R E V I E W EDC-2: The Endocrine Society’s Second Scientific Statement on Endocrine-Disrupting Chemicals A. C. Gore, V. A. Chappell, S. E. Fenton, J. A. Flaws, A. Nadal, G. S. Prins, J. Toppari, and R. T. Zoeller Pharmacology and Toxicology (A.C.G.), College of Pharmacy, The University of Texas at Austin, Austin, Texas 78734; Division of the National Toxicology Program (V.A.C., S.E.F.), National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; Department of Comparative Biosciences (J.A.F.), University of Illinois at Urbana- Champaign, Urbana, Illinois 61802; Institute of Bioengineering and CIBERDEM (A.N.), Miguel Hernandez University of Elche, 03202 Elche, Alicante, Spain; Departments of Urology, Pathology, and Physiology & Biophysics (G.S.P.), College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612; Departments of Physiology and Pediatrics (J.T.), University of Turku and Turku University Hospital, 20520 Turku, Finland; and Biology Department (R.T.Z.), University of Massachusetts at Amherst, Amherst, Massachusetts 01003 TheEndocrine Society’s first Scientific Statement in 2009providedawake-up call to the scientific community abouthow environmental endocrine-disrupting chemicals (EDCs) affect health and disease. Five years later, a substantially larger bodyof literature has solidifiedour understandingof plausiblemechanismsunderlying EDCactions andhowexposures in animals andhumans—especially during development—may lay the foundations for disease later in life. At this point in history, we have much stronger knowledge about how EDCs alter gene-environment interactions via physiological, cellular,molecular,andepigeneticchanges,therebyproducingeffects inexposedindividualsaswellastheirdescendants. Causal links between exposure andmanifestation of disease are substantiated by experimental animalmodels and are consistentwith correlative epidemiological data inhumans. There are several caveats becausedifferences inhowexper- imental animal work is conducted can lead to difficulties in drawing broad conclusions, and we must continue to be cautious about inferring causality in humans. In this second Scientific Statement,we reviewed the literature on a subset of topics for which the translational evidence is strongest: 1) obesity and diabetes; 2) female reproduction; 3) male reproduction; 4) hormone-sensitive cancers in females; 5) prostate; 6) thyroid; and 7) neurodevelopment and neuroen- docrine systems.Our inclusion criteria for studieswere those conductedpredominantly in thepast 5 years deemed tobe of high quality based on appropriate negative and positive control groups or populations, adequate sample size and experimentaldesign,andmammaliananimalstudieswithexposurelevelsinarangethatwasrelevanttohumans.Wealso focused on studies using the developmental origins of health and disease model. No report was excluded based on a positive or negative effect of the EDC exposure. The bulk of the results across the board strengthen the evidence for endocrinehealth-relatedactionsof EDCs. Basedon thismuchmore completeunderstandingof theendocrineprinciples by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability, these findingscanbemuchbetter translatedtohumanhealth.Armedwiththis information, researchers,physicians,andother healthcare providers canguide regulators andpolicymakers as theymake responsible decisions. (Endocrine Reviews 36: E1–E150, 2015) I. Introduction to EDC-2 II. Obesity, Diabetes Mellitus, and Cardiovascular A. Five years after the Endocrine Society’s first Sci- Diseases entific Statement A. Introduction B. Endocrine systems are a physiological interface B. Definition and etiology of obesity with the environment, and gene-by-environment C. Definition and etiology of type 2 diabetes mellitus interactions are perturbed by EDCs D. EDCs and type 1 diabetes mellitus C. The developmental origins of health and disease E. EDCs and cardiovascular diseases D. Epigenetics and transgenerational effects of EDCs F. Conclusions E. Dose-response characteristics of EDCs F. Identifying effects of EDCs on human health: Abbreviations: AGD, anogenital distance; AhR, aryl hydrocarbon receptor; AHS, Agricul- tural Health Study; AR, androgen receptor; ARC, arcuate nucleus; ATR, atrazine; AVP, where to start? arginine vasopressin; AVPV, anteroventral periventricular nucleus; BBP, butyl benzyl phtha- G. Review criteria for EDC-2 late; BMI, body mass index; BPA, bisphenol A; BPH, benign prostatic hyperplasia; CI, confidence interval; CPP, Collaborative Perinatal Project; CVD, cardiovascular disease; D1 ISSN Print 0163-769X ISSN Online 1945-7189 (Dio1), type 1 deiodinase; DBP, di-n-butyl phthalate; DCP, dichlorophenol; DDD, dichlo- Printed in USA rodiphenyldichloroethane; DDE, dichlorodiphenyldichloroethylene; DDT, p,p’-dichlorodi- Copyright © 2015 by the Endocrine Society phenyltrichloroethane; DEHP, di(2-ethylhexyl)phthalate; DES, diethylstilbestrol; DINP, di- Received February 24, 2015. Accepted September 1, 2015. isononyl phthalate; DNMT, DNAmethyltransferase (enzyme); DOHaD, developmental origins First Published Online November 6, 2015 of health and disease; E, embryonic day; EDC, endocrine-disrupting chemical; (continued p.2) doi: 10.1210/er.2015-1010 Endocrine Reviews press.endocrine.org/journal/edrv E1 TheE ndocrineS ociety.D ownloadedf romp ress.endocrine.orgb yi{$[ ndividualsU er.displayNameo ]} nM 70 arch2 .610 a t:60 F 10 orp ersonalu seo nly.N oo theru sesw ithoutp ermission.. A llr ightsr eserved. E2 Gore et al Second Scientific Statement on EDCs Endocrine Reviews III. Female Reproductive Health C. Chemicals with direct actions on the thyroid A. Introduction to EDCs and female reproduction gland: perchlorate, chlorate, nitrate, thiocyanate B. Effects of EDCs on the ovary D. EDCs and the thyroid C. Effects of EDCs on uterine structure and function E. Conclusions D. Effects of EDCs on the vagina VIII. Neurodevelopmental and Neuroendocrine Effects of E. Effects of EDCs on the anterior pituitary gland EDCs F. Female reproductive cycles A. Introduction to EDCs and the developing brain G. Pathophysiological reproductive conditions B. EDC effects on steroid hormone receptors and ste- H. Pregnancy and birth roidogenic enzymes I. Conclusions C. Molecular