History EDCs

TOXICOLOGICAL SCIENCES 120(S1), S93S108 (2011) doi:10.
1093/toxsci/kfq329 Advance Access publication October 25, 2010
Endocrine Disruption: Historical Perspectives and Its Impact on the Future of Toxicology Testing
Mary Sue Marty,1 Edward W. Carney, and Justin Craig Rowlands
Toxicology and Environmental Research and Consulting, The Dow Chemical Company, Midland, Michigan
Downloaded from http://toxsci.oxfordjournals.org/ at University of Nairobi on November 16, 2013
To whom correspondence should be addressed at Toxicology and Environmental Research and Consulting, The Dow Chemical Company, 1803 Building, Midland, MI 48674. Fax: (989) 638-9863. E-mail: mmarty@dow.com. Received August 13, 2010; accepted October 15, 2010
The endocrine system is one of the bodys major homeostatic control systems whose aim is to maintain normal functions and development in the face of a constantly changing environment. Working in tandem with the nervous system, which mainly is responsible for rapid and immediate responses, the approximately 30 different glands comprising the endocrine system tend to act in a slower and more sustained manner to regulate processes as diverse as the female reproductive cycle, bone growth, cell proliferation, and psychosocial behavior. Multiple endocrine glands also work in concert with one another to form complex feedback loops, which tightly regulate critical physiological processes. Like all homeostatic control systems, the capacity to maintain physiological parameters within normal bounds is nite, and when this capacity is exceeded by chemical exposures, drugs, or environmental stressors, adverse consequences can ensue. In the mid-1990s, concerns about the potential for environmental chemicals, drugs, and other stressors to alter endocrine physiology rapidly mounted and received a great deal of attention within the toxicology community as well as in the public media. These circumstances spurred a urry of research on mechanisms of toxicity, new questions about how to deal with cumulative exposures to endocrine-active compounds (EACs) from man-made, dietary, and endogenous sources, and the creation of major chemical screening and testing programs focused on endocrine-mediated modes of toxic action. In this review, we will trace the path of knowledge regarding the effects of exogenous agents on the endocrine system, the events which gave rise to the explosion of interest in this topic in the mid-1990s, and how it led to a fundamentally different paradigm in toxicity testing as well as a great deal of basic research on mechanisms of endocrine-mediated toxicity. We rst cover the history of testing for endocrine-mediated
toxicity, followed by coverage of key historical events in basic science, and then bring the two together to discuss implications of the science on testing both now and for the future.
EARLY HISTORY OF TOXICITY TESTING FOR ENDOCRINE-MEDIATED EFFECTS
A casual observer might easily be led to believe that the ability of exogenous substances to interfere with the endocrine system was unknown until relatively recently, but in fact, this is far from true. Even the ancients were familiar with the actions of various herbal preparations to modulate what we now know to be hormonally regulated processes. For centuries, farmers have observed reproductive problems in female sheep and cows grazing on pastures rich in certain clover species, which later were found to contain estrogenic compounds such as coumestrol (Adams, 1995). As early as the 1930s, the ability of both natural and synthetic chemicals to interact with endogenous hormone receptors was already well established (Schueler, 1946; Sluczewski and Roth, 1948; Walker and Janney, 1930). Starting around the 1940s, steroidal compounds began to be used in the livestock industry to modulate reproductive cycles and to enhance rate and efciency of body weight gain, while compounds such as ethinyl estradiol, mestranol, norethynodrel, and megestrol acetate were being developed as contraceptive agents and to treat specic disease conditions in humans (Newburgh, 1975). In parallel with these developments, toxicologists began to investigate these compounds for potential adverse effects. Diethylstilbestrol (DES), a synthetic estrogen with similar potency to 17b-estradiol, was rst prescribed in 1938 to
The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oup.com
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prevent miscarriage and premature births. This drug was particularly signicant as the daughters of women who took DES were found to have an increased incidence of clear-cell carcinoma, a rare vaginal cancer. As a result, in 1971, the Food and Drug Administration (FDA) advised physicians to stop prescribing DES to pregnant women. Further research has revealed an increased risk for breast cancer in DES mothers and also possible links with reproductive tract abnormalities in DES sons and daughters (Giusti et al., 1995). It has been estimated that 510 million Americans received DES during pregnancy or were exposed to the drug in utero. Other episodes of endocrine-mediated toxicity also occurred via unintentional exposure to hormonally active compounds, such as polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs). Some members of this large family of structurally related congeners can act on the estrogen and arylhydrocarbon (Ah) receptor systems and/or modulate thyroid hormone function. In Japan (1968) and Taiwan (1979), a few thousand women were exposed to PCBs and their pyrolysis products (PCDFs) following consumption of contaminated rice oil. The offspring of these mothers exposed to PCBs and PCDFs tended to be smaller at birth and exhibited delays in neurological development (Aoki, 2001). Subsequent studies on PCB exposure at lower levels have alleged other adverse effects, although these data remain somewhat controversial. In the environment, the presence of natural hormones in wastewater treatment outfalls in the United States was noted as early as 1965 (Stumm-Zollinger and Fair, 1965), and this work expanded in 1970 to include synthetic estrogens used as birth control agents (Tabak and Bunch, 1970). By the mid-1970s, scientists began to detect other pharmaceutical agents near wastewater outlets in the United States (Garrison and Pope, 1975; Hignite and Azarnoff, 1977). Concerns about dichlorodiphenyltrichloroethane (DDT) in the environment were highly publicized in Rachel Carsons book, Silent Spring, although a specic link to endocrine-mediated toxicity was yet to come. Despite the aforementioned events in both wildlife and humans, endocrine-mediated toxicity did not garner any special attention above and beyond other mechanisms of toxicity known at the time. Instead, it tended to be regarded as just one of many potential mechanisms that could lead to certain adverse outcomes, such as reproductive toxicity, developmental toxicity, or cancer.
THE BIRTH OF ENDOCRINE DISRUPTORS
This mindset changed rather abruptly in the mid-1990s following a series of publications suggesting that pesticides and other man-made chemicals were threatening the reproductive capability and intelligence of future generations of humans and wildlife (Colborn et al., 1996, 1993; Hunter and Kelsey, 1993). Of particular impact was a book entitled Our Stolen Future, Are
We Threatening Our Fertility, Intelligence and Survival? A Scientic Detective Story written by Colborn et al. (1996) with a forward penned by then Vice President Al Gore. These and other authors proposed that many EACs elicited effects at doses far lower than toxicities caused by other modes of action (MOAs) and thus required special regulation. Also around the same time were a number of more specic reports alleging associations between endocrine-disrupting chemicals and declining sperm counts (Carlsen et al., 1992; Sharpe and Skakkebaek, 1993), increases in breast cancer (Davis et al., 1993), cryptorchism and hypospadias (Barlow et al., 1999), and developmental abnormalities in wildlife species (Sumpter, 1995). The level of concern spiked even higher following a 1996 report claiming > 1000-fold level of synergy among a mixture of four weakly estrogenic agents as assessed in a yeast estrogen receptor (ER) transcriptional activation assay (Arnold et al., 1996). Ironically, the latter results could not be reproduced by others (Ashby et al., 1997; Ramamoorthy et al., 1997) and a National Institutes of Health inquiry revealed intentional falsication of the results leading to retraction of the original report, which had been published in Science (McLachlan, 1997). Despite uncertainties about the scientic validity of these reports and the true extent of the problem, both the scientic community and public reacted as if this were an approaching tsunami. Media coverage of what would previously have been considered an esoteric science topic became mainstream headline news. Prominent government and scientic organizations such as the World Health Organization, International Union of Pure and Applied Chemistry and the U.S. National Research Council reacted by gathering endocrinologists, toxicologists, and other public health professionals from around the world to capture their knowledge of the state-ofthe-science and to recommend research to address the issue. These efforts are documented in a series of comprehensive review papers (IPCS, 2002; Lintelmann et al., 2003; NRC, 1999), which also helped spur a plethora of research on endocrine-mediated toxicity. The IPCS (2002) report was particularly signicant in that it described a structured weightof-evidence approach to guide the application of new research for addressing causal relationships between exposure to EACs and adverse health outcomes. In fact, application of weight-of-evidence approaches has failed to provide convincing support for many of the early claims that initially sparked the issue, such as alleged links between EACs and declining sperm counts (Fisch, 2008) and breast cancer (summarized at: http://epi.grants.cancer.gov/LIBCSP/ ). Reported effects in aquatic species have pointed more toward natural and synthetic hormones (e.g., birth control pills) as causative agents, more so than industrial chemicals as originally suggested (Jobling et al. 2006). Nonetheless, the term endocrine disruptor has been adopted in the vernacular of toxicologists, public health professionals, and even the general public and appears here to stay.
