V. Gayrard Physiologie ENVT « Pesticides perturbateurs endocriniens »

V. Gayrard Physiologie ENVT « Pesticides perturbateurs endocriniens »
Impact des perturbateurs endocriniens sur la reproduction de la femelle et le développement de la glande mammaire V. Gayrard Physiologie ENVT « Pesticides perturbateurs endocriniens »

Les systèmes endocriniens cibles des perturbateurs endocriniens
You know that all hormone-sensitive physiological systems are vulnerable to EDCs, including brain and hypothalamic neuroendocrine systems; pituitary; thyroid; cardiovascular system; mammary gland; adipose tissue; pancreas; ovary and uterus in females; and testes and prostate in males. Following the presentation of N. Hagen, my talk will focus on the potential effect of EDC on hormone-sensitive physiological functions (female reproduction, breast development associated to lactation), breast cancer, neuronedocrinology, metabolism and obesity.

Introduction Perturbateurs endocriniens contaminants environnement, chaîne alimentaire Perturbateurs endocriniens: santé publique Perturbateurs endocriniens: pesticides, produits chimiques, plastiques... Along my talk, I will present you results from animal models, human clinical observations, and epidemiological studies that converge to implicate EDCs as a significant concern to public health. Furthermore, EDCs represent a broad class of molecules such as organochlorinated pesticides and industrial chemicals, plastics and plasticizers, fuels, and many other chemicals that are present in the environment or are in widespread use.

Plan de l’exposé 1. Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle 2. Effets des perturbateurs endocriniens sur le développement de la glande mammaire 3. Cibles neuroendocriniennes des perturbateurs de la fonction de reproduction

Observations cliniques
1. Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle Observations cliniques DES (diethylstilbestrol) Le DES administré chez la femme enceinte entre 1948 et 1971 comme thérapie anti-abortive a entraîné des cancers de l’appareil génital et des troubles de la reproduction des enfants, 20 ans après le traitement Données expérimentales: rôle du DES dans l’incidence des troubles de la reproduction In the adult female, the first evidence of endocrine disruption was provided almost 40 yr ago through a clinical observation of the human DES syndrom DES administered to pregnant women between and 1971 as an anti-abortive therapy caused genital cancers and reproductive disorders in their children 20 years later. Subsequently, DES effects and mechanisms have been substantiated in animal models. Experimental data support the causal role of DES in female reproductive disorders Thus, robust clinical observations together with experimental data support the causal role of DES in female reproductive disorders. The implications of EDCs have been proposed in other disorders of the female reproductive system, including disorders of ovulation and lactation, benign breast disease, breast cancer, endometriosis, and uterine fibroids

Développement et fonctionnement du système reproducteur femelle
Les phases précoces du développement sont critiques Les processus biologiques impliqués sont sensibles aux substances hormonalement actives L’intéraction des PE avec ces processus peut avoir des conséquences sur la reproduction de la femme adulte The human DES syndrome has shown that early development is crucial and has evidenced the plasticity of the fetus in response to environmental cues and the potential effects that other hormonally active chemicals can have on human health and reproduction. Development and function of the female reproductive tract depends on coordinated biological processes that, if altered by endogenous or exogenous factors during critical periods of development or during different life stage, could have significantly adverse effects on women’s health and reproductive function and outcomes

La fonction de reproduction femelle Production cyclique d’un ovocyte fécondable pour la préservation de l’espèce Gestation chez les mammifères Fonction ovarienne Ovogenèse Stéroïdogenèse: contrôle cycle activité ovarienne, maintien de la gestation Développement du système reproducteur initié précocément au cours de la vie foetale The female reproductive function includes the differentiation and cyclic release of oocyte for fertilization and successful propagation of the species. In mammals, the reproductive system involve the adaptation of the genital tract for supporting pregnancy. The major functions of efmale gonads is the differenciation and release of oocyte. Additionnaly, female gonads produce steroids that allow the development of feamle secondary sexual characteristics and support pregnancy. The development of the female reproductive system is initiated during the early prenatal life.

Cellules germinales Vie foetale Une couche de cellules folliculaires Migration et différenciation (1er trimestre) ovocytes Nucleus + nucleolus oocyte I Follicule primordial Folliculogenèse Naissance oocyte I Méiose The process of oogenesis is initiated precociously during the fetal life where the full complement of cell types in the human ovary depends on successful germ cell migration from the yolk sac during the first trimester and differentiation into oocytes with associated somatic cells to form the functional unit of the primordial follicle by the second to third trimesters of gestation. The folliculogenesis corresponds to the stages of development of the follicle from the staeg “primordial follicle” to the stage of mature secondary follicle required for ovulation. Factors that interfere with germ cell migration or follicle formation can result in abnormal functioning of this tissue with significant reproductive consequences. Also, the oocyte is arrested in the diplotene stage of late prophase until meitic divisions occur beginning at puberty (meiosis I) and after fertilization (meiosis II), and abnormalities in these processes can have a profound impact on reproductive outcomes, such as aneuploidy, premature ovarian failure (POF), and miscarriage. Follicule secondaire mature Puberté ovogenèse Ovulation Oocyte II Fécondation Ovule Aneuploïdie, fausse couche…