epigenetic mechanisms for EDC effects IV. Male Reproductive Health in the brain A. Introduction D. Developmental EDC effects on neuroendocrine B. Male sexual development, and Nature’s systems experiments E. Neurobehavioral effects of developmental EDCs C. Hypospadias F. Conclusions D. Cryptorchidism IX. Conclusions and Recommendations E. Testicular cancer A. Research gaps F. Semen quality B. Recommendations beyond research G. Conclusions V. Hormone-Sensitive Cancers in Females A. Introduction I. Introduction to EDC-2 B. Critical periods of mammary gland development C. Effects of EDCs on the mammary gland: rodent A. Five years after the Endocrine Society’s first Scientific models and epidemiological studies Statement D. Uterine cancer, ovarian cancer, and EDCs t has been 5 years since the Endocrine Society convened E. Cellular and molecular mechanisms of EDCs in mammary, ovary, and uterus I a group of experts to review the state of the science on F. Conclusions endocrinological effects of environmental contaminants VI. Prostate Gland Disruption that perturb hormonal systems, termed endocrine-dis- A. Prostate Development and Hormone Sensitivity rupting chemicals (EDCs). That team conducted a thor- B. EDC actions in the prostate gland ough reviewof the extant literature up to that time (2008), C. Conclusions and wrote an initial white paper that was then developed VII. Thyroid Disruption A. Characteristics of the hypothalamic-pituitary-thy- into the landmark Scientific Statement onEDCspublished roid (HPT) axis in2009,herein referred toas“EDC-1” (1). Since that time, B. Role of the micronutritional environment in thy- numerous publications have emerged. What has influ- roid hormone action enced the field most deeply since 2008 has been four types of studies: 1) those describing the consequences of EDC (continued) EPA, Environmental ProtectionAgency; EPM, elevated plusmaze; ER, estrogen receptor; EZH2, enhancer of Zeste homolog2;G, gestational day;GPER,Gprotein-coupled exposures on development and physiology (mainly con- ER; GR, glucocorticoid receptor; GST, glutathione transferase; HCB, hexachlorobenzene; ducted in rodent models); 2) those investigating themech- HCH, hexachlorocyclohexane; HDAC, histone deacetylase; HPA, hypothalamic-pituitary- adrenal; HPG, hypothalamic-pituitary-gonadal; HPT, hypothalamic-pituitary-thyroid; anistic underpinnings of these disorders (gene expression HPTE, 1,1,1-trichloro-2,2-bis(4-hydroxyphenyl)ethane; HSD, hydroxysteroid dehydroge- and epigenetic changes induced in cell and tissue culture, nase; iAs, inorganic arsenic; IVF, in vitro fertilization; KCC2, potassium chloride cotrans- porter 2; LQ, lordosis quotient; MBP, monobutyl phthalate; MBzP, mono-n-benzyl phtha- together with molecular and cellular work conducted in late; MEHHP, mono-(2-ethyl-5-hydroxylhexyl) phthalate; MEHP, mono-(2-ethylhexyl) endocrine tissues of EDC-exposed animals); 3)work seek- phthalate; MEOHP, mono-(2-ethyl-5-oxohexyl) phthalate; MEP, monoethyl phthalate; MPOA, medial POA; MWM, Morris water maze; MXC, methoxychlor; NCD, noncommu- ing to document associations between body burdens of nicable disease; NHANES, National Health and Nutrition Examination Survey; NIEHS, Na- certain EDCs to disease propensity in humans (mainly ep- tional Institute of Environmental Health Sciences; NIS, sodium/iodide symporter; NOAEL, no observed adverse effect level; NTP, National Toxicology Program; OVX, ovariectomized; idemiological work); and 4) those reports of humans with P, postnatal day; PBB, polybrominated biphenyl; PBDE, polybrominated diphenyl ether; known occupational or acute exposures to a particular PCB, polychlorinated biphenyl; PCDD, polychlorinated dibenzodioxin; PCDF, polychlori- nated dibenzofuran; PCOS, polycystic ovarian syndrome; PFOA, perfluorooctanoic acid; chemical or group of chemicals with EDC activity (eg, PFOS, perfluorooctane sulfonate; PIN, prostatic intraepithelial neoplasia; POA, preoptic pesticide applicators, or families residing near the Seveso, area; POP, persistent organic pollutant; PPAR, peroxisome proliferator-activated receptor; PR, progesterone receptor; PRL, prolactin; PSA, prostate-specific antigen; P450scc, P450 Italy, factory, site of a large dioxin leak). In 2014–2015 side-chain cleavage; PTU, propylthiouracil; PVN, paraventricular nucleus; RXR, retinoid X when this second Scientific Statement, EDC-2, was writ- receptor; SNP, single nucleotide polymorphism; StAR, steroidogenic acute regulatory (pro- tein); TBBPA, tetrabromobisphenol A; TBT, tributyltin; TCDD, 2,3,7,8-tetrachlorodibenzo- ten, there was far more conclusive evidence for whether, p-dioxin; T1D, type 1 diabetes mellitus; T2D, type 2 diabetes mellitus; TDS, testicular when, and how EDCs perturb endocrine systems, includ- tdhyysrgoeidnehsoisrmsyonnderoremce;ptToErB;,TPteOrm, tihnyarloepnedroxbiudda;seT;GVCDCR, vteitsatmicuinlaDr rgeecremptocer;llVcManHc,evre; nTthroR-, ing in humans. Thus, it is more necessary than ever to medial nucleus of the hypothalamus; WAT, white adipose tissue. minimize further exposures, to identify new EDCs as they TheE ndocrineS ociety.D ownloadedf romp ress.endocrine.orgb yi{$[ ndividualsU er.displayNameo ]} nM 70 arch2 .610 a t:60 F 10 orp ersonalu seo nly.N oo theru sesw ithoutp ermission.. A llr ightsr eserved. doi: 10.1210/er.2015-1010 press.endocrine.org/journal/edrv E3 emerge, and to understand underlying mechanisms in or- More BPA is produced annually than any other chemical, der to develop interventions. with 15 billion pounds produced in 2013 (5). It is used in Through the years there has been controversy around en- a verywide array ofmanufacturing, food packaging, toys, docrine disruptors, in part because different stakeholders, and other applications, and BPA resins are found in the some with financial incentives, may review the literature lining of many canned foods and beverages such that vir- froma very different perspective. The chemical industry and tually everyone is exposed continuously (6). In food con- environmental nongovernmental organizations have often tact materials, BPA may leach into food or water under been in conflict, and the lay press sometimes oversimplifies high heat, physical