ENDOCRINE-MEDIATED TOXICITY
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Somewhat unwittingly, the new emphasis on endocrine disruption also marked the beginning of a fundamental change away from the traditional outside-in approach to toxicology, in which adverse outcomes are identied rst followed by mechanistic research to understand these effects. With endocrine disruption came a reversed approach in which identication of mechanism came rst followed by research to dene possible consequences of activating these mechanisms. The implications of this change are quite signicant and are discussed later in this review.
GENESIS OF ENDOCRINE SCREENING AND TESTING
At around the same time that these alarming reports were emerging, the U.S. Safe Drinking Water Act was being updated while the Food Quality Protection Act was being created to enhance protection of children and other vulnerable subpopulations. This temporal convergence led to a 1996 congressional mandate requiring the Environmental Protection Agency (EPA) to implement a screening and testing program to detect endocrine disruptors. Despite the nascent state of the science at the time, Congress gave EPA just 2 years to establish the new program. To assist EPA, an advisory committee known as the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC) was formed to evaluate protocols for approximately 50 different endocrine assays and select a subset to comprise a Tier I screening battery for biological activity involving the estrogen, androgen, and thyroid (EAT) hormone systems. Although limiting screening to these three hormone systems might seem arbitrary, these three appeared to have the broadest range of potential effects as well as the greatest number of ligands with which they interact (Blair et al., 2000). Compounds that can bind steroid hormone receptors include industrial chemicals such as bisphenol A (BPA), certain alkylphenol ethoxylates, phthalates, parabens, benzophenones, and environmental contaminants like DDT, methoxychlor, some PCBs, dioxins and furans. There also are many pharmaceuticals purposely designed to have hormonal activity, such as DES, contraceptive agents, and the selective ERmodulators (SERMs) used in the treatment of diseases such as osteoporosis, as well as numerous plant-derived compounds such as genistein, daidzein, naringenin, genistin, daidzin, glycitein, and puerarin (Boue et al., 2003) and resveratrol, a constituent of red wine (Klinge et al., 2003). Finally, exposure to synthetic and plant-derived estrogens occurs against a background of endogenous estrogens, such as estradiol-17b (E2), estrone (E1), and estriol (E3), the levels of which in blood and tissues can vary dramatically depending on gender, age, and physiological state (e.g., pregnancy, pre-, or postmenopause). Beyond need to cover so many different chemistries and sources, the challenge laid before EDSTAC was further complicated by the desire to screen for both receptor agonists
and antagonists, as well as compounds which act indirectly. Indirect mechanisms are plentiful and include the inhibition of steroid biosynthesis or modulation of the hypothalamicpituitary-gonadal (HPG) axis. An even more complex example begins with activation of the Ah receptor (AhR), which can ultimately oppose estrogen signaling. Ironically, some compounds bearing the very properties that are the targets of screening can actually be benecial. Studies of endocrine-disrupting AhR ligands have led to the development of selective AhR modulators, such as ring-substituted diindolylmethanes (DIMs), for chemotherapy of breast, endometrial, pancreatic, and prostate cancer (Safe et al., 2008). Substituted furans and indoles have been licensed by a pharmaceutical company as antitumor development compounds (Safe, personal communication). Some of the DIMs have been further modied into anticarcinogenic AhR-inactive analogs, which activate orphan nuclear receptors that induce genes associated with growth inhibition and cancer cell death.