Exposition foetale continue au BPA :un xénestrogène 2007: Canada interdit l’utilisation du BPA dans les biberons There is a type of exposure that needs to be addressed: the inadvertent and continuous exposure of fetuses to environmentally active chemicals, such as bisphenol A. BPA (Bisphenol A) is a chemical used in certain food contact materials. Indeed, bisphenol A (BPA) is the monomer that is polymerized to manufacture polycarbonate plastic products used in many hard plastic bottles and resins, such as those used to line cans containing food and beverages BPA Plastic containers have recycle codes on the bottom. In general, plastics that are marked with recycle codes 1, 2, 4, 5, and 6 are very unlikely to contain BPA. Some, but not all, plastics that are marked with recycle codes 3 or 7 may be made with BPA. In recent years, concerns have been raised about BPA's safety The recent evaluations of BPA toxicity did not lead to a consensus regarding at what exposure level BPA poses a health risk. Canada banned the use of BPA in baby feeding bottles, a same proposal was considered in several states in the US while in 2008 FDA and EFSA have concluded that BPA does not pose a threat to human health On January 15, 2010, the FDA issued an interim update on BPA1. At this interim stage, FDA supports reasonable steps to reduce exposure of infants to BPA in the food supply 50mg/(kg.j) LOAEL =50µg/(kg.j) DJA= 1000 EFSA, FDA, 2006; 2008

BPA is the most highly produced chemicals worldwide

Exposure to BPA is thought to result primarily from ingestion of food containing BPA. BPA is ubiquitous in our environment and can be detected in the majority of individuals in the US BPA was detected in 92.6% of persons ≥ 6 years of age with total concentrations ranging from 0.4 μg/L to 149 μg/L BPA and tOP are of concern to environmental public health because of the high potential for exposure of humans to these phenols and their demonstrated animal toxicity .

Configuration normale de la métaphase I Anomalies de la méiose des femelles exposées au BPA Chr éjecté du fuseau 10%–25% des ovocytes humains fécondés sont aneuploïdes Des anomalies de nombre des chromosomes sont à l’origine d’avortements, de retards mentaux... Défaut de congression Configuration de type télophase Authors have described deleterious effects of accidental fetal exposure to BPA on meiosis in mice. Accidental exposure of mice to BPA exposure via damaged caging material was accompanied by highly significant increases in meiotic chromosome abnormalities, including nondisjunction; thus, bisphenol A was implicated as a potent disruptor of meiosis Specifically, adult exposures result in abnormalities in alignment of chromosomes on the meiotic spindle and aneuploidy, which lead to aneuploid gametes and offspring The figure shows confocal images of intact mouse oocytes in normal metaphase I configuration (A) The oocytes were immunostained with an antibody to β-tublin to visualize the meiotic spindle (green) and counterstained with propidium iodide to visualize the chromosomes (red) (B–D) Representative meiotic abnormalities from exposed females. (B) Congression failure in an MII-arrested oocyte [C] metaphase I cell with chromosomes that have been ejected from the spindle [D] a cell that should be undergoing the first meiotic division but appears to have two separate groups of chromosomes in a telophase-like configuration The deleterious effects of fetal exposure to EDC on meiosis could contribute to the high percentage of fertilized human oocytes that are aneuploid (10%–25%), the numerical chromosome abnormalities are the leading cause of miscarriage, congenital defects, and mental retardation. Hunt et al., Curr Biol 13: 546 BPA: puissant perturbateur de la méiose

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle Genistein: phytoestrogen MCX: Metothoxychlor HPTE: MCX metabolite BPA: Bisphenol A DES: Diethylstilbestrol DDE: DDT metabolite An overview of ovarian folliculogenesis which comprises stages of division of the follicle cells to form multiples layers, differenciation of the peripheral theca cells, formation of a cavity the antral cavity that is considerably enlarged at the stage antral preovulatory follicle. After ovulation, the follicle cells are luteinized to form the corpus luteum. The stimulatory (+) and inhibitory (−) effects of environmental endocrine disruptors are shown. You can see that potentially these EDC can act at different stages of the folliculogenesis. DDE is a DDT metabolite 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene DDT (dichlorodiphenyltrichloroethane) is one of the most well-known synthetic pesticides. First synthesized in 1874, DDT's insecticidal properties were not discovered until 1939, and it was used with great success in the second half of World War II to control malaria and typhus among civilians and troops. After the war, DDT was made available for use as an agricultural insecticide, and soon its production and use skyrocketed In 1962, Silent Spring by American biologist Rachel Carson was published. The book catalogued the environmental impacts of the indiscriminate spraying of DDT in the US and questioned the logic of releasing large amounts of chemicals into the environment without fully understanding their effects on ecology or human health. The book suggested that DDT and other pesticides may cause cancer and that their agricultural use was a threat to wildlife, particularly birds. Its publication was one of the signature events in the birth of the environmental movement, and resulted in a large public outcry that eventually led to DDT being banned in the US in 1972.[4] DDT was subsequently banned for agricultural use worldwide under the Stockholm Convention, but its limited use in disease vector control continues to this day and remains controversial.[5] Methoxychlor is an organochlorine pesticide, which is currently used as a replacement for DDT. HPTE is the major MXC metabolite Genistein is a flavanoid phytoestrogen with proven estrogenic activity in several mammalian species, including humans and pigs

