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Endocrinology. First published ahead of print June 25, 2012 as doi:10.1210/en.2012-1422
Endocrine-Disrupting Chemicals and Public Health
Protection: A Statement of Principles from The
Endocrine Society
R. Thomas Zoeller, T. R. Brown, L. L. Doan, A. C. Gore, N. E. Skakkebaek,A. M. Soto, T. J. Woodruff, and F. S. Vom Saal Biology Department and Molecular and Cellular Biology Program (R.T.Z.), University of Massachusetts,Amherst, Massachusetts 01003; Johns Hopkins Bloomberg School of Public Health (T.R.B.), Baltimore,Maryland 21205; The Endocrine Society (L.L.D.), Chevy Chase, Maryland 20815; Division ofPharmacology and Toxicology (A.C.G.), The University of Texas at Austin, Austin, Texas 78712;University Department of Growth and Reproduction (N.E.S.), Rigshospitalet, 2100 Copenhagen,Denmark; Department of Anatomy and Cell Biology (A.M.S.), Tufts University School of Medicine,Boston, Massachusetts 02111; Program on Reproductive Health and the Environment (T.J.W.), Institutefor Health Policy Studies, University of California San Francisco, Oakland, California 94612; andBiological Sciences Division (F.S.V.S.), University of Missouri, Columbia, Missouri 65211 An endocrine-disrupting chemical (EDC) is an exogenous chemical, or mixture of chemicals, that can
interfere with any aspect of hormone action. The potential for deleterious effects of EDC must be
considered relative to the regulation of hormone synthesis, secretion, and actions and the vari-
ability in regulation of these events across the life cycle. The developmental age at which EDC
exposures occur is a critical consideration in understanding their effects. Because endocrine systems
exhibit tissue-, cell-, and receptor-specific actions during the life cycle, EDC can produce complex,
mosaic effects. This complexity causes difficulty when a static approach to toxicity through endo-
crine mechanisms driven by rigid guidelines is used to identify EDC and manage risk to human and
wildlife populations. We propose that principles taken from fundamental endocrinology be em-
ployed to identify EDC and manage their risk to exposed populations. We emphasize the impor-
tance of developmental stage and, in particular, the realization that exposure to a presumptive
“safe” dose of chemical may impact a life stage when there is normally no endogenous hormone
exposure, thereby underscoring the potential for very low-dose EDC exposures to have potent and
irreversible effects. Finally, with regard to the current program designed to detect putative EDC,
namely, the Endocrine Disruptor Screening Program, we offer recommendations for strengthening
this program through the incorporation of basic endocrine principles to promote further under-
standing of complex EDC effects, especially due to developmental exposures. (Endocrinology 153:
0000 – 0000, 2012)
Foundedin1916,TheEndocrineSocietyistheworld’s basic,applied,andclinicalinterestsinendocrinology.In-
oldest, largest, and most active organization devoted to cluded among The Endocrine Society’s members are the research on hormones and the clinical practice of endo- world’s leading experts on hormones and the endocrine crinology. Today, The Endocrine Society’s membership consists of more than 15,000 scientists (basic and clinical Drawing on the expertise of its members, The Endo- researchers, physicians, educators, nurses, and students) crine Society published a Scientific Statement on endo- in more than 100 countries. Society members represent all crine-disrupting chemicals (EDC) in June of 2009 (1). The Abbreviations: AR, Androgen receptor; BPA, bisphenol A; DEHP, diethylhexyl phthalate; DES, diethylstilbestrol; DHT, 5␣-dihydrotestosterone; ED, endocrine disruptor; EDC, en- Copyright 2012 by The Endocrine Society docrine-disrupting chemical; EDSP, ED Screening Program; EDSTAC, ED Screening and doi: 10.1210/en.2012-1422 Received April 16, 2012. Accepted June 4, 2012.
Testing Advisory Committee; EPA, Environmental Protection Agency; ER, estrogen recep-tor; PCB, polychlorinated biphenyl; PND, postnatal day; T, testosterone.
Endocrinology, September 2012, 153(9):0000 – 0000 Copyright (C) 2012 by The Endocrine Society
Endocrinology, September 2012, 153(9):0000 – 0000 statement resulted from a great deal of scientific interest also discuss the current validated assays employed for the and research among The Endocrine Society members com- purpose of characterizing chemicals that can disrupt thy- bined with concern about the consequences of widespread roid hormone, estrogen, and androgen action and focus on exposure of human and wildlife populations during all life the principles of endocrinology that would strengthen stages to chemicals that can interfere with hormone ac- tion. Therefore, The Endocrine Society and its membersare keenly interested in applying their collective knowl-edge and expertise to improve human and wildlife health What Is an ED?
through the effective chemical safety assessment that isfundamental to successful public health policies. It is im- The definition of an ED is critical, because it will dictate portant to consider the issue of EDC within the context of the evidence required to identify a chemical as an EDC and normal endocrine function, which is described in a very will inform the subsequent steps of assessing the risk of large body of literature. Fundamental principles of endo- EDC exposures. Various agencies worldwide have defined crinology must be applied to the design and execution of an EDC, and we review these definitions with their studies that characterize the ability of chemicals to inter- fere with hormone action. The Endocrine Society is in a The Food Quality Protection Act of 1996 mandated unique position to help inform the ongoing debate about that the United States Environmental Protection Agency the health effects of endocrine disruptors (ED), and the (EPA) “develop a screening program, using appropriate purpose of this article is to outline (from an endocrine validated test systems and other scientifically relevant in- perspective) key issues related to identifying EDC and pro- formation, to determine whether certain substances may tecting humans and wildlife from their adverse effects.
have an effect in humans that is similar to an effect pro- The current statement of principles is a commentary duced by a naturally occurring estrogen, or other such that builds upon the groundwork laid in the Scientific endocrine effect as the Administrator may designate. . . .