EPAS ENDOCRINE DISRUPTOR SCREENING PROGRAM TIER I ASSAYS
In 1998, EDSTAC released its report outlining a screening and testing program to assess the potential of compounds to interact with the endocrine system. EDSTAC recommended that a tiered approach be adopted to examine endocrine activity with a focus on the potential for compounds to interact with the EAT hormone systems. The Tier I assays were designed to screen for potential interaction with these endocrine systems and designed to be an efcient screening process to screen a large number of chemicals for endocrine activity. In Tier I, EDSTAC opted to prioritize assays with greater sensitivity (but possibly lower specicity) to avoid false-negative results. EDSTAC described the importance of using a weight-ofevidence approach to evaluate Tier I screening results. Tier II tests, such as the multigeneration reproductive toxicity study, were to be used to characterize dose-response relationships, whether endocrine-mediated adverse effects for EAT occur, as well as providing data for risk assessment. During this time period, EPA also took on an extensive revision of the multigeneration and some other Tier II tests in order to enhance their sensitivity to endocrine disruptors. The Tier I assays included in vitro, in vivo mammalian, and in vivo nonmammalian screening assays. EDSTAC noted that EACs may alter endocrine function by affecting the availability of a hormone at a target tissue (e.g., synthesis, transport, metabolism, HPG/hypothalamic-pituitary-thyroid [HPT] axis function, etc.) or by affecting the cellular response to a hormone (e.g., receptor binding). The original Tier I battery proposed by EDSTAC (EDSTAC, 1998) included the following: (1) ER binding/transactivation assay, (2) androgen receptor (AR) binding/transactivation assay, (3) steroidogenesis assay with minced testis, (4) rodent 3-day uterotrophic assay (sc route),
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TABLE 1 U.S. EPA EDSP Tier I Assays
In vitro assays ER bindingrat uterine cytosol ER(hERa) transcriptional activationhuman cell line (HeLa-9903) AR bindingrat prostate cytosol Steroidogenesishuman cell line (H295R) Aromatasehuman recombinant microsomes In vivo assays Uterotrophic (rat) Hershberger (rat) Pubertal female (rat) Pubertal male (rat) Amphibian metamorphosis (frog) Fish short-term reproduction Note. http://www.epa.gov/scipoly/oscpendo/pubs/assayvalidation/ tier1battery.htm#assays.
(5) rodent 20-day pubertal female assay with thyroid, (6) rodent 5- to 7-day Hershberger assay, (7) frog metamorphosis assay, and (8) sh gonadal recrudescence assay. In addition, four alternate assays were identied: (1) placental aromatase assay, (2) modied rodent 3-day uterotrophic (ip), (3) rodent 15-day intact adult male assay with thyroid, and (4) rodent 20-day thyroid/pubertal male assay. These alternative assays, if validated, could be used to replace other Tier I assays, namely, either the intact adult male assay or the pubertal male assay would replace the pubertal female and Hershberger assays. Per the Food Quality Protection Act (1996), the endocrine screening program was required to use appropriate validated test systems; consequently, the EPA launched the endocrine disruptor screening program (EDSP) validation program. Based on results of methods development and validation work, some of the proposed Tier I in vitro assays were adopted, whereas others have undergone some modications. The nal list of EPA Tier I EDSP screening assays appears in Table 1. Both the ER binding and ER transactivation assays have been adopted into Tier I. The ER binding assay measures the ability of a radiolabeled ligand ([3H]-17b-estradiol) to bind to rat uterine cytosolic ER in the presence of increasing test material concentrations. A compound is positive for ER binding if it effectively competes with [3H]-estradiol binding in a concentrationrelated manner, and an inhibitory concentration that decreases radioligand binding by 50% (IC50) is obtained. The ER competitive binding assay cannot distinguish agonists from antagonists. Test guidelines also were issued for the Stably Transfected ER Transcriptional Activation assay using HeLa9903 cells (Organization for Economic Co-operation and Development [OECD] TG 455; OPPTS 890.1300), although detection of agonists is all that is required. Although having limited metabolic capability, the ER transactivation assay reportedly has good concordance with in vitro ER binding and in vivo uterotrophic responses for active parent compounds (Takeyoshi, 2006); however, the test guideline criteria for