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle FSH: Follicle Stimulating Hormone LH: Luteinizing Hormone LH FSH LH The pituitary gonadotropins are key hormones in the regulation of folliculogenesis and steroidogenesis. LH stimulated the production of T by theca cells while FSH stimulated the synthesis of E2 from T provided by theca cells It is clear that the ability of gonadotropins to modulate ovarian function depends not only on the circulating levels of the gonadotropins, but also on the expression of appropriate receptor proteins by potential target cells in the ovary FSH and LH act through stimulatory G protein-coupled receptors expressed on target cells and transduce their signal, at least in part, by the activation of adenylyl cyclase and the production of the second messenger cAMP. The expression of receptors for LH is one of the major markers of the FSH-induced differentiation of granulosa cells The results of the actions of LH upon the mature granulosa cell include steroidogenesis, luteinization, and ovulation. As such, the induction of the LH receptor in granulosa cells is a critical step in reproductive physiology. + Chol E2: estradiol T: testosterone Chol: cholesterol P: progesterone Chol P + + Follicular fluid P Blood E2 T T E2 Granulosa cell

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle 2,3,7,8-Tetrachlorodibenzodioxin (TCDD): polychlorinated dibenzodioxin (dioxin) 2,3,7,8-Tetrachlorodibenzodioxin (TCDD) is a polychlorinated dibenzodioxin (dioxin). It is the most potent compound of the series and became known as a contaminant in Agent Orange, a herbicide used in the Vietnam War.[1] It was detected and treated in Viktor Yushchenko after he presented with severe poisoning in TCDD levels in his blood serum were 50,000-fold greater than those in the general population Dose-related effect of TCDD on FSH-induced LH receptor mRNA. (A) Granulosa cells from DES-primed immature rats were cultured for 24 h alone, and were then cultured with 30 ng/ml FSH alone and then FSH (30 ng/ml) plus increasing concentrations of TCDD for 48 h. LH receptor mRNA levels were measured using Northern blot analysis Autoradiographs of LH receptor (5.4 kb) mRNA were quantified by densitometric scanning. To examine the possible effect of TCDD on the acquisition of LH receptor, granulosa cells were cultured in the absence or presence of FSH (30 ng/ml), with or without TCDD 10 pM for 72 h. Basal LH receptor mRNA expression was negligible but increased in the presence of FSH Treatment with FSH produced, a substantial increase in LH receptor mRNA, whereas concurrent treatment with TCDD resulted in a significant decrease in LH receptor mRNA after being cultured from 24 to 72 h. As shown in cotreatment with FSH 30 ng/ml and increasing doses of TCDD (1–100 pM) inhibited the levels of FSH-induced LH receptor mRNA in a dose-dependent manner, and 1 pM of TCDD inhibited the effect of FSH significantly after 48 h. Le co-traitement des cellules de la granulosa avec FSH et des doses croissantes de TCDD (1–100 pM) inhibe de façon dose dépendante les niveaux de mRNA du récepteur à LH induits par FSH (Minegishi et al., Mol Cell Endocrinol 202: 123)

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle Control of theca cell steroidogenesis. LH binds to the LH receptor, stimulating the mobilization of stimulatory guanine-nucleotide binding proteins (Gs). The subsequent activation of adenylyl cyclase and generation of cAMP leads to the phosphorylation of cAMP-dependent protein kinase (pPKA). Substrates for pPKA include StAR protein and a cohort of steroid pathway enzymes and other molecules, which collectively mobilize intracellular cholesterol and guide the formation of progesterone from cholesterol and the synthesis of androstenedione and testosterone. Androstenedione and testosterone are secreted, and within granulosa cells, are aromatized into estrogens as described in the text and Fig. 2. The inhibitory (−) effects of genistein and inhibitory and/or stimulatory (+/−) effects of DES are shown. DHEA: dehydroepiandrosterone; HDL: high-density lipoprotein; P5: pregnenolone DHEA: dehydroepiandrosterone; HDL: high-density lipoprotein; P5: pregnenolone

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle Control of granulosa cell steroidogenesis. FSH binds to the FSHR, stimulating the mobilization of stimulatory guanine-nucleotide binding proteins (Gs). The subsequent activation of adenylyl cyclase and generation of cAMP leads to the phosphorylation of cAMP-dependent protein kinase (pPKA). Substrates for pPKA include StAR protein and a cohort of steroid pathway enzymes and other molecules that collectively mobilize intracellular cholesterol and guide the formation of progesterone from cholesterol and the aromatization of androstenedione and testosterone into E2. Progesterone, estrone (E1), and E2 represent the predominant steroids, which are secreted. The stimulatory (+) and inhibitory (−) effects of the environmental endocrine disruptors reviewed are shown. HDL: high-density lipoprotein; P5: pregnenolone. HDL: high-density lipoprotein; P5: pregnenolone.

