion of testosterone [51]. In Sertoli cells,it induces an impairment of junctional proteins [52] and stimu-lates in vitro human seminoma cell proliferation [53,54]. In uteroexposure in rats, leads in adult male to impaired spermatogene-sis [55]. Few human epidemiological studies have been able tocorrelate urinary BPA and sperm quality. Recently, Ly et al. [56]

Fig. 2. Human bisphenol A (BPA) cord blood levels. Free BPA cord blood levelswere compared in male born after 35 weeks of amenorrhea with or withoutundescended testis (UDT).

From Fnichel et al. [48].

could find a correlation between urinary BPA, number, motilityand sperm vitality.

6. Hormone dependent cancer studies

6.1. Breast cancer

Breast cancer is the most frequent cancer in women withone million of new cases every year all over the world includ-ing 40,000 in France corresponding to 32% of feminine cancers[57]. It represents 2% of all causes of women deaths and thefirst cause by cancer. Its incidence is still increasing (two-foldincrease within the last 20 years), 2 to 5% more every yeardepending on the part of the world, with a great disparity betweenon the one hand North America, Europe and Australia which are

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high-incidence areas, versus on the other hand, Asia or Africawith a lower incidence [58]. These geographical differences sug-gest environmental factors supported by the data obtained frommigrant populations from Asia to America and the fact that lessthan 10% of breast cancers are associated with germinal muta-tions [59,60]. For all these reasons, nutritional and/or toxicantfactors including EED with estrogenic effects have been sug-gested. Indeed, the carcinogenic effect of fetal programmingby exposure to low doses of some of them, including BPA, hasbeen clearly established in rodent models. Fetal exposure to BPAinduces in mice at the peripubertal period, an impaired devel-opment of the histological architecture of the mammary glandswith an enhanced sensitivity to estrogens, an increase in area andnumber of terminal buds when normalized to the total canalarstructures, a decrease of apoptotic activity, an increase of thenumber of epithelial cells expressing the progesterone receptorand an increase of lateral canalar ramifications [61,62]. Theseobserved abnormalities suggest that development and morpho-genesis have been disturbed during the fetal exposed period.This was identified and confirmed during this period in fetalmice [63]. Moreover, in Wister rats, prenatal exposure to BPAwas associated in adults with changes in epithelial and stromaltissues, an increased ratio proliferation/apoptosis and a cleardecrease of the doses of carcinogens necessary to induce neo-plastic lesions, doses which remain without any effect in controlanimals non exposed in utero to BPA. All these elements sup-port an increased susceptibility to develop a mammary cancer.BPA is able to stimulate in vitro human breast cancer cell pro-liferation [64]. Strikingly, BPA is also able at very low doses(nM) similar to the blood level found in the general population,to induce in vitro a chemo-resistance on several human malig-nant breast cell lines whether these cells are ER" positive ornegative, while it increases expression of several anti-apoptoticproteins [65]. Nevertheless, up to now, no epidemiological studyhas been reported correlating chronic exposure to BPA with anincreased risk of breast cancer as it has been shown for someEED [59], although one has to consider the methodologicaldifficulties associated to such a demonstration [66].

6.2. Prostate cancer

Prostate cancer is the most frequent cancer of the elderly manmore than 50 years and the second cause of male death by can-cer. Its incidence is still increasing independently of age [67].Despite a better screening and treatment, its mortality remainshigh when the androgen dependent phase is over. Among therisk factors, only nutritional (high fat diet) and genetic (famil-ial and ethnic) have been well identified but here too the roleof EED has been suggested. The prostate gland is classicallyandrogen dependent, develops during puberty under androgencontrol. Androgen dependence of prostate cancer allows anti-androgenic treatment. But additive hormonal control throughestrogens in normal and malignant prostate development is sup-ported by many experimental data [68]. Aromatase and ER(both ER" and ER!) are expressed in human prostate [68].Maternal estrogens allow at the third trimester of pregnancythe growth of the prostate gland in men. When exposed to DES