Statement by introducing specific guidelines for the appli- ” (reviewed in Ref. 2). As a result, the ED Screening and cation of principles and practices of the discipline of en- Testing Advisory Committee (EDSTAC) was established docrinology to the process of chemical safety assessment.
The cornerstone of this process is chemical risk assess- in 1996 to advise the EPA on methods of screening and ment, which is a systematic approach to organizing and testing individual chemicals for endocrine-disrupting ac- analyzing scientific knowledge and information about po- tivity. To accomplish its goals, the EDSTAC described an tentially hazardous activities or substances that might ED as “an exogenous chemical substance or mixture that pose risks. Risk assessment is typically divided into four alters the structure or function(s) of the endocrine system steps: hazard identification, dose-response assessment, ex- and causes adverse effects at the level of the organism, its posure assessment, and risk characterization. Endocrine progeny, populations, or subpopulations of organisms, practices and principles should be applied to the design, based on scientific principles, data, weight-of-evidence, implementation, and interpretation of screening and test- and the precautionary principle” (3). Subsequently, other ing programs intended to identify EDC (hazard identifi- entities have also defined an ED in similar terms as follows.
cation) and to the analysis of data to assess the health risks 1) United States EPA (4). An ED is an exogenous agent from EDC exposure (hazard identification, dose-response that interferes with the production, release, trans- assessment, and risk characterization). Moreover, when port, metabolism, binding, action, or elimination of attempting to link exposures to outcome in the field of natural hormones in the body responsible for the EDC research, it is important to be cognizant of the waysin which hormone action changes over the lifetime of an maintenance of homeostasis and the regulation of Considering these issues, the goal of this document is to 2) European Union (5). An ED is an exogenous sub- provide a concise and cogent justification for the perspec- stance that causes adverse health effects in an intact tive that EDC must be evaluated within the context of organism, or its progeny, secondary to changes in fundamental principles of endocrinology. EDC cannot be endocrine function. A potential ED is a substance evaluated as if they are general toxins. Therefore, in this that possesses properties that might be expected to document, we discuss the definition of an EDC and frame lead to endocrine disruption in an intact organism.
the principles of endocrinology that need to be incorpo- 3) World Health Organization (6). An ED is an exog- rated into studies designed to identify EDC and to char- enous substance or mixture that alters function(s) of acterize their risk to human and wildlife populations. We the endocrine system and consequently causes ad- Endocrinology, September 2012, 153(9):0000 – 0000 verse effects in an intact organism, or its progeny, or hormone action are a significant risk. Risk will depend on the exposure and the potency of the chemical. However,estimating the potency of a chemical in terms of its ability When considered in the context of hazard and risk char- to cause adverse effects is as complicated as studying the acterization, these definitions are complicated and prob- role of the endocrine system in development and adult lematic. Currently, in the regulatory process, chemicals physiology. Therefore, screening and testing for EDC and are first evaluated for their potential to cause some overt estimating potency require insight derived from principles harmful effect (hazard identification). Subsequent to the of endocrinology that have been developed over decades identification of a chemical as a hazard, quantitative dose of research on hormones, their effects, and the conse- response is developed, and risk characterization (defined quences of endocrine dysregulation and disease.
as dose response ϫ exposure) (7) is conducted. The con-cept of risk management is that toxic chemicals can bereleased into the environment and the human population Principles of Endocrinology Relevant to
safely provided releases and exposures are minimized to the extent that adverse effects in humans and wildlife areaverted (8). However, estimating the exposure level that Endocrinology is the study of the mechanisms by which will cause no harm requires an accurate measure of the hormones coordinate and control the functions of multi- dose response (i.e. how sensitive are populations or sub- ple organ systems and processes throughout life. Because populations to the substance in question). Thus, the crit- hormones produce different effects at different times dur- ical first step of hazard identification must be properly ing the life cycle, the timing and duration of EDC exposure designed and implemented to ensure accurate evaluation are important elements of their effects on endocrine sys- of the sensitivity of human and wildlife populations to tems. The mechanisms by which EDC may interfere with chemicals that pose potential risks.
hormone action can be quite complex. Chemicals may All but one of the definitions above define an EDC in bind to hormone receptors and exert direct agonist or an- terms of both the mode of action (i.e. the ability to interfere tagonist actions, they may exert indirect agonist or antag- with hormone action) and the ability to produce adverse onist actions, or they may bind to allosteric sites and pro- effects (cause harm). This conflates the process by which duce unexpected effects at very low concentrations (1). In a chemical is identified as an EDC with the process by addition, chemicals are known to interfere with hormone which its potency is characterized. We propose that the synthesis or metabolism, transport (in serum or across ability of a chemical to interfere with hormone action is a membranes), or degradation. Therefore, chemicals must clear predictor of adverse outcome, much like mutagen- be examined for EDC activity in the context of fundamen- icity is a predictor of carcinogenicity. The only uncertain- tal endocrinology that has arisen from decades of careful ties relate to exposure dose, duration, and whether expo- research into the mechanisms and consequences of hor- sure occurred during critical periods of increased mone action under normal and pathological circum- sensitivity during the life cycle so that the risk will not be stances. In addition, our understanding of normal endo- underestimated. Thus, the definition of an EDC must fo- crine function is evolving rapidly as researchers apply cus on its ability to interfere with hormone action rather sophisticated and insightful experimental approaches and than stipulate adverse outcome, and this is precisely what state-of-the art technologies to the study of endocrinology.
the EPA definition does. The EPA defines an EDC as an So too, our understanding of EDC actions on endocrine exogenous agent that interferes with some aspect of hor- signaling is evolving rapidly. This endocrine literature, mone action and then spells out those aspects known at the largely described in The Endocrine Society Scientific State- time to be affected by environmental chemicals. We there- ment on EDC (1), highlights several important features of fore simplify the language of the EPA definition to account the endocrine system that must be considered in the design, for current and future information about the range of ac- execution, and interpretation of studies attempting to tions through which chemicals may influence the endo- identify EDC hazards and to define the risks to human and crine system but without changing the definition itself.