Pathogenèse des troubles de la reproduction femelle ?
Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle Pathogenèse des troubles de la reproduction femelle ? Question clé: Contribution des PE aux mécanismes physiopathologiques? Données expérimentales: rôle des PE dans la pathogenèse du syndrome des ovaires polykystiques, des anomalies du tractus génital, insuffisance ovarienne, des fibromes, de l’endométriose … Most female reproductive disorders are well described with regard to clinical presentation, histological evaluation of involved tissues where applicable, and diagnostic classification. However, whereas few are polygenic inherited traits and some are due to infections, the pathogenesis of the vast majority of female reproductive disorders is not well understood. This has hindered a preventive strategy to their development and/or exacerbation, and in some cases limited the development of effective therapies for symptoms and associated morbidities. A key question arises as to whether EDCs contribute to the development of female reproductive disorders, particularly those occurring during a critical window of susceptibility: in utero, neonatally, in childhood, during puberty, and during adulthood. There are increasing data from wildlife studies and laboratory studies with rodents, ungulates, and nonhuman primates that support a role of EDCs in the pathogenesis of several female reproductive disorders, including polycystic ovarian syndrome, aneuploidy, POF, reproductive tract anomalies, uterine fibroids (benign smooth muscle tumors of the myometrium), endometriosis, and ectopic gestation

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle Signes ou symptomes Femmes PCOS Singes femelles androgénisés prénatalement Hyperandrogénisme ovarien + +a Anovulation +b Enlarged polyfollicular ovaries +c LH hypersécrétion +d Diminution du rétrocontrôle négatif stéroïdien +b,e Résistance à l’insuline +g Sécrétion insuline perturbée +h Augmentation du diabète de type II Augmentation du gras abdominal +I Syndrome des ovaires polykystiques (PCOS) Accumulation de petis follicules Polycystic ovary syndrome (PCOS) is one of the most common female endocrine disorders affecting approximately 5%-10% of women of reproductive age (12-45years old) and is one of the leading causes of infertility PCOS is a heterogeneous syndrome characterized by persistent anovulation, oligo- or amenorrhea, and hyperandrogenism At the level of the ovary, there is recruitment and growth of follicles to the small antral stage, without selection of a dominant, preovulatory follicle, leading to accumulation of multiple, small, antral follicles (An accumulation of incompletely developed follicles in the ovaries is seen in polycystic ovarian disease ) Obesity and insulin resistance occur in about 50% of women with PCOS, and obese women have a 12% risk of having PCOS The physiological processes associated to PCO are regulated by hormonal and metabolic parameters. Hence, endocrine disruption by environmental chemicals may indeed contribute to the pathogenesis of PCOS. Prenatally androgenized female rhesus monkeys have proved experimentally-induced phenotypic mimics of PCOS signs and symptoms Hence, in rhesus monkeys, prenatal exposure to high levels of testosterone results in fetal programming of PCOS traits. Specifically, high levels of testosterone exposure at gestational d 40–60 result in rhesus monkey females who, in adulthood, have anovulatory infertility, hypersecretion of LH, elevated circulating levels of testosterone, neuroendocrine feedback defects, central adiposity and compensatory insulin resistance, and polycystic ovaries with ovarian hyperandrogenism and follicular arrest in adulthood Le plus commun des désordres endocriniens féminins (5%-10% des femmes): infertilité Nécessité de comprendre la contribution des PE pour minimiser l’exposition et permettre la prévention

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle Bisphenol A: a xenoestrogen Les femmes atteintes de PCOS ont des niveaux élevés de BPA (Takeuchi et al., Endocr J 53: 485) Other pathways may be involved in endocrine disruption of PCOS. BPA is an estrogenic compound widely used in the production of polycarbonate plastics and epoxy resins Women with PCOS have higher levels of the EDC BPA Although adult exposures do not necessarily imply earlier exposures in life, there are data demonstrating nearly 5-fold higher levels of BPA in amniotic fluid compared with other body fluids, suggesting significant prenatal exposure Although a cause and effect of BPA and PCOS have not been demonstrated definitively, the biological plausibility is interesting and worthy of further consideration.

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle Di-(2-ethylhexyl)-phthalate (DEHP): plastifiant du polychlorure de vinyl (PVC) Endometriosis, a common cause of female infertility of unknown aetiology, occurs almost exclusively in menstruating women of reproductive age Endometriosis is a disease that affects females in their reproductive years. It is a painful, chronic disease that affects more than 5 1/2 million women and girls in the USA, and millions more worldwide. The endometrium is the tissue that lines the inside of the uterus, which builds up and sheds each month in the menstrual cycle. With Endometriosis this tissue is found in locations outside of the uterus, and develops into nodules, lesions, tumors, growths, or implants. This misplaced tissue develops into growths or lesions which respond to the menstrual cycle in the same way that the tissue of the uterine lining does Emerging evidence suggests a possible role for ubiquitous environmental contaminants in the physiopathology of endometriosis. In particular, polyhalogenated aromatic hydrocarbons (PHAH), a class of widespread environmental contaminants including dioxins, have been postulated to be linked to endometriosis and unbalanced expression of sex hormones or growth factors as a consequence of immune response can be recognized as the result of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin) exposure Di-(2-ethylhexyl)-phthalate (DEHP) is the most commonly used plasticizer in flexible polyvinylchloride (PVC) formulations. However, DEHP does not bind with the plastic and leaches with time and use from vinyl products, thus becoming an ubiquitous environmental contaminant (Mayer et al., 1972 ; Giam et al., 1978 ; Griffiths et al., 1985 ; Sharman et al., 1994 ; Bauer and Hermann, 1997 ) with potential adverse effects both on fertility and reproduction in animal models correlative findings of phthalate levels in plasma and endometriosis. For example, Cobellis et al. (113) found high plasma concentrations of di-(2-ethylhexyl)-phthalate in women with endometriosis, and an association of phthalate esters with endometriosis was found among Indian women (114). Thus, the evidence is accumulating of correlations between EDCs in the circulation of women with endometriosis, although a cause-and-effect relationship has yet to be established, which is not uncommon in reproductive environmental toxicity. Cobellis et al., Human Reprod 18: 1512