given during the pregnancy to their mothers the newborn pre-sented prostate hyperplasia [69]. A recent study performed in theFrench Antilles, have clearly illustrated the likely risk factor ofchronic exposure to chlordecone, an estrogenic organochloridepesticide in the occurring of prostate cancer [70]. Chlordeconeor kepone has been widely used in the French Antilles dur-ing 1973 and 1993 in the culture of banana. Its degradationis very slow and it will persist in groundwater during severaldecades. In rodents, it acts as a carcinogen able to induce livercancer. In men this case/control study has been performed ina limited geographical area with a controlled exposure, whichhas allowed to support the relation between chlordecone expo-sure and prostate cancer risk independently of ethnical originor other statistical bias or confounders. The risk was positivelycorrelated to chlordecone blood levels and enhanced in mengreater than 60 years, with familial antecedent and/or in pres-ence of a specific polymorphism for the chlordecone reductasegene coding for an enzyme involved in hepatic detoxification[70]. Moreover, tumoral aggressiveness was correlated withchlordecone blood levels [70]. These epidemiological data aresupported by experimental ones. Fetal or perinatal BPA or DESexposure in rodents, are associated in adults with prostate hyper-plasia and pre-malignant lesions [68]. This perinatal exposureto BPA or to estradiol increases the susceptibility to develop aprostate cancer either spontaneously or after a second estrogenicshot [71]. In order to explain the therapeutic escape to anti-androgens in prostate cancer becoming androgen independent,several mechanisms have been proposed including the occur-ring of secondary mutation of the androgen receptor gene [72].In some of these cases, it has been shown in vitro that cancercells became sensitive to BPA, activating proliferation throughthis mutated receptor [73].

6.3. Testicular cancer

Testicular germ cell cancers (TGCC) represent only 2% ofall cancers but they are the most frequent cancer in young manwith an increasing incidence all over the world estimated as 2to 3% every year [74]. This increase remains unexplained butthe possible role of in utero exposure to EEP interfering in themalignant transformation of fetal stem germ cells, the gonocytes,into TGCC [75], are supported by several epidemiological andexperimental data. In the USA, among the agriculture work-ers exposed to pesticides, an odd ratio of 6 has been found fortesticular cancer when compared to control [76,77] especiallyfor atrazine, an organochloride estrogenic pesticide which is stillwidely used in the West of France. Hardell et al. [78] have studiedin Sweden the correlation between TGCC and the blood levelsof several EED in patients and their mothers. Strikingly, no cor-relation was observed with the patients levels but the motherslevels when elevated were associated with an increasing riskwith an odd ratio of 4 for PCB (bisphenyls polychlors), HCB(hexachlorobenzene), nonachlordane, all estrogenic EED, butnot with ppDDE a stable anti-androgenic derivative of DDT sup-porting the role of fetal exposure to xenoestrogens [78]. Howeveris TTGC really estrogen dependent as other classical estro-gen dependent cancers? In fact we and others have shown that

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Fig. 3. Bisphenol A (BPA) regulates human seminoma cell proliferation. Very low doses of BPA (pM) stimulates human seminoma cell proliferation in vitro. Noticethat the dose/response curve is not monotonic but an inversed U shape, which can be explained by two different effects mediated by two different receptors. FIRSTBPA stimulates at low doses cell proliferation through a non-classical membrane estrogen receptor, GPR30 for whom it presents a very high affinity; secondly BPAtriggers a suppressive effect through ER! at high doses because of a relative low affinity.

From Bouskine et al. [53].

seminoma, the most frequent testicular germ cell cancer,expresses both aromatase and ER! but not ER" [79]. At theopposite of prostate or breast cancer, it is not possible to induceexperimentally a TGCC in rodents by fetal exposure to BPA orother estrogenic EED and seminoma rodent models do not existat all. For this reason we have used a human seminoma cell line todemonstrate that estrogens and xenoestrogens were able to stim-ulate cell proliferation in vitro [79,80]. Estradiol 17!, the naturalestrogen induced a suppressive effect on seminoma cell prolifer-ation through the classical estrogen receptor ER! [79]. Estradiolwhen coupled to bovine serum albumin which cannot cross thecell membrane, stimulate at the opposite of the free molecule,cell proliferation through a non-classical G protein coupledestrogen receptor (GPR30/GPER) [54,80]. Human seminomaare therefore estrogen dependent and xenoestrogens may actthrough two different receptors depending to their respectiveaffinity for them. We have screened several EED such as atrazine,DES and BPA and observed different effects depending on theresultant of the two receptors. Concerning BPA (Fig. 3), we wereable to demonstrate that very low doses of BPA (pM) under thelevels found in male cord blood [48] stimulate seminoma cellproliferation through GPR30, a membrane associated estrogenreceptor, different from classical ERs [48]. The dose-responsecurve was not monotonic but showed a inversed U shape (Fig. 3),which could be explained by the resultant of the double oppo-site effect on ER! and GPR30 [53,81]. At low doses (nM orpM), BPA acted only through GPR30 by a promotive effect.At higher doses (mM), BPA acted also through ER!, whichcounteracted the promotive GPR30 mediated effect [79]. Lowdoses of BPA (nM) are similar to the one able in rodents whenexposed in utero, to induce mammary or prostatic malignantlesions in adult [82,83] as described before. We have recentlyshown that seminoma over express GPR30 [81] likely through aspecific polymorphism abnormally present in men with TGCCconferring likely a genetic susceptibility to such cancers.