a positive response (i.e., if a compound achieves a PC10, which is 10% of the positive control response at 1nM 17b-estradiol) may result in numerous false-positive results and jeopardize the predictiveness of this assay. AR binding using rat prostate cytosolic preparations also is included in Tier I (OPPTS 890.1150), although an AR transactivation assay for use in Tier I screening is still being developed. The AR binding assay shares many of the same advantages and limitations as the ER binding assay. To detect compounds affecting steroidogenesis, the minced testis assay was originally proposed. This assay was designed to detect enzyme inhibition in the steroid biosynthesis pathway by measuring decreased testosterone production; however, the assay was removed from consideration because of high falsepositive and false-negative responses and difculty assessing Leydig cell cytotoxicity in the mixed population of cells present in minced testis preparations (Powlin et al., 1998). The steroidogenesis assay was replaced with a cell-based system that uses a human cell line, the H295R cells (Hecker et al., 2006, 2008). This assay is designed to measure production of testosterone and 17b-estradiol by the H295R cells in the presence of varying concentrations of test compound to identify alterations in steroid biosynthesis. Because of the limited metabolic capacity of these cells, the assay primarily examines the ability of the parent material to interfere with steroidogenesis. A positive result occurs if there is a signicant difference in steroid production in the presence of less than 20% cytotoxicity. Aside from steroidogenesis alterations, a separate in vitro assay was developed specically to identify compounds affecting aromatase (CYP19), the enzyme that converts androgens to estrogens. The aromatase assay examines effects of the parent compound on recombinant human aromatase activity by measuring the production of heavy water (3H2O) during the conversion of 3H-androstenedione to estrone. Compounds that competitively inhibit aromatase activity will decrease estrone production and consequently 3H2O levels in a dose-related manner. Interlaboratory validation has been conducted with these Tier I in vitro assays to verify each assays ability to detect known EACs operating via relevant MOAs, although the size of the validation programs (i.e., number of test compounds, number of laboratories, etc.) has differed by assay. One issue with the in vitro EDSP assays is that the assays have limited ability to metabolize substances; thus, if a metabolite is the EAC, it may not be detected by these assays. Options to improve metabolic capacity are under consideration. Although in vitro assays provide useful information on potential MOA and can help clarify in vivo assay results, previous studies have suggested that in vivo data should carry more weight than in vitro data when determining the need for Tier II testing (e.g., Charles et al., 2005). The uterotrophic assay went through an extensive validation program, which was led by the OECD Endocrine Disruptor
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Testing and Assessment (EDTA) Task Force and the Validation Management Group (VMG)Mammalian. This validation process, led by Japanese representatives on the EDTA, included a detailed background document prepared by the OECD (2003) and multiphase intra- and interlaboratory validation studies involving approximately 20 laboratories to show transferability, reproducibility, and reliability. This validation program tested a potent estrogen (ethinyl estradiol), weak estrogen agonists in both a coded (blinded) and doseresponse manner to evaluate sensitivity, and negative compounds to verify specicity. This work culminated in a series of publications (Ashby et al., 2003; Kanno et al., 2001, 2003a,b; Owens and Ko eter, 2003; Owens et al., 2003) and an OECD test guideline in 2007. The U.S. EPA test guideline was released in 2009. The assay uses female rats (or mice) with nonfunctional HPG axis to yield sensitive and consistent increases in uterine weight in response to stimulation by estrogenic materials. Ultimately, the test guideline outlines the application of the uterotrophic assay for the detection of compounds with estrogenic activity; use of this assay to identify antiestrogens is supported by the test guideline, but not required. Overall, the uterotrophic assay appears to perform well for the identication of estrogenic compounds, although the ovariectomized model has been recognized as more specic for identication of estrogenic activity than the immature model. The immature model, which has an intact HPG axis, can detect aromatizable androgens and is more sensitive to body weightmediated changes in uterine weights. The alternate uterotrophic assay, which used ip dosing, was not included in the validation because of potential localized effects of the test compounds on uterine tissue. The Hershberger assay also underwent an extensive multilaboratory validation process, led by U.S. EPA through the OECD EDTA VMGmammalian group (Owens et al., 2006, 2007). The Hershberger assay uses castrated adult male rats with a nonfunctional HPG axis. Although originally proposed with a 5- to 7-day exposure period, rats are treated with the test compound for 10 days by oral gavage or sc dosing in the adopted version of the assay. Increases in accessory sex tissue (AST) weights (i.e., glans penis, ventral