manipulation, or repetitive use. Due to the research results. It is also notable that the goals of indus- its ubiquitous nature and continuous exposure, 93% of try-funded studies and federal granting agency-funded stud- Americans have a measurable amount of BPA in their ies can differ both in design and in desired outcomes. The urine (7, 8). It is also detected in breast milk of some former(industry)areoftendonetoprovesafety,andnegative women (9). BPA is so prevalent in our daily environment results are considered a favorable outcome and are pub- that elimination of BPA contamination during carefully lished. By contrast, government-funded research is usually controlled quantitative procedures has proven difficult hypothesis-driven, seeking underlying mechanisms, and not (10, 11). BPA is rapidly metabolized to nonbioactive necessarily intended to prove or disprove safety. As a result, formsandhas a short half-life of approximately 4–5hours such studies may be omitted from the risk assessment pro- in adult humans, with lower metabolic rates in the fetus cess, something that ought to be overcome by better integra- and infants (12, 13). Measurements of bioactive or free tion of the different types of studies. BPA inhuman serum is controversial at present,with some Here, our goal was to present relevant research inde- documenting nanograms per milliliter quantities in sam- pendent of the results, and we considered both industry- ples using contamination-free conditions (13–15), and government-fundedwork.Aswediscuss later, there is whereas others report that ordinary exposures result in always a potential bias toward positive study findings, ie, picograms per milliliter levels or lower (16). Although rel- finding of adverse effects rather than reporting no effects. evant internal exposure remains a critical issue that is still However, there is also, unfortunately, a possibility of the unresolved, it is noteworthy that industrial exposures, vul- opposite kind of bias that might be in the best interests of nerable populations, and individual variations in metab- a producer to show that its product is safe, and therefore olism and susceptibility must be taken into consideration a negative test result is desirable. Thus, readers of papers (17). Currently, the US Environmental Protection Agency on EDC effects should consider whether articles are peer- (EPA) safety level of BPA is set at 50 ␮g/kg/d, whereas the reviewed and whether there might be a conflict of interest European Food Safety Authority’s temporary tolerable of reviewers, editors, or publishers with industry connec- daily intake was recently lowered to 4 ␮g/kg/d. Several tions, and readers should use their scientific judgment in studies in the present report will document BPA effects in evaluating the strength of the scientific work. Consider- mammalian systemsatorbelow these current safety levels. ation of the quality of research should also include b. Phthalates. Phthalates and phthalate esters are a large whether or not experiments were done in a blind or dou- group of compounds used as liquid plasticizers found in a ble-blind fashion to avoid inadvertent experimental bias. wide range of products including plastics, coatings, cos- An Executive Summary of this statement has been pub- metics, and medical tubing. These compounds were first lished separately (2), summarizing thekeypointsof the full introduced as additives in the production of plastic in the Scientific Statement. 1920s and resulted in the rapid widespread use of poly- 1. Definition of EDCs and prototypical examples vinyl chloride plastic in the 1930s and later. Because they For the purposes of EDC-2, we provide an operational are not chemically bound to the plastic, phthalates can working definition of an EDC as: “an exogenous chemi- leach into the environment. Moreover, a variety of con- cal, ormixtureof chemicals, that interfereswithanyaspect sumer products use various phthalates, including personal of hormone action” (3). Although there may be hundreds care products, medical tubing, vinyl flooring materials, or more environmental chemicals with EDC activity, sev- and toys. In one case, food companies in Taiwan began eral classes are most commonly studied and will be intro- deliberately using a variety of phthalates as emulsifiers at duced briefly here; these and several others are summa- very high concentrations (18). When this was discovered, rized in Table 1. the government notified the population that certain kinds of foods were contaminated, including sports drinks, fruit a. Bisphenol A. Bisphenol A (BPA) was first synthesized in beverages, tea drinks, fruit jam or jelly, and health foods 1891 and was discovered to be estrogenic in 1936 (4). or supplements in powder or tablet form. But it was later The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 March 2016. at 06:01 For personal use only. No other uses without permission. . All rights reserved. E4 Gore et al Second Scientific Statement on EDCs Endocrine Reviews Table 1. Classifications, Histories, Chemical Properties, and Physiological Effects of Common EDCs Introduction Restricted/ Route of Effects/Body EDC General Chemical Structure Group date Ban Date Exposure Sources Half-Life Burden ATR Chlorotriazine 1959 European Union Ingestion, Pesticide/ herbicide, 10–12 h Endocrine, respiratory herbicide ban 2004 inhalation contaminated and nervous water and soil system targets, liver damage BPA Bisphenols 1960s Restricted 2012 Ingestion, Polycarbonate plastics, 4–5 h Estrogenic, inhalation, epoxy resins, obesogenic, dermal plastic toys and neurological absorption bottles, lining of effects, adverse food cans thyroid hormone action, reproductive and developmental effects DDT Organochloride 1940s Banned 1972 Ingestion, Contaminated water, 6–10 yr Carcinogen, central inhalation, soil crops, fish nervous system, dermal kidney, liver and absorption peripheral nervous system effects DES Non-steroidal 1941–1947 Restricted Ingestion, Pharmaceutical 2–3 days Transplacental synthetic 1971–1975 injection, carcinogen, estrogen vaginal teratogen suppository EE2 Synthetic 1943 Oral Oral contraceptives, 13–27 h Cardiovascular derivative of contaminated disease, 17␤-estradiol water