7. Exposure to BPA and thyroid function

BPA may interfere with thyroid hormone action [84]. Arecent human epidemiological study reported an inverse relation

between BPA concentration in maternal urine and maternalserum total T4, during pregnancy [85]. The association wasstronger when maternal urinary BPA and serum total T4 weremeasure closer together, suggesting a transient effect of BPA,and when measured in the third trimester of pregnancy [85].They also reported an inverse correlation between maternal BPAand neonatal TSH but only in male, which could be explainedby a sexual differentiation of UDP-glucuronosyl transferaseexpression as shown in rat [86]. A negative correlation betweencord blood fT4 and maternal milk PCB118, PCB180 and DDEhas also been reported [87].

8. Metabolism, storage, waste, dosage

Free BPA, the active form, is theoretically in humans rapidlyglucurono or sulfo conjugated by the liver and evacuatedby the kidney as demonstrated by the pharmacokinetic studyafter absorption of an unique oral dose [88]. This kinetics isdifferent from rodents and this difference has been used to con-test any chronic potential deleterious effect [89,90]. However,lots of studies have nevertheless identified relatively high lev-els of free BPA in cord blood, amniotic fluid, plasma or urineof adults from general populations [11]. Its blood persistenceduring fasting [28] suggests that BPA is stored in adipose tis-sue and unless a relative lipophility [28]. Moreover daily oralabsorption contributes to maintain constant high blood levels.Moreover sulfatases and/or glucuronidases are present in sev-eral adults organs and highly expressed in the placenta whichcould explain that relative high free BPA levels are present inthe blood flow, which is the only form able to induce delete-rious effects as EED. The fetus and the newborn seem to beparticularly exposed [91,92] in part due to hepatic immaturity ofdetoxification enzymes. Chromatography and mass spectrome-try represent the reference in term of free or conjugated BPAin blood or urine. Moreover a RIA (roter interaction analy-sis) has been proposed and validated by H. Dchaud at Lyon[48,93]. It allowed us to confirm the presence of free BPA in cordblood and pregnant women between 1 and 10 ng/mL [48]. How-ever, one must be very careful to avoid plastic contaminationfrom transport, storage and manipulation of the samples at the

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Table 1Interactions bisphenol A (BPA)/hormone receptors.Receptor Location Effect References

ERK Nuclear Agonist [14,63,90,94]ERK Membrane Agonist [14,39]ERK Nuclear Agonist [14,39,90]GPR3Q Membrane Agonist [36,37,52,53,63,77]ERRK Nuclear Agonist [9294]AR Nuclear Antagonist [95]PPARK Nuclear Antagonist [96]TR Nuclear Antagonist [81,97]

laboratory and the simple perfusion of the patient beforeincreases the BPA concentrations due to release from the tubu-lures and perfusion bottles.

8.1. BPA, an endocrine disruptor: which receptors?

One important question concerning the endocrine disruptionmediated by BPA is whether it acts really as a weak estrogensince its affinity for the classical nuclear ER" is 10,000 timesweaker as for estradiol 17 ! and 1000 weaker than affinity forER! [94]. For this reason BPA at the nanomol concentrationfound in humans should not been considered as an estrogeno-mimetic at all [95]. Different explanations have been proposedfor this apparent paradoxal criteria, especially the involvementof non-classical membrane estrogen receptors. One of this recep-tor G protein coupled, GPR30 have been identified. This GPCRis present in breast cancer cells, ! Langerhans islets, adipocyteswith a very high affinity for BPA and mediates activation ofsignaling pathways classically involved in the control of cellproliferation, apoptosis and survey. In our laboratory, we haveshown its promoter role on human seminoma cells [53,54].Another receptor, ERR# (estrogen-related receptor-gamma), anuclear receptor, has also a good affinity for BPA [96]. It ispresent on adipocytes [97]. GPR30 and ERR# are both expressedby ER" and ER! negative breast cancer cells which respond toestrogens and BPA [98]. An anti-androgen effect has also beenidentified at the AR level for BPA [99]. It can also interact withPPAR# as an antagonist [100] able to inhibit adipocyte prolif-eration and also as an antagonist for thyroid hormone receptorsTR (Table 1) [101].