prostate, seminal vesicles with uid, Cowpers glands, and levator ani-bulbocavernosus muscles) indicate potential androgenicity. To evaluate antiandrogenic potential, rats are given the test compound (oral gavage or sc) concurrent with 0.2 or 0.4 mg/kg/day testosterone propionate sc. If the test compound inhibits the testosterone-induced increase in AST weights, then the compound has potential antiandrogenic activity or is a potential 5a-reductase inhibitor. Two or more organ weights must be signicantly altered to yield a positive result. Overall, the Hershberger assay appears to perform well for the identication of potential androgens, antiandrogens, and 5a-reductase inhibitors. During the Hershberger validation studies, a weanling (non-castrated) animal model was included;
however, the weanling model did not detect weak antiandrogens as consistently as the castrated adult model (Freyberger and Schladt, 2009) and therefore was not included in either the EPA or the OECD test guideline. Initially, the 20-day female pubertal assay was recommended, and the 20-day male pubertal assay was an alternate EDSP screen. Prior to initiation of the interlaboratory validation work, the exposure period for the pubertal male assay was increased from 20 to 30 days, and ultimately, both assays were adopted into the Tier I EDSP battery. One of the strengths of the male and female pubertal assays is that these assays look for changes in young animals during a dynamic period of endocrine activity. The pubertal assays are the most apical of the Tier I in vivo mammalian assays, but these assays are multimodal, capable of detecting EACs that operate through a variety of MOAs (i.e., strong and weak (anti)estrogenic and (anti)androgenic compounds, steroid biosynthesis inhibitors, aromatase inhibitors, 5a-reductase inhibitors, thyroid-active agents, and compounds affecting the HPG or HPT axis). Some of these MOAs are not detected by other Tier I in vitro or mammalian assays; thus, it may be difcult to dismiss a positive result in the pubertal assays even if there is some suspicion that the primary MOA is not endocrine mediated. One limitation of the pubertal assays is that it may be difcult to determine the MOA for potential EACs. Some endpoints assessed in these assays (puberty onset, estrous cycle, and organ weights) can be altered by chemicals that operate via an endocrine MOA or by nonspecic/systemic toxicity. During the validation program, some compounds were not identied for their primary MOA. For example, phenobarbital, which is a weak thyroid-active compound, did not alter thyroid weights, thyroid histopathology, or serum T4 or thyroid stimulating hormone levels in the male pubertal assay during the validation program but rather showed signs of antiandrogenicity, which included delayed preputial separation and decreased reproductive and AST weights (U.S. EPA, 2007). There remains some question regarding the specicity of the pubertal assays. The Interagency Coordinating Committee on the Validation of Alternative Methods denes specicity as the proportion of inactive substances that are correctly identied. During assay validation, 2-chloronitrobenzene (2-CNB) was selected as a negative control chemical for the male and female pubertal assays; however, 2-CNB caused thyroid effects, delayed vaginal opening, and altered adrenal and uterine weights in females, and delayed preputial separation, decreased serum testosterone levels, decreased androgen-dependent organ weights, and had testis histopathological effects in males. The EPA selected hydroxyatrazine as an alternate negative control compound and recently reported that it was negative in both the male and the female pubertal assays (Stoker and Zorrilla, 2010). Only a subset of these data has been published. Once available, the full data set is likely to provide additional information on setting maximum tolerated dose levels and interpreting pubertal assay results (i.e., provide perspective for
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hydroxyatrazine results reported in Laws et al., 2003). With few negative compounds included in the pubertal assay validation, it is unclear whether the male and female pubertal assays are prone to nonspecic effects, which will produce a high frequency of false-positive results. One alternative assay proposed as part of the EDSP was the 15-day intact adult male rat assay. Approximately 30 EACs, across a variety of MOAs, have been evaluated using the 15-day intact male assay including estrogen agonists/antagonists, androgen agonists/antagonists, steroid biosynthesis inhibitors, thyroid-active compounds, and prolactin modulators. The validation program also examined assay specicity by evaluating the impact of body weight effects on assay endpoints (OConnor et al., 2000) and including a negative control compound, allyl alcohol (OConnor et al., 2008). The intact male assay was specically designed as an MOA screening study, whereby researchers could compare the ngerprint of the