cerebrovascular disease, thromboembolic disease, gallbladder disease, carcinogenic MXC Organochlorine 1948 United States Ingestion, Contaminated soil, Aerobic soil Central nervous insecticide 2003 inhalation, water, and food ⬎100 system depression, banned use dermal days damage to liver as pesticide absorption and kidney, developmental and reproductive effects in animals, transgenerational kidney and ovary disease, obesogen PCBs Organochloride 1927 Banned 1979 Ingestion, Contaminated air and 12 days to Carcinogen, inhalation, food, skin contact 16 yr chloracne, dermal with old electrical stomach and liver absorption equipment damage, reproductive and nervous system effects and thyroid injury Phthalates Plasticizers 1920s Restricted 2009 Ingestion, Contaminated food, ⬃12 h Carcinogen, liver inhalation, PVC plastics and damage, dermal flooring, personal reproductive and absorption care products, developmental medical devices effects, asthma, and tubing obesogen (Continued) The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 March 2016. at 06:01 For personal use only. No other uses without permission. . All rights reserved. doi: 10.1210/er.2015-1010 press.endocrine.org/journal/edrv E5 Table 1. Continued Introduction Restricted/ Route of Effects/Body EDC General Chemical Structure Group Date Ban Date Exposure Sources Half-Life Burden PFOA Fluorosurfactant 1940s United States Ingestion, Contaminated food 2–4 yr Liver, and mammary 2015 inhalation and water, dust, gland voluntary floor waxes, fire developmental, production fighting foam, and immune restiction electrical wiring, system toxicant, lining of food carcinogen wrappers, stain resistant carpeting TCDD Polychlorinated Synthesized Ingestion, By-product of 7–11 yr Liver damage, weight dibenzo- 1872 inhalation chlorinated loss, atrophy of p-dioxin herbicide thymus gland, production, immunosuppression, smelting, chlorine reproductive bleaching of paper effects and cancer Vinclozolin Dicarboximide 1981 Ingestion, Diet and occupational Aerobic soil Antiandrogenic fungicide inhalation, 28 days, activity, male dermal plasma reproductive and absorption 20 h neurological effects, transgenerational reproductive effects, potential carcinogen Abbreviations: EE2, ethinyl estradiol; PVC, polyvinyl chloride. shown that the contamination was more widespread, in- wide varietyof applicationsused thesemixtures, including cluding ice cream, frozen food, and cake mixes (19). In plasticizers in rubber and resins, carbonless copy paper, fact, phthalates are detectable in human urine, serum, and adhesives, and paints and inks. The varied nature of their milk samples (20–22), and the estimateddaily exposure to use resulted in widespread environmental contamination, one major phthalate, di(2-ethylhexyl)phthalate (DEHP), including buildings and schools (30). These persistent or- ranges from 3–30 ␮g/kg/d (23). ganic pollutants (POPs) bioaccumulate in the environ- ment and are stored in body fat, and they therefore have c. Atrazine. Atrazine (2-chloro-4-ethylamino-6-isopropyl- continued potential for adverse health effects (31). Some amino-s-triazine) (ATR) is a widely used chlorotriazine PCBs are classified as EDCs because they have thyroido- herbicideused to control broadleaf andgrassweedgrowth genic, estrogenic, and antiandrogenic actions (32, 33). on crops such as commercial corn, sorghum, and sugar The commercial production of polybrominated diphenyl cane. Christmas tree farms, parks, and golf courses also ethers (PBDEs) began in the late 1970s (34), just about the use ATR. ATR has been the major herbicide used world- time that PCB production was banned. They were used as wide since its registration in 1959 because of its ability to flame retardants in upholstered products, mattresses, and remain active for extended periods of time, its economical clothing. In 2001, approximately 33 000 metric tons of price, and its broad spectrum of weed control (24). ATR PBDEs were produced, most of which was used in North and its metabolites are commonly reported groundwater America (34). As a result, NorthAmericans have the high- contaminants and the most commonly detected pesticide est blood levels of PBDEs, compared to those living in in US surface waters, including drinking water. For this other regions of the world (35, 36). PBDEs consist of pre- reason, the EPA is particularly concerned about the po- dominantly three congener mixtures, pentaBDE, oct- tential of ATR and related chlorotriazines to affect aBDE, and decaBDE. The first two have been banned in aquatic organisms and the carcinogenic risk from ex- Europe and Asia, but decaBDE mixtures continue to be posure (25, 26). widely used globally (37). Of the 209 possible congeners, d. Polychlorinated biphenyls and polybrominated diphenyl the five that account for over 90% of body burden in ethers. Polychlorinated biphenyls (PCBs) are a class of in- human tissues are tetraBDE47; pentaBDE99, -100, and dustrial chemicals with paired phenolic rings and variable -153; and decaBDE209. degrees of chlorination. They were synthesized by expos- ing the biphenyl molecule to chlorine gas in the presence e. DDT and DDE. p,p’-Dichlorodiphenyltrichloroethane of a catalyst, resulting in complexmixtures of 209possible (DDT) is a synthetic industrial and household insecticide congeners (27–29). PCBs were mass-produced globally with a long half-life, extensive use, and lipophilic nature from the late 1920s until they were banned in 1979. A that have made it a prominent environmental contami- The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 March 2016. at 06:01 For personal use only. No other uses without permission. . All rights reserved. E6 Gore et al Second Scientific Statement on EDCs Endocrine Reviews nant. The United States banned DDT in 1972 due to its ervoir of POPs that are liberated into blood, especially effects on the environment and potential human health during weight loss periods. At the same time, it is a mech- effects, despite the benefit of decreased incidence of ma- anismofprotectionaswell because it limits the availability laria and typhoid (38, 39). DDT and its metabolites, di- of chemicals to enter the blood and access other tissues, chlorodiphenyldichloroethylene (DDE) and dichlorodi- thereby eliciting detrimental effects. In fact, retention of phenyldichloroethane (DDD), have been associated with POPs in WAT may limit the ability to estimate the real endocrine-related diseases such as testicular tumors (40), burden of these chemicals frommeasurements in serumor endometrial cancer (41), pancreatic cancer (42), type 2 urine levels, as occurs in most animal and human studies. diabetes mellitus (T2D) (43), and breast cancer (44, 45). We must note that in the list of chemicals described B. Endocrine systems are a physiological interface with above, there are EDCs, such as BPA and phthalates, that the environment, and gene-by-environment interactions are commonly detected in most of the population because are perturbed by EDCs of their widespread use. These latter EDCs have relatively The endocrine glands are distributed throughout the low accumulation in body fat tissue; therefore, serum or body and produce the hormones that act as signalingmol- urine levels of these chemicals, their metabolites, or spe- ecules after release into the circulatory system (Figure 1). cific reaction products likely reflect the so-called “body Development, physiological processes, and homeostatic burden,” defined as the total amounts of these chemicals functions are regulated andmaintained by hormones. Sev- that are present in the humanbody at a given point in time. eral functions of natural hormones are critical for both On the contrary, PCBs, PBDEs, DDT, andDDE are POPs. health and disease and are relevant to EDCs. First, many These are highly lipophilic and accumulate in the food hormones bind to receptorswith remarkable affinity, hav- ⫺12 ⫺9 chain and in white adipose tissue (WAT) with important ing dissociation constants between 10 and 10 M, consequences. This storage depot can contain a large res- which approximates their very low concentrations in the Figure 1. Figure 1. Diagram of many of the body’s endocrine glands in females (left) and males (right). The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 March 2016. at 06:01 For personal use only. No other uses without permission. . All rights reserved. doi: 10.1210/er.2015-1010 press.endocrine.org/journal/edrv E7 circulation. The maximal response of a cell is achieved at markedly increased during the last few decades (48). The ligand concentrations at which most of the receptors are four principal NCDs are cancers, cardiovascular diseases still not occupied. Second, the amount of a hormone that (CVDs), chronic respiratory diseases, and diabetes melli- is synthesized and released is determined by the interplay tus. Cumulatively, they kill 36 million people each year, of numerous molecular and physiological processes, in- 80% in low- and middle-income countries (49). Both ge- cluding regulation of gene and protein expression, exocy- netics and environment play a role in the incidence of these tosis of vesicles containing peptide or protein hormones, NCDs, and increasing interest is turning toward finding metabolism and steroidogenesis of lipophilic hormones, how chronic, low-dose lifelong exposures to EDCs con- transport through circulation (often in association with tribute to these statistics. binding partners), actions at target receptors,metabolism, An individual’s genes play important roles in determin- degradation, excretion, and many others. Third, a funda- ing health and physiological parameters, such as insulin mental property determining hormone levels involves the sensitivity and blood pressure. Single nucleotide polymor- ability of the gland and its targets to interact, most often phisms (SNPs) in the genome are related to incidence and by negative feedback of a hormone on its receptor in the severity of CVDs, diabetes mellitus, obesity, and abnor- original target gland. Fourth, levels of any hormone must malities inmetabolism, reproduction, andother endocrine be within a physiologically relevant range to be most ef- systems. Such polymorphisms may also contribute to vari- fective. Excursions outside of that range to superphysi- ability in responses to chemical exposures. It is important to ological (elevated) or subphysiological (depressed) levels note that a SNP is rarely responsible for aNCD; rather, there for any extended period nearly always result in dysfunc- are a large number of SNPs, each with a small effect that tion or disease. This latter concept is exemplified by the accumulates and is associated with these diseases (50, 51). thyroid system: normal (euthyroid) levels of the thyroid For example, there are 65SNPsknown tobe associatedwith hormones are needed for appropriate metabolic health. the risk of one of the most common NCDs, T2D (52). The Hyperthyroidism is associated with a range of symptoms interactions between EDC exposure and SNPs associated due to elevated metabolism, and hypothyroidism, with a withNCDsare still greatly unknown, and further studies are very different disease phenotype, results from depressed necessary. The rise in common NCDs, therefore, is thought hormone levels. to be attributable to environmental and social factors, in- EDCs interfere with the action of hormones, disrupt cluding EDCs (53), more than any common gene variant. homeostasis, and may alter physiology during the whole Themechanisms for these changes, involvingmolecular epi- life span of an individual, from fetal development to adult- genetic processes, are discussed in Section I.D. hood (1). Understanding how EDCs affect physiological Although much of the focus of EDC-2 is on develop- processes and initiate pathophysiology is essential in un- mental exposure,EDCsalso interactwith receptorsduring derstanding the etiology of hormone-related diseases. adulthood.Aswill be discussed later, thismay elicit effects Some EDCs, at environmentally relevant doses, bind to such as weight gain (“obesogens”) and/or insulin resis- hormone receptors and act either as agonists or antago- tance and hyperinsulinemia (“diabetogens”). Although nists, thus enhancing, dampening, or blocking the action actions in adults may be reversible if the insult is removed, of hormones. They also alter the number of hormone re- exposure in the realworld ismore often continuous and