8.2. Molecular mechanisms programming in utero thedevelopmental origin of adult diseases induced by EED

Fetal or perinatal developmental origin of adult diseases hasbeen nicely analyzed for bisphenol A concerning mammary car-cinogenesis consecutive to very low doses administrated duringthis critical period in rats and mice which have later developedpre or malignant lesions. Bromer et al. in 2009 have characte-rized the imprinted molecular mechanisms, which have led afterin utero exposure to DES, to uterus cancer in mice. They identi-fied one gene HOXA10 coding for a transcription factor involvedin uterus growth and which promoter region was hypomethy-lated through BPA exposure [102]. This epigenetic modificationis traduced into adult overexpression of HOXA10, influencing

Fig. 4. Fetal exposure to environmental exposure disruptors (EED), epigeneti-cal modifications and hormone dependent cancers. Hormonal disruptions duringdevelopment such as for example exposure to EED are able to induce epi-genetical modifications, which will later at adult participate to the malignantcell transformation when associated to de novo mutations or new epigeneticalalterations.

From Chiam et al. [109].

carcinogenesis. Other DES induced epigenetic changes havebeen reported for the oncogene c-fos which showed a similargene hypomethylation leading to its overexpression in adultuterus stimulating cell proliferation [103]. Similar modifica-tions concerning HOXA10 gene have been reported with BPA[104] and methoxychlore an estrogenic organochloride pesticide[105]. DNA methylation is an epigenetic hallmark involved inthe initiation and/or promoting phase of carcinogenesis [106].Ho and Prins [71,83] have studied the epigenetic changesinduced in prostate rat by BPA or estrogens fetal or perinatalexposure. Out of 50 gene sequence changes they identified a genecoding for a particularly interesting enzyme phosphodiesterase4D4, which degrades cAMP. Hypomethylation of this gene iscorrelated with the overexpression of the protein. Hypomethy-lation could be identify before adult age before any histologicalmodification and could therefore serve as an early biomarker[83]. Hypomethylation of this, gene is also present in humanprostate cancer. These modifications observed after EED expo-sure are likely induced through the action of DNA methyltrans-ferases (DNMT), enzymes which control DNA methylation onthe CpG islets [102]. There are some data suggesting that theseenzymes are regulated by classical estrogen receptors [103]and/or other nuclear or not nuclear receptors [37,107,108]. Fig. 4summarizes the proposed mechanism through which EED willimprint epigenetic changes after fetal in utero exposure which

218 P. Fenichel et al. / Annales dEndocrinologie 74 (2013) 211220

will contribute with others deleterious factors to malignant trans-formation by enhancing chromosomal instability [109].

9. Conclusion

In conclusion there are solid arguments to support a dele-terious role for bisphenol A after chronic exposure but its realparticipation in human health remain to be demonstrated. BPAis ubiquitously present in the current environment. Low dosesof BPA environmentally relevant might interfere in the develop-ment of chronic metabolic diseases, reproductive pathologies,hormone dependent cancers (prostate, breast, testes). However,prospective longitudinal studies are needed to confirm such aresponsibility. It is also necessary to identify biomarkers of dura-tion and intensity of exposure and to recognize early inducedmolecular imprintings (gene methylation, histone modifications,microRNAs). ANSES works has led first to the prohibition ofbaby bottles in 2011 in France and for 2014 to the completeprohibition of BPA use.

Disclosure of interest

The authors declare that they have no conflicts of interestconcerning this article.

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Bisphenol A: An endocrine and metabolic disruptor1 Introduction2 Historical record3 Obesity and metabolic syndrome4 Diabetes5 Reproductive studies5.1 Polycystic ovaries and BPA exposure5.2 Cryptorchidism5.3 Male hypofertility

6 Hormone dependent cancer studies6.1 Breast cancer6.2 Prostate cancer6.3 Testicular cancer

7 Exposure to BPA and thyroid function8 Metabolism, storage, waste, dosage8.1 BPA, an endocrine disruptor: which receptors?8.2 Molecular mechanisms programming in utero the developmental origin of adult diseases induced by EED

9 ConclusionDisclosure of interestReferences