Impacts des perturbateurs endocriniens sur le développement et le fonctionnement du système reproducteur femelle 8 semaines de grossesse Troubles du développement associés au DES : Utérus en T, anatomie anormale des trompes utérines et du col de l’utérus In addition, whereas Mullerian tract formation begins at 8 wk gestation with fusion of the Mullerian ducts and subsequent differentiation into the uterus (endometrium, myometrium), cervix, and upper vagina, uterine differentiation with regard to formation of luminal epithelium, glandular epithelium, and stromal components is mostly a postnatal event, with functionality of response to steroid hormones beginning at puberty. Interference with these processes can predispose women to infertility, ectopic gestation, poor pregnancy outcomes, and other reproductive disorders that may be programmed during development (e.g., endometriosis, uterine fibroids). Thus, abnormal development or alterations at other times in the life cycle can alter anatomy and functionality of the female reproductive tract and thus can alter the reproductive potential of affected individuals and their offspring. Infertilité, gestation ectopique, endometriose, fibromes

Effets des perturbateurs endocriniens sur le développement de la glande mammaire
Augmentation significative de l’incidence du cancer du sein au cours des 50 dernières années Facteurs de risque du cancer du sein: durée de l’exposition aux hormones ovariennes: âge à la puberté, première grossesse, ménopause, lactation et parité Risque accru chez les jumelles: exposition importante aux oestrogènes pendant la gestation Windows of vulnerability to carcinogenic agents and "natural" risk factors The standard risk factors for developing breast cancer include age at menarche, first pregnancy, menopause, lactation, and parity. All of these factors are related to lifetime exposures to ovarian hormones. It is also known that there are developmental periods of enhanced vulnerability (see Section I). For example, sensitivity to radiation is highest during puberty. More recently, epidemiological studies have revealed that the intrauterine environment may also influence the risk to develop breast cancer later in life. Studies comparing human dizygotic twins and single births revealed that the propensity to breast cancer is enhanced in female twins, and this outcome was attributed to excess estrogen exposure in dizygotic twins during gestation (120). Rôle hypothetique des xénoestrogènes dans la carcinogenèse de la glandemammaire

Stéroïdes placentaires
Mère Placenta Foetus Surrénales PRG Prégnénolone-S Cholestérol Prégnénolone DHA DHA DHA-S Androstenedione DHA-S Foie E2 E3 16-OH DHA-S Rôle protecteur de la conjugaison des androgènes par le fœtus Synthèse oestriol (E3) indicateur de la vitalité du fœtus et du placenta

In the February 1st issue of the New England Journal of Medicine, Henley and colleagues from the University of Colorado report on 3 children whose gynecomastia resolved after stopping the use of lavender and tea tree oils. Both oils were tested on breast cancer cell lines to determine their interaction with the estrogen and androgen signaling pathways. Henley et al., 2007

Mammogenèse Puberté: développement canaliculaire Pregnancy: Développement lobulo-alvéolaire Rôle des changement des sécrétions hormonales (oestrogènes, progestérone, PRL, hPL) During postnatal life, the mammary gland undergoes massive architectural changes, comparable to those usually associated with organogenesis. These changes occur in response to alterations in endogenous hormone levels such as those associated with puberty and pregnancy and can be induced experimentally by endocrine manipulation. Schematic (Aa–d) and wholemount (Ba–d) presentation of the different stages and the principal hormones that control development. A rudimentary ductal design within the mammary fat pad is visible at birth, which grows at the same rate as the animal until the onset of puberty. During puberty, the cyclical production of ovarian oestrogen and progesterone promotes and accelerates ductal outgrowth (Aa, Ba). At this stage, conspicuous club-shaped structures (terminal end buds (TEB)), where the highest levels of cell division occur, appear at the ductal tips. In the mature virgin, the entire fat pad is filled with a regularly spaced system of primary and secondary ducts, with side branches that form and disappear in each oestrous cycle (Ab, Bb). Hormonal changes that occur when pregnancy begins (the release of prolactin, placental lactogens and progesterone) increase cell proliferation and the formation of alveolar buds (Ac, Bc), which grow and differentiate into milk-secreting alveoli at the end of pregnancy (Ad, Bd). During lactation, alveoli are fully matured and the luminal cells synthesize and secrete milk components into the lumina. ERBB4 (EGF-R) RANK-L, receptor activator of nuclear factor B (NF- B)-ligand. Many studies of endocrine disruptors have illustrated that developmental exposure to these exogenous hormone mimics can alter normal patterns of tissue organization and hence disrupt stromal-epithelial interactions Une exposition aux PE au cours du développement peut altérer l’organisation tissulaire