organ weight, histopathology, and hormonal endpoints for an unknown compound with data generated for a series of positive controls to help characterize the MOA (OConnor et al., 2002). There was some concern regarding the use of numerous serum hormone measurements in this assay as serum hormone levels are inherently variable. However, data developed during the validation process have shown that serum hormone measurements are reliable with the group sizes employed (n 15 per dose). Of greater concern for the U.S. EPA was the inability of the intact male assay to reliably detect weak antiandrogens (OConnor et al., 1999), which would require that the assay be paired with an alternative assay (e.g., Hershberger assay) for Tier I screening. Because of this weakness, the 15-day intact male assay was not included in the EPA EDSP. In addition to the in vitro and in vivo mammalian assays, two nonmammalian assays also are included in the Tier I EDSP: the amphibian metamorphosis assay and the 21-day sh reproduction screen. The amphibian metamorphosis assay (OPPTS 890.1100) provides an environmental screen to detect compounds that have the potential to interfere with the HPT axis. Xenopus laevis tadpoles are exposed for 21 days starting at Nieuwkoop and Faber stage 51 (Nieuwkoop and Faber, 1994) and examined for mortality, developmental stage, hind limb length, snout-vent length, wet weight, and thyroid histopathology. Because the rate of metamorphic development is controlled by thyroid hormone in X. laevis, developmental stage (which is determined by the inspection of various morphological changes associated with metamorphosis) and hind limb length (which is normalized by snout-vent length) are used to determine if development is accelerated, asynchronous, delayed, or unaffected by the test compound after either 7 or 21 days of exposure. Many of the endpoints assessed in this assay are apical and can be altered by MOAs that are not specic for thyroid effects. Some endpoints (e.g., snout-vent length and wet weight) provide information on whether the test compound alters growth and/or causes systemic toxicity. If any changes in developmental stage are observed
(i.e., advanced, delayed, or asynchronous development), thyroid histopathology is an important endpoint to conrm a specic thyroid effect. To facilitate interpretation, laboratories are encouraged to include positive control compounds to verify assay performance and build historical data. Thus, the amphibian metamorphosis data should be considered in a weight-of-evidence approach to determine potential thyroid activity. Originally, the sh gonadal recrudescence assay was proposed as an EDSP Tier I screening assay. In this assay, sh were examined for compound effects on maturation from a regressed state (recrudescence) in response to an increased photoperiod and temperature regime. Endpoints included secondary sex characteristics, ovary and testis weight increases, gonadosomatic index, gamete maturation, and induction of vitellogenin. However, feasibility studies indicated that there was high variability in the gonadal recrudescence response in continuous spawning species like the fathead minnow (Touart, 2005); thus, the recrudescence assay was not included in the EDSP. Instead, the U.S. EPA has opted for the 21-day sh reproduction screen (OPPTS 890.1350) in the Tier I battery as opposed to the OECD 21-day sh assay (OECD TG 230). This screen uses a minimum of 96 reproductively mature adult fathead minnows (alternate models allowed per OECD) exposed continuously for 21 days to a minimum of three test concentrations and a control. The EPA 21-day sh reproduction screen includes additional parameters not measured in the OECD 21-day sh screen, including fecundity, fertility, gonadal histopathology, and sex steroid concentrations. In addition, adult survival, secondary sex characteristics (such as coloration patterns and tubercle scores), reproductive behavior, vitellogenin, and gonadal-somatic index (GSI) are evaluated. Secondary sex characteristics, vitellogenin, and plasma sex steroids are more predictive of an endocrine MOA, although the high variability seen in plasma sex steroids makes them less robust and a lower priority than vitellogenin when blood plasma quantities are limited. In contrast, fecundity is an indicator of general reproductive condition and not endocrine specic. Thus, evaluations in the sh reproduction screen are a combination of endocrine-specic endpoints and apical endpoints that can be altered by non-endocrine MOAs. With these apical endpoints and the variability in some of the observations, weight of the evidence will be employed in determining the potential of the compound to interact with the endocrine system of fathead minnow. Both environmental screens must manage issues related to the physicochemical properties of various test materials, the need to develop a solvent-free test system (if possible), development of analytical methods for dose verication, and the need to generate range-nding data to assist in dose selection. Appropriate dose setting is critical to differentiate systemic toxicity from genuine endocrine-mediated effects. In 2009, the U.S. EPA released its rst list of priority compounds for endocrine screening. The initial priority list