Therefore, we propose the following version of the EPAdefinition: Hormone effects are mediated by receptors
An ED is an exogenous chemical, or mixture of chem- A central tenet of endocrinology is that hormones exert icals, that interferes with any aspect of hormone action.
their physiological actions through receptors (9). This sim- It is important to recognize that this definition does not ple fact has several implications. First, hormone action is imply that all chemicals that interfere with any aspect of saturable, in terms of both ligand-binding and effect. The Endocrinology, September 2012, 153(9):0000 – 0000 magnitude of the effect and the sensitivity of the receptor tions. First, the curves are never linear, although they may to ligand (ligand efficacy) depend in part on the affinity of contain linear portions. Instead, they tend to be sigmoidal the hormone for its receptor and in part on receptor abun- in shape (Fig. 1A) but with important departures from this dance (e.g. Ref. 10); other, less well understood variables basic form, as in the case of nonmonotonic dose responses also affect ligand efficacy (11). Moreover, the maximum (Fig. 1C). It is the nature of sigmoidal-shaped dose re- effect of the hormone typically occurs at ligand concen- sponses that an equivalent change in hormone level (or trations well below those that result in receptor saturation, action) at the low end of the curve will have a proportion- a phenomenon that has been referred to as “the spare ally greater effect than at the high end of the curve; in fact, receptor hypothesis” (12 and review in Ref. 13). These once functional receptor saturation is reached, no further observations impose several consequences for the ex- increase in the response will be observed (see Fig. 1). Fur- pected shape of dose-response curves induced by hor- thermore, overstimulation of hormone receptors (binding mones and by chemicals that interfere with hormone ac- saturation) can down-regulate the receptor, leading to FIG. 1. A, Typical sigmoidal dose-response curve for hormones. As the dose of hormone increases, the response increases in a logarithmic manner
until the point of saturation of the response. Different hormone-receptor interactions will have differences in the dose of hormone or the dynamic
range of the log-linear portion of the curve or the maximal response. Some receptors are down-regulated by the hormone, so the dose-response
curve will decline at the high dose (this will be a function of both dose and time). Note that a small change in hormone concentration at the low
end of the curve (box) will have much greater effects on the response than a similar change in hormone concentration at the high end of the curve
(box). It is also important to note that saturation of the response can occur at levels of receptor occupancy in the range of 10%; thus, there are
“spare receptors” (e.g. Ref. 73). B, The dose response to the hormone depends on receptor concentration. These data show clearly that as the
receptor concentration increases, the hormone becomes “more potent”; that is, it takes significantly less hormone to produce the same response.
In fact, at low hormone receptor levels, the maximum response does not achieve the “EC ” response of the high receptor level (from Ref. 10). C,
Nonmonotonic dose response curve. The inverted U dose-response curve may have many different mechanisms underlying it. For example,receptor down-regulation at high concentrations of hormone is an important mechanism. However, the addition of separate monotonic doseresponses also provides an important mechanism. This issue is reviewed extensively by Vandenberg et al. (13).
Endocrinology, September 2012, 153(9):0000 – 0000 changes in abundance of the receptor and a decrease in Hormones exert very specific effects on development sensitivity of the cell to the hormone. This process often and adult physiology, precisely because they act through results in “high-dose inhibition”; that is, a dose-response receptors that exhibit specific patterns and intensities of curve in which low doses increase the response, and high distribution. For example, during sexual differentiation, doses decrease the response. This “inverted U” dose re- testosterone (T) secreted from the testis acts on the an- sponse is an example of a nonmonotonic dose response drogen receptor (AR) expressed in fetal tissues to cause the (Fig. 1C). These observations are universally acknowl- development of the male reproductive tract. It is the loca- edged by the endocrine community and have been exten- tion-specific expression of the AR that permits this inter- action (17–19). However, the direct action of T in the Because of the role of receptors in mediating hormone development of the male reproductive tract (Wolffian duct effects, and because hormones exhibit nonlinear dose-re- derivatives) differs considerably from its indirect action in sponse characteristics, EDC will necessarily replicate these the fetal and adult male brain, which for some areas of the characteristics. This has several implications. First, the ef- brain is mediated by the estrogen receptor (ER) after T is fect of a high dose of a chemical may not predict the effect converted to estradiol by the action of the aromatase en- of the chemical at a low dose. The most obvious reason for zyme. Therefore, T acts in the male by interacting with this is the existence of a nonmonotonic dose response.
either the AR or ER (after aromatization). Furthermore, However, at very high doses, chemicals can also produce the AR can be activated by T itself or by its derivative a number of interacting effects that obscure what would be 5␣-dihydrotestosterone (DHT) through actions of the en- most important for low-dose exposures. Second, because zyme, 5␣-reductase. Recent work indicates that T and low doses of endogenous hormones are present and fluc- DHT impose different structural constraints on the AR tuating, small additions (or subtractions) to their actions that may explain the different effects of T and DHT on different tissues (20). These examples also highlight the These implications are problematic for risk assess- fact that actions of EDC may be at the level of the steroid ments, because it cannot be assumed that high doses biosynthetic or metabolizing enzyme, instead of or in ad- always provide information relevant to low-dose expo- dition to actions on the receptor itself (21). Furthermore, sures, and because it cannot be assumed that there is a hormone action can be prevented by enzymatic conversion threshold. The absence of a threshold for EDC has also of the hormone from one form to another. For example, been demonstrated experimentally in animals (14, 15), cortisol is prevented from acting on the mineralocorticoid in epidemiological research (16), and theoretically receptor in the distal convoluted tubules of the kidney by based on mechanisms of hormone action (14). Directly the action of 11␤-hydroxysteroid dehydrogenase, and lic- related to this issue is that the human population is orice can block this activity producing hypertension in chronically exposed to low doses of EDC, which even further necessitates a “no-threshold” approach to risk An important point is that hormone actions during de- velopment are often permanent. They affect elements of In addition, because some endpoints are more sensitive organ development that have lifetime consequences. Like- than others to the actions of endogenous hormones, it is wise, hormone disruption during development can pro- also clear that some endpoints of EDC effects on hormone duce effects that are permanent, some of which do not action will be more sensitive than others. Thus, establish- become manifested until adulthood. These developmental ing the potency of a chemical’s ability to interfere with origins of health and disease are exemplified by the effect hormone action (a key element in the risk assessment pro- of the drug diethylstilbestrol (DES) on cancer incidence.