Control 25µg/kg BPA 250 µg/kg BPA (J9-J20 gestation) Histoarchitecture of mammary glands from 6-mo-old mice. from mice exposed to DMSO (a; control), 25 μg/kg of BPA (b), and 250 μg/kg of BPA (c) during in utero development from Days 9 to 20 of gestation. Note the proliferation of ducts (D), terminal ducts (TD), and terminal end buds (TEB) indicated in a, and of alveolar buds (AB) indicated in b, which are apparent in both BPA-treated groups. By 6 mo of age, the mouse mammary glands showed a dramatic expansion of the ductal network, such that it filled the entire fat pad. Typically, virgin CD-1 mice of this age develop a ductal tree that comprises terminal ducts, terminal end buds (though far less bulbous than those seen during the pubertal period), and very few alveolar buds. Quantitative analysis of these epithelial structures revealed that in utero exposure of mice to both 25 and 250 μg/kg of BPA resulted in a significant increase of all ductal and alveolar structures relative to the control group Histograms representing tissue changes in whole-mounted mammary glands. Quantitation of ductal-alveolar structures reveals increases in the relative area of ducts (a), terminal ducts (b), terminal end buds (c), and alveolar buds (d) in mammary glands of mice exposed in utero to 25 or 250 μg/kg of BPA relative to DMSO These changes may disturb important regulatory mechanisms and enhance the potential for neoplastic lesions Markey et al., 2001, Biol Reprod, 65, 1215

Susceptibilité de la glande mammaire au cours ed la période périnatale Incidence X par 2 chez les femmes DES Susceptibility of the breast during puberty and adulthood Several epidemiological studies explored the link between exposure to endocrine disruptors and breast cancer incidence. In general, these are case-control studies that usually measure exposure to a single chemical at the time of breast cancer diagnosis. This type of study has produced inconsistent results. Prospective studies that measured exposure several years before cancer diagnosis revealed a positive link between breast cancer and chemical exposure to toxaphene (129) and DDT (130). In particular, a study linked DDT with an increased risk of breast cancer when the exposure was measured before 14 yr of age Susceptibility of the mammary gland during the perinatal period Direct evidence of prenatal estrogen exposure and breast cancer risk is being gathered from the cohort of women born to mothers treated with DES during pregnancy As shown in Fig. 1 , results differed by age: the IRR (incidence rate ratio) for women ages <40 years was 0.61 (95% CI, ) whereas the IRR for ages ≥40 years was 1.91 (95% CI, ), and the interaction was statistically significant (P = 0.03). There was a further increase for women ages ≥50 years, among whom the IRR was 3.00 (95% CI, These women are now reaching the age at which breast cancer becomes more prevalent. In the cohort of these women who are aged 40 yr and older, there is a 2.5-fold increase in the incidence of breast cancer compared with unexposed women of the same age (134, 135), suggesting that indeed, prenatal exposure to synthetic estrogens may play an important role in the development of breast neoplasms. Consistent with this, experiments in rats showed that prenatal exposure to DES resulted in increased mammary cancer incidence during adulthood (136, 137). Both the epidemiological and experimental data are consistent with the hypothesis that excessive estrogen exposure during development may increase the risk of developing breast cancer. Susceptibilité de la glande mammaire au moment de la puberté Niveaux sériques de DDT élevés (17 ans avant le diagnostic): risque de cancer du sein X par 5 (femmes < 14 ans en 1945, Cohn and al., Environ health perspect 115: 1406)

Cibles neuroendocriniennes des perturbateurs de la fonction de reproduction
Brain The central neuroendocrine systems of the body serve as an interface between the brain and the endocrine systems in the rest of the body. These neuroendocrine systems control diverse functions such as reproduction, stress, growth, lactation, metabolism and energy balance (including thyroid), osmoregulation, and other processes involved in homeostasis. Considering that these neuroendocrine systems mediate the ability of the organism to respond to its environment through rapid (neuronal) and more sustained (endocrine) responses, it is not surprising that they are targeted by environmental EDCs Furthermore, neuroendocrine cells in the brain have both neuronal and endocrine properties, which is important in the context of endocrine disruption because EDCs can have neurobiological and neurotoxic effects along with the endocrine effects discussed in this Scientific Statement. The physiological processes controlled by central neuroendocrine systems are highly complex, making an understanding of neuroendocrine disruption a particular challenge. Each of these neuroendocrine systems comprises several interdependent levels of organization: the brain (specifically the hypothalamus), the pituitary gland, and often a target organ. These levels of organization may each produce a unique hormone(s) or a complex protein (e.g., breast milk), and each level also responds to the hormones produced by the other levels via feedback mechanisms Here, we will discuss the evidence for central neuroendocrine systems as targets for EDCs (Fig. 1). The bulk of the literature to date has studied primarily the reproductive (HPG) syste