to ceptors in different cell types and the concentration of a mixture of chemicals. In such circumstances as chronic circulatinghormones (46,47).These effects, dependingon lifelong exposure, EDCs may predispose individuals to timing and exposure dose, give rise to alternative pheno- pathologies such as T2D (54) or thyroid dysfunction. types, which may lead to increased disease susceptibility. If exposure alters hormoneactionsduringontogenesis, the C. The developmental origins of health and disease effects are often permanent and can affect organ develop- During embryonicdevelopment, organogenesis and tis- ment and function. Furthermore, these effects could have sue differentiation proceed through a series of tightly reg- lifetime consequences that are both complex and difficult ulated and temporally coordinated events at the cellular, to predict. The extent and nature of long-term conse- biochemical, and molecular levels, ultimately resulting in quences depend on the interaction of genes and environ- a functional, mature structure. Development is an Ein- ment and involve many variables, including the develop- bahnstrasse (one-way street), and thus natural substances mental window of exposure, the individual’s metabolism, such as hormones as well as environmental changes, in- and his or her genetic background. cluding exposures to exogenous environmental chemicals, Long-termpermanent physiological changes that result alter this unidirectional process. These latter perturba- fromearlyEDCexposure alter one’s susceptibility to com- tions may impart structural and functional changes that mon noncommunicable diseases (NCDs), which have canprofoundlydeflect thedevelopmental trajectory, often The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 March 2016. at 06:01 For personal use only. No other uses without permission. . All rights reserved. E8 Gore et al Second Scientific Statement on EDCs Endocrine Reviews leading to lifelong phenotypic changes such as increased 1. Diethylstilbestrol and beyond endocrine disease propensity. Overt toxicant exposures Perhaps the best-studied endocrine-based example is in duringgestationhavebeen recognized fordecades to cause utero exposure to diethylstilbestrol (DES), a potent syn- adverse outcomes in exposed children,with examples that thetic nonsteroidal estrogen taken by pregnant women include links between thalidomide and limb malforma- from the 1940s to 1975 to prevent miscarriage and other tions, folate deficiency and spina bifida, methyl mercury complications.DESwas prescribed at doses from less than andMinamata disease, as well as fetal alcohol syndrome, 100 mg (in most cases) upward to 47 000 mg, with a me- to name a few (55). Childhood and puberty are also pe- dian dose of 3650 to 4000 mg in the United States (IARC riods of rapid change in endocrine-dependent organ sys- 2012). Most women received low doses (ie, 5 mg) and tems and are beginning to be recognized as additional sen- increased their intake (up to125mg) as symptomsorpreg- sitive periods (56–59). nancy progressed, translating to doses of about 100 ␮g/kg The targets of endocrineglands typically exhibit height- to 2mg/kgDES per day (66). In 1953, a study provedDES ened sensitivity to hormones during specific developmen- was ineffective (67). Its use was discontinued when a sub- tally critical windows. During these periods, hormonal set of exposed daughters presented with early-onset vag- signals cause changes to cells at the molecular (often gene inal clear-cell adenocarcinoma (68), with a 40-fold in- expression and/or epigenetic) level (see Section I.D.) and crease in risk compared to unexposed individuals (69) dictate or modify structural and functional organization (Table 1). A highly significant incidence ratio for clear-cell of the tissues. Such early-life programming events are best adenocarcinomawas also found in theDutchDES cohort, exemplified in studies on brain development where an- a population that may have had lower exposures than US drogens and estrogens play early, essential roles in im- women (70). Itwas subsequently determined that exposed printing sexual dimorphisms in structure, gene expres- offspring of both sexes had increased risk for multiple sion, and signaling that determine behaviors throughout reproductive disorders, certain cancers, cryptorchidism life (60). Likewise, there is growing appreciation that de- (boys), and other diseases (71–73), although the risk for velopment during the critical period is particularly vul- sons is more controversial (74). New data are emerging to nerable to the effects of exogenous EDCs that can repro- implicate increased disease risk in grandchildren (75).Not gram essential signaling/differentiation pathways and surprisingly, a plethora of examples is emerging for in- lead to lifelong consequences (61). In fact, molecular creased disease susceptibility later in life as a function of changes in response to EDCs often precedemorphological developmental exposures to EDCs that include BPA, consequences, sometimes by weeks, years, or decades (de- phthalates, PCBs, pesticides, dioxins, and tributyltin pending upon life span), and experimental studies show- (TBT), among others. ing gene or protein expression changes in response to EDCs may be sentinels for disease propensity later in life. D. Epigenetics and transgenerational effects of EDCs This reprogramming process is best appreciated within Themechanisms of action of EDCs are varied (Table 2) the framework of the developmental origins of health and and not entirely understood, but recent evidence suggests disease (DOHaD) hypothesis, which posits that an ad- that some EDCs may cause epigenetic changes, which in verse environment experienced by a developing individual turn may lead to transgenerational effects of EDCs on can increase the risk of disease later in life (62, 63). As numerous organ systems (76–80). Epigenetic changes are originally formulated, the DOHaD paradigm initially fo- described as heritable changes in gene expression that are cused on multiple studies that documented links between not due to changes in DNA sequence (ie, not due to mu- poor nutrition in utero and increased risk in offspring of tation). Several possible