cess) will require that several of the most sensitive end- Specifically, the female children of women who were pre- points of hormone action be evaluated.
scribed DES during the first trimester of pregnancy have ahigher incidence of breast cancer, clear cell adenocarci- Endpoints of hormone action
noma of the vagina and cervix, and reproductive anomalies Many hormones exert widespread actions in the body.
(23). These effects of fetal DES exposure occur in adulthood, However, the specific actions of individual hormones often and there is good experimental evidence that chemical ex- change throughout life, they may be different in males and posures can produce similar actions (24 –26). Thus, the ad- females, and they may be mediated by different receptors or verse effects of EDC exposures during development may re- receptor isoforms expressed in different tissues or at different quire an extended period to be manifested, a period during life stages. To list the extensive examples of these phenomena which a generation of people will have been continuously is beyond the scope of this article, but there are some funda- exposed. This kind of effect is clearly important to incorpo- mental patterns that should be highlighted.
rate in a screening and testing paradigm.
Endocrinology, September 2012, 153(9):0000 – 0000 Taken together, it is clear that hormones have very ical and technical error that can obscure meaningful re- complex actions simultaneously in different tissues, and it sults, such as unplanned events in the animal colony as is important to recognize that EDC will exert similarly well as the various measurement errors that can occur.
complex actions but perhaps in patterns that do not ex- Thus, if the experiment is unable to identify effects of the actly replicate the effects of the native hormone. Several positive control, failure to identify effects of the EDC characteristics of endocrine systems can explain how EDC would not be meaningful. Considering this goal, it is im- can produce selective effects on hormone action. One portant to employ a dose of the positive control that would mechanism is that chemicals can influence hormone me- challenge the limit of detection for effects.
tabolism in a tissue-specific manner that can directly in-terfere with normal hormone actions only in some tissues.
In addition, the chemicals themselves may be metabolized Characterizing the Endocrine-Disrupting
(e.g. hydroxylated) in a tissue-specific manner, and the Properties of Environmental Chemicals
metabolites may directly interfere with hormone actiononly in those tissues where they are generated. Chemicals, The final report of the EDSTAC, published in 1998, rec- or their metabolites, may also interact with hormone re- ommended a two-tiered system, in which an initial battery ceptors in a tissue-specific manner, either because some of relatively short-term in vitro and in vivo assays was tissues exhibit greater receptor density, or because differ- proposed to screen chemicals for potential ED activity, ent receptor isoforms are expressed in different tissues (27, followed by a second tier of “definitive” tests (3). Since 28). Considering these possibilities, it is unrealistic to ex- then, the United States EPA further developed the Tier 1 pect or require completely consistent results of EDC ef- assays and engaged in a validation process intended to fects on hormone action across all hormone-sensitive end- ensure that the Tier 1 data would be reliable and repro- points, as EPA’s Weight of Evidence document for the ED ducible across laboratories. The assays that make up the Screening Program (EDSP) Tier 1 recommends (http:// Tier 1 battery (Table 1) were developed on the basis of www.regulations.gov/#!documentDetail;DϭEPA-HQ- several criteria: to “a) maximize sensitivity which serves to OPPT-2010-0877-0021). In addition, EDC are imperfect minimize false negatives, b) include a range of organisms ligands of hormone receptors and should be expected to representing differences in metabolism, c) detect all interact with them in ways that do not perfectly replicate known modes of action by the endocrine endpoints of the actions of the endogenous hormone (29). Thus, it is concern, d) include a sufficient range of taxonomic groups possible that some EDC can cause a hormone receptor to among the test organisms, and e) incorporate sufficient do something that it would not normally do. Each of these diversity among the endpoints, permitting weight-of-evi- events is a likely explanation for the observation that dence conclusions” (3). Thus, the assays in Tier 1 were many EDC influence a subset of a given hormone’s effects designed to identify chemicals that may interfere with es- trogen, androgen, or thyroid hormone action, and there- In addition, it is known that differences in husbandry, fore, would require additional testing (in Tier 2) for the such as phytoestrogen content of different types of animal EPA to determine the degree of risk to human and wildlife feed, can impact the outcome of an experiment (31). Un- health. Details of the assays are described by the EPA in a planned events in an animal colony, such as fire alarms or series of test guidelines available on the EPA website at construction noise, can also influence the physiological http://www.epa.gov/ocspp/pubs/frs/publications/Test_ status of the animal and thus the outcome of studies on EDC. Although rats and mice are the basis of most basic Considering the complexity of hormone action and the animal research, differences in species and strains also known complexity of EDC effects on hormone action, the contribute to differences across studies. Because these en- goal of identifying all EDC in a rapid animal-based screen vironmental contributors introduce inconsistencies, it is is impossible to achieve. However, the United States EPA important to include positive and negative controls within has developed a first step toward accomplishing that goal and between studies so that worthy results will not be in the current EDSP and is engaged in a promising new ignored simply because they cannot be fully explained strategy of using high throughput in vitro assays that (32). The purpose of including animals exposed to an ap- would be faster and more efficient (33), with the aim of propriate low dose of control chemical is to demonstrate replacing animal testing and evaluating more chemicals to that: 1) the test system is sensitive to the class of chemical which the human population is already routinely exposed being examined and 2) the sensitivity of the technical ap- (34). These important steps would be strengthened by the proach is sufficient to identify meaningful effects. This incorporation of specific endocrine principles to support latter issue relates to all the sources of introduced biolog- the design of future EDC assays, as well as to support the Endocrinology, September 2012, 153(9):0000 – 0000 Receptor binding
Steroidogenesis
Screening assay
Test guideline
Complementary endpoints across assays are indicated (X) within each column.