GnRH (Gonadotropin-Releasing Hormone)
Cibles neuroendocriniennes des perturbateurs de la fonction de reproduction KISS GnRH (Gonadotropin-Releasing Hormone) - + Estradiol - The control of reproductive neuroendocrine function involves a group of neurons in the basal hypothalamus that synthesize and release the decapeptide GnRH (281). GnRH release drives reproduction throughout the life cycle, and this is the primary stimulus to the rest of the reproductive axis (the pituitary and gonads). GnRH release stimulates gonadotropin release from the anterior pituitary gland, which in turn activates steroidogenesis and gametogenesis in the ovary and testis. sex steroids also control the hypothalamic GnRH neurons, but this involves indirect effects because GnRH neurons do not express most of the receptors for steroid hormones This introduces the important point that other cells in the brain that express steroid hormone receptors and that regulate GnRH cells through afferent neural inputs are targets for EDCs. These points also relate to evidence that EDCs can act upon neurotransmitter systems that, at first glance, may not seem to have relevance to neuroendocrine control. For example, EDCs have been shown to cause neurotoxicity of noradrenergic, serotonergic, dopaminergic and other neurotransmitter-containing neurons (reviewed in Refs. 2 and 279). Considering that all of these neuronal types have been shown to express steroid hormone receptors and all of these cell types can project to and regulate GnRH neurons (281), this is a mechanism for convergence of effects of EDCs on the link between neural and endocrine systems. The hypothalamic-releasing hormones are not released in sufficiently high quantities to be detectable in peripheral circulation LH FSH NA: noradrenalin DA: dopamin 5-HT: serotonin

GT1 model cell organochlorine pesticide, chlorpyrifos (Dursban), has been reported to cause severe neurological defects (Campbell; Chanda and Whitney). Banned in North America only recently (summer, 2000), chlorpyrifos was the most commonly used pesticide in urban apartments and on rural farms chlorinated pesticides, including DDT and its analogs (e.g. methoxychlor), GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration Gore, Mol Cell endocrinol 192: 157

Soy (Genistein) Mammalian in vivo studies also implicate GnRH neurons as targets for EDCs. O'Byrne’s laboratory (289) has shown that coumestrol suppresses LH release (a proxy for GnRH) and the GnRH pulse generator. Coumestrol is a natural organic compound in the class of phytochemicals known as coumestans. It has garnered research interest because of its estrogenic activity and its prevalence in some foods, such as soybeans (germes ed soja) and herbs such as Pueraria mirifica. Coumestrol was first identified by E. M. Bickoff in alfalfa (luzerne) in 1957.[2] It has since be found in a variety of legumes, soybeans, brussels sprouts, and spinach. Clover and soybeans have the highest concentrations.[3] Genistein is one of several known isoflavones. Isoflavones, such as genistein and daidzein, are found in a number of plants, with lupin, fava beans, soybeans, kudzu, and psoralea being the primary food source[1]. Besides functioning as antioxidants, many isoflavones have been shown to interact with animal and human estrogen receptors, causing effects in the body similar to those caused by the hormone estrogen. Examples illustrating the effects of iv administration [0.3-ml bolus injection ( ) followed by continuous 0.45 ml/h infusion for 8.5 h] of 17 -estradiol (0.14-µg bolus plus 0.21 µg/h), coumestrol (1.6-mg bolus plus 2.4 mg/h), genistein (1.6-mg bolus plus 2.4 mg/h), or vehicle (0.1% DMSO in 45% cyclodextrin) on pulsatile LH secretion and GnRH (500 ng/kg, iv bolus injection) induced LH release in ovariectomized rats. Note that estradiol and coumestrol suppressed LH pulses, that estradiol attenuated the GnRH-induced LH response, and coumestrol completely blocked the GnRH challenge. *, LH pulse as defined by ULTRA (17 ). Alfafa (Coumestrol) McGarvey et al., Endocrinology 142: 1202

Plasma BPA (ng/mL) BPA 5 mg/(kg.j) 37.9 ng/mL exposition humaine maximale BPA 80 mg/(kg.j) 551 ng/mL Temps (h)

Données règlementaires DJA = 50 µg/(kg.d) Effet du BPA pour des doses inférieures chez les rongeurs Différence de toxicocinétique du BPA entre l’Homme et les rongeurs DJA = Exposition externe Dose Clairance = Concentration Cl=20mL/(kg.min) Concentrations BPA de l’ordre du ng/mL ( ng/mL) Vandenberg et al., 2010