mechanisms of epigenetic change obesity, CVD, and diabetes mellitus over a life span (62). exist, includingmethylation of cytosine residues onDNA, The most notable example is the Dutch “hunger” winter post-translational modification of histones, and altered during World War II when maternal starvation—in a tri- microRNAexpression.Todate,most studieson the effects mester-specific manner—correlated with increased car- of EDCs on epigenetic changes have focused on DNA diovascular and metabolic diseases of the offspring in methylation, but recent studies have also addressed the adulthood (64, 65). Importantly, the DOHaD hypothesis effects of EDCs on histone modifications and microRNA readily expands to accommodate perturbations in the en- expression (77, 78, 81). docrine system during early development, including ab- DNA methylation is a process in which methyl groups errations in endogenous hormones (in timing, sequence, are attached to cytosine residues by DNA methyltrans- and levels), maternal intake of synthetic hormones, and ferase enzymes (DNMTs), usually in cytosine-guanosine inadvertent exposures to environmental chemicals includ- dinucleotide pairs (CpG sites), although DNA methyl- ing EDCs. ation canoccuronnon-CpGresidues (82, 83).DNAmeth- The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 March 2016. at 06:01 For personal use only. No other uses without permission. . All rights reserved. doi: 10.1210/er.2015-1010 press.endocrine.org/journal/edrv E9 Table 2. Mode of Action for EDCs EDC Mechanism Mode of Action BPA Nuclear receptor ER agonist (859, 1246); strong affinity for ERR␥ (860, 1247); antiandrogen (1248); increased PR expression (477, 1249); hPXR agonist (1250) ER-mediated nongenomic Activates membrane-associated ER␣, ER␤ signaling cascades through PI3K-pAkt and pathway MAPK-pErk and GPER-pErk pathways (960, 1251–1255) Nonsteroidal receptor Antagonist of ThR (1095); binds to GPR30 (861) 2⫹ 2⫹ Ion channels Activates membrane ER␤-Ca pathway; activates ER␤-KATP and Ca mobilization (293); 2⫹ up-regulation of Ca ion channel gene and protein, Orai1 (966, 326, 1256, 1257) Uninhibited growth Alters MaSC gene expression and induces early neoplastic lesions (348); induces beaded ducts and increases hyperplasia (362, 1258, 1259) Inflammation Induces proinflammatory cytokines and chemokines (1260) DDT and Nuclear receptor Binds and transactivates ER␣ and ER␤ (1246, 1261); DDE binds AR and represses metabolites transcription (1262) Microenvironment/stroma Induced estrogenic microenvironment in breast adipose tissue (865) DES Nuclear receptor ER␣ agonist (1246, 1263); AR binding (1264); suppresses activation of ERR ␣, ␤, and ␥ (1265) ER-mediated non-genomic Activates MAPK and PI3K and induces phosphorylation of ERK (1266, 1267) pathway Epigenetic Hypermethylation of HOXA10 (1268); DNA methylation (1269) Dioxins Nonsteroidal receptor Binds to AhR (1270) Coactivator recruitment Recruitment of coactivator p300 (1270) PCBs Steroid hormone biosynthesis Inhibits sulfotransferase (1271), inhibits aromatase (1272); increases T4 glucuronidation, competes with thyroid hormone binding proteins (1273) Nuclear receptor Weak binding to ER (1246), weak binding to AR (1264) PFOA Nuclear receptor Binds to ER and EREs (845, 846) Nonsteroidal receptor PPAR␣ agonist (157, 1274) Uninhibited growth Increased hyperplasia and stromal density (853) Phthalates Nuclear receptor DBP weak affinity for ER (874) Microenvironment/stroma MEHP induced PPAR␤ in adipose (1274) Abbreviations: EREs, estrogen response elements; ERR, estrogen-related receptor; PI3K, phosphatidylinositol-3-kinase. ylation is important for several normal developmental and MicroRNAs often bind to the 3⬘ end of gene transcripts and reproductive processes such as gametogenesis and em- initiate mRNA degradation or suppression of protein trans- bryogenesis. Hypermethylation in a promoter region is lation (93). Studies also suggest that microRNAs can affect thought to repress gene transcription because the methyl- theexpressionofotherepigenetic regulators suchasDNMTs ated promoter region has a decreased affinity for tran- and histone-modification enzymes (94). scription factors and an increased affinity for methylated Both hormones and EDCs cause DNAmethylation, hi- DNA-binding proteins, methyltransferases, histone deacety- stone modifications, and altered microRNA expression lases (HDACs), and/or corepressors (84, 85). (95). These epigenetic changes often cause phenotypic Histone modification is a process in which specific changes in organisms, which may appear immediately or amino acids in the N-terminal ends of histones undergo long after EDC exposure. These properties are dictated by post-translational modification, including acetylation, the timing of exposure. When EDCs introduce epigenetic methylation, phosphorylation, sumoylation, and ubiq- changes during early development, they permanently alter uitination by enzymes such as histone acetyltransferases, the epigenome in the germline, and the changes can be deacetylases, methyltransferases, and demethylases (86, transmitted to subsequent generations. When an EDC in- 87). These modifications determine whether the DNA troduces epigenetic changes during adulthood, the wrapped around histones is available for transcription changeswithin an individual occur in somatic cells and are and play roles in determining the rate of transcription. not permanent or transmitted to subsequent generations Histone modifications also help regulate replication, re- (76, 77, 81, 96). For an EDC to have truly transgenera- combination, and higher-order organization of the chro- tional effects, exposure must occur during development, mosomes (88). Changes to these modifications are often and the effects need to be observed in the F3 generation. found in diseases such as cancer and are best studied for This is because when a pregnant F0 female is exposed to those diseases (89, 90). The molecular mechanisms by which microRNAs and anEDC, germ line cells in her F1 fetus are directly exposed other noncoding RNAs affect gene expression are not en- to the EDC.These exposed F1 germ line cells are then used tirely understood, but it is likely that microRNAs play a to produce