a 5␣-Reductase inhibition only. OCSPP, Office of Chemical Safety and Pollution Prevention; OECD, Office of Economic and Cooperative Development.
execution of current assays and interpretation of the cur- high dose and examining a few lower doses that are all very rent data. Specifically, incorporating endocrine principles high by endocrine standards. The initial (reference) high can help integrate the large battery of high throughput in dose of the chemical is required to be near the maximum vitro assays into risk assessment procedures for EDC.
tolerated dose (determined to cause some sublethal effect, These principles are not targeted to the risk assessment of typically indicated by a decrease in body weight) or a dose any particular chemical but are broadly applicable to of no more than 1 g/kg body weight. This strategy is based EDC, nor are they specific to the EDSP itself. Academic on the concept that toxic effects will appear at maximum studies focused on understanding mechanisms of EDC ef- doses (e.g. LD50, which is the dose that kills 50% of the fects on hormone action that could account for disease animals) and that there is a linear relationship between trends likewise should incorporate these principles. How- dose and effect. In addition, there are typically only three ever, the assays described in Tier 1 of the EDSP (Table 1) doses tested that cover about a 50-fold range. Although provide a useful focal point, around which to illustrate this approach has been effective in identifying classic tox- practical application of endocrine principles to improve icants, the EDSP was mandated to identify EDC, which do screening for EDC, thereby enhancing strategies to protect not behave like toxicants. Two problems with this ap- proach are immediately clear for EDC. First, it is impos-sible to assess the shape of the dose-response curve withonly three doses; and second, the dose-response curve can- Tier 1 of the EDSP
not be assumed to be monotonic (or linear), which is the The assays described in Table 1 represent a combination core assumption underlying this “top down” approach to of in vitro and in vivo assays. They have certain strengths, dose selection. The lowest dose tested is assumed to be but they also have identifiable weaknesses. We describe within 10-fold of the no effect level, even if adverse effects below the relative merits of these assays using specific ex- are found at the lowest dose tested, and the calculated no amples to help focus the ways in which our current knowl- effect dose (which is 10-fold less that the lowest dose edge can capitalize on the strengths and minimize the tested) is declared a “threshold” dose. This 10-fold as- weaknesses. However, two initial points of concern that sumption is not based on the known differences in hor- apply to all of the following examples are that first, the mone potency based on receptor abundance alone, be- current endpoints being examined in the EPA’s EDSP stud- cause this can change by 10,000-fold (e.g. Fig. 1B).
ies to determine the hazard of EDC do not meet the cri- Therefore, by endocrine standards, the assumed threshold terion of using the most sensitive outcomes to assess haz- dose is always very high, because it is based on a dose that ard; and second, the in vivo assays will use the traditional is sublethal rather than on mechanistic information about approach in regulatory toxicology of starting with a very the biochemical and molecular actions of the EDC that Endocrinology, September 2012, 153(9):0000 – 0000 may be observed at doses more than a million-fold lower than 4%. The intraassay variability may have contrib- uted to the wide range of total T4 values that would be Finally, people differ in their baseline exposures to EDC considered normal and undoubtedly contributed to the and in their sensitivities to endocrine disruption. In addi- overall variability of hormone measurements observed tion, because some endpoints are more sensitive than oth- ers to the actions of endogenous hormones, it is also clear Another limitation of the EDSP is the EPA’s guidance that some endpoints of EDC effects on hormone action that the primary endpoint for consideration of thyroid will be more sensitive than others. Thus, establishing the hormone action in Tier 1 should be histopathological potency of a chemical’s ability to interfere with hormone changes within the thyroid gland itself (36). Chemical- action (a key element in the risk assessment process) will induced changes in thyroid histopathology mostly reflect require that several of the most sensitive endpoints of hor- chemical-induced changes in serum TSH. However, many chemicals reduce serum total and serum-free T4 without Thus, the data derived from the traditional approach eliciting an increase in serum TSH, although it is not clear just described will have a high probability of underesti- how this happens (37). Therefore, it remains possible that mating potency and may miss important effects alto- these chemicals are disrupting thyroid hormone action at gether. As a result, the risk assessment process will come sites other than the thyroid gland through mechanisms to conclusions that could have negative impacts on public that do not require changes in TSH. A good example of this health. We describe specific examples below.
scenario is that of polychlorinated biphenyls (PCB).
PCB are a class of industrial chemicals, the production of which was banned by the United States Congress in the 1970s (38). Because of their stability and persistence, PCBremain ubiquitous contaminants in the environment and Tier 1 has three in vivo assays that measure chemical ef- in the human population even today (38). They are well fects on the thyroid system, the amphibian metamorphosis known to cause a reduction in circulating total and free T assay, and the male and female pubertal assays. For these but do not cause an increase in serum TSH, nor do they mammalian assays, according to the EPA protocol, change elements of thyroid histopathology (39). How- Sprague Dawley rats are treated with test chemical frompostnatal day (PND) 22 (female) or 23 (male) to PND 42.
ever, PCB interfere with thyroid hormone action in the The dose of the chemical is dictated to be near the maxi- periphery and the brain of experimental animals and are mum tolerated dose (or no more than 1 g/kg) as indicated linked to neurobehavioral effects in humans (40 – 42).
by effects on body weight. At PND 42, the animals are Some PCB, or their hydroxylated metabolites, can bind to euthanized and serum collected. The endpoints for thyroid the thyroid hormone receptor in a competitive binding assay (43) and may exert allosteric effects on the thyroid 4 and TSH, and thyroid histopathology.