Exposition prénatale au BPA: perturbation de la différenciation sexuelle du cerveau The difference in TH neuron number in the AVPV represents one robust anatomical marker of brain sexual differentiation that has been documented in rats and mice (50, 51, 52). Sexual dimorphism in the population of TH neurons in the AVPV appears to result from the influence of sex steroids during the perinatal period of development (50, 64) and is reportedly observed in rat pups by postnatal d 10 (as reviewed in Ref. 38). Perinatal and/or postnatal exposure to testosterone or estradiol is effective in reducing TH neuron number in the AVPV Photomicrographs of sections through the rostral periventricular preoptic area of control and BPA-exposed mice. TH-positive neurons are shown in representative sections through the mid AVPV (A) and through the caudal AVPV (B) of female and male offspring born to control dams and born to dams treated with 250 ng BPA/kg BW/d. III V, Third ventricle; OC, optic chiasm. Bar, 100 µm. As expected, the total number of TH-positive neurons in sections through the rostral-caudal extent of the AVPV was significantly higher in control females relative to males. Offspring born to mothers exposed perinatally to the low dose of BPA (25 ng/kg BW/d) also revealed significant sex differences in total TH neuron number. In contrast, offspring born to mothers exposed to a 10-fold higher dose of BPA (250 ng/kg BW/d) failed to show significant differences in total TH-positive neuron number. When the analysis of TH neuron number was restricted to seven consecutive sections extending from the caudal AVPV through the midregion of the nucleus in all subjects, only the control animals showed a significant sex difference. Additional analysis of the data suggested that TH neurons in the midregion of the AVPV may be particularly vulnerable to perinatal BPA exposure. The loss of sex differences in TH neuron number in BPA-exposed offspring can be attributed primarily to a decrease in TH neuron number in female offspring, which would be consistent with BPA’s actions as an estrogen. Total number of TH-positive neurons in sections though the AVPV. A, Data are shown for offspring born to control dams and offspring born to dams treated with two doses of BPA (mean ± SEM). Significant sex differences are noted in TH neuron number in control offspring (***, P = 0.001) as well as offspring born to dams treated with the lowest dose of BPA (*, P = 0.024). A significant decline in TH neuron number is noted in 250-ng females relative to controls (**, P = 0.004). Black bars, females (n = 7–8 per treatment); gray bars, males (n = 7–8 per treatment). B, The mean female to male ratio of the total TH-positive neuron number was calculated for each pair of littermates examined. As shown, control females have approximately twice the number of TH-positive neurons in the AVPV relative to control males, and the female to male ratio is markedly reduced with exposure to the higher dose of BPA. The x-axis label refers to the level of BPA exposure of the mothers (per kg BW per day). Beverly et al., Endocrinology 147: 3681.

Conclusion Exposition humaine aux xénoestrogènes significative: signification biologique, risque Périodes de sensibilité Problème de l’extrapolation à l’homme des données de toxicologie obtenues chez les rongeurs: prise en compte de la toxicocinétique Etudes prospectives difficiles Effets de co-exposition: additifs, antagonistes, synergiques It should be clear from this Scientific Statement that there is considerable work to be done. A reconciliation of the basic experimental data with observations in humans needs to be achieved through translation in both directions, from bench to bedside and from bedside (and populations) to bench. An example of how human observation and basic research have successfully converged was provided by DES exposure in humans, which revealed that the human syndrome is faithfully replicated in rodent models. Furthermore, we now know that DES exposure in key developmental life stages can have a spectrum of effects spanning female reproduction, male reproduction, obesity, and breast cancer. It is interesting that in the case of breast cancer, an increased incidence is being reported now that the DES human cohort is reaching the age of breast cancer prevalence. The mouse model predicted this outcome 25 yr before the human data became available. In the case of reproductive cancers, the human and mouse data have since been confirmed in rats, hamsters, and monkeys (463). This is a compelling story from the perspective of both animal models and human exposures on the developmental basis of adult endocrine disease. Another estrogenic compound, BPA, is also linked to a wide variety of endocrine dysfunction. BPA exposure, particularly in development, increases the risk of mammary cancer, obesity, diabetes, and reproductive and neuroendocrine disorders. The human evidence for BPA is mounting; recently, Lang et al. (464) published a cross-sectional analysis on the relationship between concentrations of urinary BPA and chronic disease states in over 1400 adults in the United States. They found a significant correlation between BPA concentrations in urine with cardiovascular disease and abnormal concentrations of liver enzymes. It would be really interesting to be able to relate the relationship of these outcomes with developmental/fetal exposure to BPA and other xenobiotics. However, epidemiological research on fetal exposure would be logistically difficult and costly because exposures must be measured at several different time points, including gestation, whereas the outcome may not be manifest in some cases until 50 or more years after the initial fetal exposure. Given the reproducibility of the human DES syndrome in rodents and recent evidence for commonalities in a relationship between BPA and cardiovascular endocrine disease, it is obvious that more research in animal models is necessary to enrich our knowledge of the mechanisms by which endocrine disruptors increase the risk of disease. A challenge to understanding the relationship between EDCs and health abnormalities is that EDCs are a "moving target." Individuals and populations are exposed to ever-changing patterns of production and use of these compounds. They also tend to be released into the environment as mixtures, rather than individual chemicals. Therefore, it is important to understand the effects of simultaneous coexposures to these chemicals, which may interact additively, multiplicatively (synergistically), or antagonistically (48). There are limited data on the interactions between chemicals within a class or across classes of chemicals. Presently, there are good analytical methods for measuring exposures to a variety of endocrine disruptors in humans. An increased understanding of the potential human health risks of exposure to mixtures of EDC is important but remains very understudied. Hence, measurement of body burden of the most prevalent xenobiotics would probably be the best strategy for finding a link between exposure and effect. Once known, this could be related to mechanistic studies in laboratory models, and future experiments could be designed to evaluate the effects of combinations of common EDCs in the laboratory, with the obvious caveat that it will not be possible to mimic every possible combination and dose. Despite these challenges, evolving and innovative technologies designed to improve the assessment of human exposure and reproductive and endocrine health endpoints should provide enhanced opportunities for improving our understanding of these relationships.

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