the F2 generation, and thus, the F2 generation role in gene regulation and chromatin organization (91, 92). was directly exposed to the EDC via the germ cells. This The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 March 2016. at 06:01 For personal use only. No other uses without permission. . All rights reserved. E10 Gore et al Second Scientific Statement on EDCs Endocrine Reviews exposure scenario makes the F3 generation the first gen- (117, 118). Again, the mechanism is proposed to involve eration that was not directly exposed to the EDC (97–99). hypo- or hypermethylation of DNA (116, 117, 119). Im- EDC-induced epigenetic changes are also influenced by portantly, work using this model relied on high-dose ex- dose of exposure, and they are tissue specific (77, 78, 81). posures to the pregnant dams (50–100mg/kg/d) andmust Thus, it is important to consider both dose of EDCand the be replicated using environmentally relevant doses. tissuebeforemaking firmconclusions about the epigenetic Although there is less research on other EDCs, evidence effects of EDCs. DNA methylation changes are the best- suggests that they can cause DNA methylation changes studied mechanism in this regard. For example, prenatal (80, 120, 121). Exposure tomethoxychlor (MXC) altered exposure to DES caused hypermethylation of the Hoxa10 DNAmethylation in multiple CpGs in the ER␤ promoter gene in the uterus of mice and was linked to uterine hy- (80). DEHP exposure caused global increases in cytosine perplasia and neoplasia later in life (100). Beyond the ef- methylation in the testes (122) and modified DNA meth- fects of prenatal exposure to DES on the daughters ex- ylation of imprinted genes in F1 and F2 oocytes in mice posed in utero are suggestions that this leads to (120). Furthermore, whereas prenatal exposure to DEHP transgenerational effects of the chemical on the reproduc- delayed puberty in the F1 and F3 generations and de- tive system (101–103), although whether this is linked to creased sperm counts, testicular germ cell function, and DNA methylation changes in humans is unknown. the number of normal seminiferous tubules in the F3 gen- The two EDCs that are best studied for transgenera- eration (121), studies are required to determine whether tional epigenetic effects, especially DNAmethylation, are DEHP-inducedDNAmethylation changes are responsible BPA and vinclozolin (Table 1). Prenatal exposure to BPA for these adverse transgenerational outcomes. decreased CpG methylation in agouti mice (104), altered Little is known about the ability of EDCs to cause hi- the methylation status of the Hoxa10 gene in the rodent stone modifications and whether this leads to transgen- uterus (100), and changed DNAmethylation of key genes erational effects in animals or humans. The herbicides associatedwithprostate cancer in rats (105,106). Prenatal paraquat and dieldrin caused histonemodifications in im- BPA exposure at physiologically relevant levels altered mortalized rat mesencephalic dopaminergic cells (123, DNAmethylationof imprintedgenes in themouse embryo 124), and the insecticide propoxur causes histone modi- and placenta (107). Furthermore, urinary concentrations fications in gastric cells in vitro (125). DES caused histone of BPA were associated with less genomic methylation of deacetylation in the promoter region of the cytochrome genes involved in immune function, transport activity,me- P450 side chain cleavage (P450scc) gene (126). Further tabolism, and caspase activity in bisulfite-converted saliva studies, however, need to be conducted to identify other DNA from girls aged 10–13 years, and BPA levels (50 EDCs causing histone modifications in animals and hu- ng/kg to 50 mg/kg) were linked with hypermethylation in mans and todeterminewhether suchmodifications lead to tail tissue inmice (108, 109). Additional studies, however, transgenerational effects. are required to directly determine whether the BPA-in- There is also little knowledge about the ability of EDCs duced changes in DNA methylation result in abnormal to alter microRNA expression. Studies show that BPA ex- phenotypes in subsequent generations. Furthermore, posure induced expression of themicroRNAmiR-146a in whereas a few studies showed that BPA induced testicular human placental cell lines (127), down-regulated several abnormalities in the F1–F3 generations of rats (110) and microRNAs in ovaries of ewes (128), and altered expres- caused changes in social interaction tasks in the F2 and F4 sion of microRNAs in the rat penis (129). Another study generations (111), it is not clear whether these effects are showed that both BPA and DDT altered the expression due to changes in DNA methylation patterns, meriting profile of microRNA in MCF-7 breast cancer cells (130). further work. Future studies are needed to identify the specific effects of Prenatal exposure to vinclozolin in rodents has also EDCs on microRNA expression and to determine whether been shown to induce transgenerational effects on phys- EDC-induced changes in microRNA expression lead to ad- iological and behavioral phenotypes in the F3 generation versephenotypes in theexposedandsubsequentgenerations. (112–114), but whether these outcomes are due to altered In summary, a prominentmechanism for increased dis- DNA methylation changes occurring in the germline, as ease risk in adulthood as a function of early-life EDC ex- shown in the sperm (115), is not known. Inmales, prenatal posure is attributed to epigenomic reprogramming, a re- exposure to vinclozolin caused germ cell death and the sult of high plasticity as the epigenetic code is installed appearance of disease-like phenotypes in the prostate during development (131, 132). Furthermore, the envi- through the F3 generation (116, 117). In females, vinclo- ronment-gene interface must be considered as a basis for zolin caused more ovarian cysts and a reduced number of individual disease susceptibility whereby EDC-induced oocytes and primary follicles through the F3 generation modifications of the epigenetic code early in life permit The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 March 2016. at 06:01 For personal use only. No other uses without permission. . All rights reserved.