Thyroid histopathology is evaluated subjectively using a hormone receptor as well (28, 44). In the Tier 1 EDSP, PCB five-point scale, and commercial kits are used to evaluate would cause a decrease in serum T4 but would cause nei- ther an increase in TSH nor a change in thyroid histopa- There are several design and guidance features of the thology. The EPA’s guidance to more heavily weigh thy- pubertal assays that limit their ability to identify chemicals roid histopathological changes rather than proper serum known to interfere with thyroid hormone action. First, the T4 measurement would result in the interpretation that, hormone levels are considered highly variable and there- despite their effects on thyroid hormone levels, PCB need fore not reliable. The performance criteria required for not be further examined in Tier 2, because they do not hormone measurements according to the protocol would cause the adverse effect of thyroid histopathological changes. In this case, the evaluation of PCB would end at about 4 to 30 and still be considered normal. This range of Tier 1 with no measure of the other adverse effects they have been shown to cause. However, the effect of PCB on 4 values in untreated Sprague Dawley rats is not known to exist but appears to be based on the variability the thyroid system may be detected in the amphibian meta- reported in the validation studies leading to adoption of morphosis assay. In this case, the combination of a positive these assays for thyroid endpoints. The intraassay vari- in the amphibian assay and the reduction in circulating T4 ation in the validation studies was reported to be 25– in the pubertal assays, may send PCBs to Tier 2. However, 35% (35), but performance criteria for these kit assays Tier 2 does not have an amphibian assay, nor does it con- routinely conducted by thousands of clinical laborato- tain endpoints of thyroid hormone action. Therefore, it is ries require an intraassay coefficient of variation of less quite likely that PCBs would not be identified as antithy- Endocrinology, September 2012, 153(9):0000 – 0000 roid agents capable of producing population effects at en- Although two commonly used phthalates, diethylhexyl phthalate (DEHP) and dibutyl phthalate, and their in vivo, Regulatory processes in the 1970s were insufficient to bioactive metabolites monoethylhexylphthalate and identify PCB as harmful, thus necessitating an act of Con- monobutylphtalate, disrupt male reproductive develop- gress to ban them (38). Considering the discussion above, ment in an antiandrogenic fashion, the activities of these the Tier 1 battery of screens within the EDSP does not compounds are not manifest via the classic antiandrogenic differ enough from regulatory standards of the 1970s and mechanism of an antagonist with high affinity for the AR would be significantly enhanced by incorporating devel- (48, 49) and would not be detected in in vitro AR binding opmental exposures and additional endpoints of thyroid assays. Rather, endocrine-disrupting activity of phthalates hormone action. A number of standard assays have been is directed at early development of the fetal testis. Expo- proposed that could ameliorate these weaknesses (e.g. cer- sure of rats to DEHP (or monoethylhexylphthalate) and ebellar histogenesis among many possible endpoints) (45).
dibutyl phthalate (or monobutylphthalate) during gesta- Many chemicals to which the human population is ex- tion causes a significant reduction in fetal T levels during posed will exhibit a similar profile of effects in the current the critical masculinization window between embryonic d 15 and 19. In utero exposure of rats to phthalates causeshistological evidence of testicular dysgenesis consisting ofreduced numbers of Sertoli and germ cells, malformed Androgens
seminiferous cords/tubules with intracordal/intratubularLeydig cells, and immature Sertoli cells. These effects can The Tier 1 assays (Table 1) evaluate chemicals for their only be ascertained by histologic examination during tes- ability to interfere with androgen action primarily in three ticular development and are not predicted by AR binding assays, the AR binding assay (rat prostate), the Hersh- assays with rat prostate cytosol or steroidogenic assays berger assay (rat), and the male rat pubertal assay. The binding assay uses the cytosol fraction from rat ventral Malformation of reproductive tissues in male rats is prostate as a source of the AR in an in vitro displacement most dramatic after fetal and lactational exposure to assay using the synthetic androgen [3H]-methyltrienolone phthalates with significant postnatal developmental as the tracer. The Hershberger bioassay is intended to anomalies, including reduced anogenital distance, nipple serve as a mechanistic in vivo screening assay for androgen retention, presence of a vaginal pouch, cleft phallus, hy- agonists, antagonists, and 5␣-reductase inhibitors. Devel- pospadias, epididymal agenesis, undescended testes, and oped in the 1930s and 1940s, it is a short-term screening reduced accessory sex gland (prostate, seminal vesicles) assay using changes in weight of five androgen-dependent weights. Pubertal administration of DEHP can result in tissues of castrated peripubertal male rats: the ventral delayed onset of puberty assessed by age of preputial sep- prostate, seminal vesicle, levator ani-bulbocavernosus aration, alterations in testis histopathology, reduced se- muscle, Cowper’s glands, and glans penis. Similarly, the rum T and elevated LH levels, and decreased accessory male rat pubertal assay incorporates measures of andro- sex gland weights. Although these effects would be ob- gen-dependent organ weights, age and body weight at time servable in the pubertal male and Hershberger assays, of preputial separation, testis histology, and serum T lev- they occur at doses higher than those that elicit effects els. Thus, the concept of using these three assays is that if during the fetal period, an observation that could lead a chemical directly interacts with the AR, it will be iden- to the inaccurate interpretation that lower exposure lev- tified in the binding study, and if it has functional conse- els are safe because the most sensitive period for expo- quences on the AR or T biosynthesis, it will affect male Distinct differences have been shown in the sensitiv- The design of this approach is based on the premise that ity of Long-Evans and Sprague Dawley rat strains to the binding assays as described will provide a comprehensive pubertal administration of DEHP, with Long-Evans view of chemical interactions with the AR and that repro- rats being more sensitive to DEHP effects on some end- ductive organ weight is a sensitive proxy measure of an- points and less sensitive on other endpoints (50). This drogen disruption at all endpoints. However, these assays demonstrates that selection of the model system is an are not sensitive to some types of antiandrogens, such as important consideration when testing for endocrine-dis- phthalates. Phthalates (phthalic acid esters) are a family of rupting properties. Moreover, various reports indicate chemicals commonly used as plasticizers, and their pres- that one primate model, the marmoset, may be resistant to ence in a large number of consumer products makes their the deleterious testicular actions of phthalates, in that re- distribution in the environment ubiquitous (46, 47).
duced T biosynthesis, abnormal testicular histology, and Endocrinology, September 2012, 153(9):0000 – 0000 altered accessory sex gland development were not ob- doses between 100 and 800 mg/kg body weight, suggest- served in this species. However, epidemiological studies ing that environmental BPA exposure poses no problems suggest that humans are sensitive to the antiandrogenic as the environmental exposure levels are orders of mag- actions of phthalates. Differences in the pharmacokinetics nitude lower than those needed to induce a significant and pharmacodynamics of phthalates in this monkey increase of the wet weight of the uterus. However, long- model likely explains the lack of sensitivity of the mar- term adverse effects on the female mouse reproductive moset to phthalate exposure (51). The apparent dichot- system, including the uterus, due to exposure to very low omy between the human and nonhuman primate data has doses of BPA during early development have been re- yet to be resolved. Despite these outstanding questions, it ported by the United States National Toxicology Program, is clear that DEHP has the potential to act as an androgen and the lowest dose tested (0.1 ␮g/kg ⅐ d) showed the high- disruptor and that it, in fact, does so under a number of est percent of uterine tumors (55). These effects of expo- sure to BPA for only 7 d during fetal life would not beidentified or accounted for in the EDSP due to the absenceof assays that involve exposure during fetal and neonatal Estrogens
life, when the animals are most sensitive to BPA (56). Oneof a number of reasons for the high sensitivity of fetuses Estrogenicity (and antiestrogenicity) is more intensively and neonates to EDC such as BPA is the maxim in pediatric tested in the battery of Tier 1 EDSP than effects on either medicine that “babies are not little adults,” and they have androgen or thyroid hormone actions. The in vitro assays a limited capacity to metabolize xenobiotics. BPA also include an ER binding assay using rat uterine cytosol, hu- produces significant effects in the adult, with some of the man ER␣ transcriptional activation using a human cell effects being mediated by the ER␣ (57) and others by ER␤ line (HeLa-9903) stably transfected with human ER␣ and (58). However, the effects in adults occur at higher doses an assay for the enzyme aromatase (estrogen synthetase) than those that impact the fetus (59), and there is evidence activity using human recombinant microsomes. In addi- that the dose-response curve is not monotonic (55–58).
tion, there is a fish short-term reproduction assay, female Chronic oral exposure of the mouse mammary tumor rat pubertal assay, and rat uterotrophic assay. As de- virus-erbB2/neu mouse model to BPA during adult life scribed above for the androgen assays, these assays are not (from PND 56 to killing) increased breast tumor multi- optimally sensitive to chemicals that interfere in some plicity, decreased the latency period, and increased the ways with estrogen action. We provide two examples number of metastases only at the lowest doses (2.5 and 25 ␮g/kg ⅐ d), whereas higher doses did not affect these end-points (60). There is also now considerable evidence fromstudies with rats and mice, as well as human breast cells, Bisphenol A (BPA)
indicating that BPA increases the risk of breast cancer andinterferes with breast cancer therapy. In addition, there is Nuclear ER binding assays as well as transcriptional ac- evidence for nonmonotonic dose-response curves for ef- tivation assays indicate that BPA has at least a 10,000-fold fects on the mammary gland (61, 62).
lower affinity for the two estrogen nuclear receptors than Specifically, in several rat models, perinatal administra- 17␤-estradiol. In isolation, these results would suggest tion of BPA (2.5–500 ␮g/kg ⅐ d) resulted in the development that BPA at environmentally relevant levels of exposure of preneoplastic mammary lesions (63, 64) and increased would not pose a public health problem. However, ex- neoplastic outcomes when animals were treated with a chem- periments published by the Kortenkamp group indicate that, ical carcinogen during adult life (65, 66). As stated previ- in conditions resembling the living condition, BPA and other ously, the EDSP is not structured to examine low-dose effects xenoestrogens act additively with ovarian estrogens, and this elicited in a nonlinear or nonmonotonic fashion.
phenomenon was observed at very low xenoestrogen levelswithin the range of environmental exposure [Kortenkampand co-workers (52)]. Similarly, in vitro experiments ad- Additional Considerations of Tier 1
dressing the role of ER embedded in the plasma membraneindicate that for some endpoints, BPA is equipotent to ovar- Dioxin-like activities
ian estradiol, and significant effects of BPA at a dose of 0.01 An example of EDC that might not be easily identified in the EDSP toxicological testing is dioxin-like com- Depending on the rodent species and strain, uterotropic pounds, such as PCB, which have pleotropic effects on effects of BPA have been observed at the relatively high multiple endocrine systems. Discussed in the section on Endocrinology, September 2012, 153(9):0000 – 0000 ● EDC exposures during development can have effects on hormone action that cannot be corrected, leaving ● Basic scientists actively engaged in the development of permanent adverse impacts on cognitive function and new knowledge in relevant disciplines should be involved in evaluating the weight-of-evidence of EDC studies, as ● People are exposed to multiple EDC at the same time, and well as in the design and interpretation of studies that these mixtures can have a greater effect on the hormone inform the regulation of EDC. Endocrinologists and specialists in other relevant disciplines should be involved in these processes as applicable (i.e. neurologists should ● As a battery of tests, the Tier 1 of the EDSP will only be involved if the affected biological process involves the The route of exposure and dosing strategy are not optimized ● Endocrine principles, such as those outlined in this to identify EDC. Moreover, the timing of exposure does not document, should be incorporated into programs by the EPA and other agencies charged with evaluating ● The weight-of-evidence guidance developed by the EPA chemicals for endocrine-disrupting potential.
must be strengthened by adhering to principles of ● State-of-the-art molecular and cellular techniques, and endocrinology outlined here, including low-dose effects highly sensitive model systems, need to be built into and nonlinear or nonmonotonic dose-response curves.
current testing, in consultation with the appropriatesystem experts.
thyroid hormone, PCB are a family of structurally stable, ● The design and interpretation of tests must incorporate synthetic compounds that were widely used in industry the biological principle that EDC act through multiple beginning in the 1930s. The United States Congress mechanisms in physiological systems.
● Testing needs to include models of developmental banned manufacture of PCB in the United States in 1977 exposure during critical life periods when organisms may due to the dioxin-like activity of congeners used in com- be most vulnerable to even very low-dose exposures.
mercial mixtures of PCB. However, significant quantities Endocrine Principles Applied to EDC Research of persistent PCB are still detectable in both the environ- ● An ED is an exogenous chemical, or mixture of chemicals, ment and in the food chain due to bioaccumulation and that interferes with any aspect of hormone action.
Principles of the biology of endocrine systems biomagnification (67, 68). A recent study estimating PCB ● Hormones play direct and essential roles in many aspects concentrations in humans shows that people born as re- of development and in adult physiology. Hormones cently as 2010 have PCB body burdens (69).
represent the means by which development progresses in PCB are classified as dioxin-like coplanar, or nonco- an orderly and coordinated manner and by which majorphysiological processes are coordinated.
planar, based upon the arrangement of chlorine atoms ● Environmental chemicals that interfere with any aspect of around the biphenyl core. Structural differences between hormone action should be presumed to produce adverse the classes of PCB influence the binding affinity to hor- mone and neurotransmitter receptors and their ability to ● Hormones act on receptors, and as a consequence, act as an agonist or antagonist. This means that the ability hormone receptor distribution and abundance represent to predict effects of PCB requires knowledge of the effects important characteristics defining hormone action.
of the individual PCB in a mixture, because exposure in- ● An EDC can interfere with hormone action on the receptor by affecting any number of steps in the biochemical Because of mixed properties of PCB and the weak bind- pathway. This includes affecting the amount of hormonesproduced and interfering with the ability of a hormone to ing of some PCB congeners to ER (71), there is high prob- reach the right receptor at the right time and right location.
ability that PCB would be missed in Tier 1 screens for ● Hormone-receptor systems are “tuned” such that very estrogenic activity. Furthermore, assays that involve low doses of hormones effectively alter development and transfection of ER (such as the transcriptional activation adult physiology. Accordingly, chemicals can interfere assay with transfected HeLa cells) have relatively low sen- with hormone action in very low doses, producingirreversible effects on development and critical sitivity, making it unlikely this assay would detect PCB or other estrogenic compounds that in mixtures can result in ● Some hormones exert their actions through more than additive effects (52). Assays involving uterotrophic activ- one receptor. Therefore, different elements of the ity and the timing of puberty in females also have low spectrum of effects produced by those hormones are sensitivity, making them poor proxies for estrogenic ac- attributable to the different individual receptors.
● Likewise, chemicals that interact with only a subset of the tivity that may be exerted at the cellular/molecular level, endogenous hormone’s receptors will produce a mosaic something that would not be discerned from just measur- of effects that does not reproduce an endocrine disease ing uterine size or timing of the onset of secondary sexual Endocrinology, September 2012, 153(9):0000 – 0000 Given the specific assays in Tier 1 and EPA’s guidance Because hormone actions are pleiotropic, a single ex- document, it is likely that PCB would not be considered an perimental design will not be optimal for testing the ability EDC in the estrogen assays, as just described, or in the of an EDC to interfere with all hormone actions. There- thyroid assays, as described earlier in this document. This fore, screens and tests for EDC need to be optimized for is concerning, because there is an abundance of experi- sensitive endpoints of hormone action, many of which are mental evidence showing that PCB interfere in complex developmental. In addition, because academic research on ways with various hormone systems, and there is evidence EDC has often been both optimized to critically evaluate in humans of the impacts of PCB exposures to reproduc- EDC actions on important hormone actions and inten- tion, cognitive function, immune function, and other sively scrutinized in peer review, including grant, journal, health outcomes (73). It may be relevant to remember that and institutional panels, this research needs to be incor- PCB production was banned by an act of Congress, not porated into the processes agencies engage to protect pub- through the regulatory processes in place in the 1970s. It lic and wildlife health. In turn, this requires that experts in is troubling that, despite the advances in science over the the basic biology of the system under investigation (i.e.
past 40 yr, the current assays and guidelines in the regu- proposed to be affected by an EDC) must be active par- latory domain would continue to underestimate PCB tox- icity. Moreover, the absence of any assay that woulddetect a compound with dioxin-like activity in a sensi-tive manner in the EDSP Tier 1 assay protocols is a Acknowledgments
Address all correspondence and requests for reprints to: R.
Thomas Zoeller, Biology Department, University of Massachu-setts, 611 North Pleasant Street, Amherst, Massachusetts 01003.
Conclusions
This work was funded in part by grants from the NIEHS The Endocrine Society’s Scientific Statement published in (ES10026 to R.T.Z., ES01839 to F.S.v.S., and ES08314 to A.S.), 2009 (1) provided an exhaustive summary of the scientific the NICHD (HD05574 to T.R.B.), The New York Community background that justifies concern for the effects of EDC Trust (P12-000024) and the Forsythia Foundation (T.J.W.), and exposures to human and wildlife population health. In that document, a number of recommendations are pro- Disclosure Summary: The authors have nothing to disclose.
posed for research and practice guiding the understandingof EDC in four categories: clinical research, basic science,epidemiology, and clinical practice. These recommenda- References
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