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consisted of 67 chemicals, primarily pesticide-active ingredients (58) and some pesticide inerts (9). For pesticide-active ingredients, substantial hazard data are available because of registration requirements. As part of the EPA EDSP program, companies were allowed to submit other scientically relevant information (OSRI), describing available toxicity data for a compound and known health risks with an emphasis on endocrine-related effects. OSRI information for many of the priority compounds is currently under review by the EPA; however, it is not clear what information will be viewed as functionally equivalent to the Tier I assays. If functionally equivalent data are available for a compound, companies may be allowed to omit one or more of the Tier I EDSP assays based on these existing data. The goal of the Tier I EDSP is to identify substances that have the potential to interact with the estrogen, androgen, or thyroid hormone system for further testing in Tier II. Given that there are apical endpoints in Tier I assays that may be altered by nonspecic effects and there is some redundancy to detect endocrine MOAs across different Tier I assays, it is benecial to use a weight-ofevidence approach to determine whether there is sufcient data to warrant Tier II testing (Fig. 1). Previous toxicity data generated with standardized test guidelines and/or validated methods should be included in this weight-of-evidence analysis. One example of applying weight of evidence can be illustrated with methoxychlor, an estrogenic compound included in validation studies for several of the Tier I EDSP screening assays. It is recognized that methoxychlor is metabolized in vivo to an estrogenic metabolite, 2,2-bis (p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE). Thus, methoxychlor was not included as part of the estrogen binding assay validation work; however, other binding assays using competitive binding to uterine cytosol ERs were negative for methoxychlor (e.g., Shelby et al., 1996). Methoxychlor was included in the ER transactivation assay validation, wherein its estimated EC50 was 1 3 105M. The authors concluded that methoxychlor exhibited a response curve that was consistent with other weak estrogens. Methoxychlor was positive in some AR binding assays (Charles et al., 2005), but it was negative in the aromatase assay. In vivo, methoxychlor was positive in the uterotrophic assay (Kanno et al., 2003a,b), although it was more readily detected with oral exposure than sc injection and sc injection is the preferred route for administration in the EPA uterotrophic test guideline. Methoxychlor was negative in the Hershberger assay at doses that caused a signicant increase in liver weights (Charles et al., 2005) and positive in both the male and the female pubertal assays. The estrogenic properties of methoxychlor also were identied in the sh reproduction assay. There were no data on methoxychlors effects in the steroidogenesis or amphibian metamorphosis assays. Based on a weight-of-evidence approach, methoxychlor would be identied as an estrogenic compound with possibly a mixed mode of action that affects androgen-dependent endpoints. Except where indicated by other citations, results of these
FIG. 1. This diagram illustrates some of the redundancy in Tier I EDSP assays. In a weight-of-evidence approach, a pattern of effects across related assays is easily interpreted (e.g., positive ER binding, ER transactivation, and uterotrophic assays coupled with estrogenic responses in the pubertal and sh short-term reproduction assays). However, in many cases, the pattern will not be as easily discerned. If results of in vitro and in vivo assays disagree, weight of evidence requires careful consideration of the in vivo results and previous toxicity data when determining whether the compound is a potential EAC and warrants Tier II testing. Aside from the expected MOA relationships depicted in this gure, other, more complex MOA relationships are not shown (e.g., the ability to detect agents with mixed MOAs like methoxychlor or HPG axis compounds like atrazine).
validation studies can be located online (http://www.epa.gov/ scipoly/oscpendo/). Based on a weight-of-evidence approach, compounds deemed positive in Tier I will undergo Tier II testing to characterize hazards and develop data for risk assessment. The specic tests that will be included in Tier II have not been denitively identied. Possible tests under consideration for inclusion in Tier II include the two-generation mammalian (rat) reproductive toxicity study, the two-generation amphibian (frog) reproductive toxicity study, the two-generation avian reproductive toxicity study, sh life cycle toxicity study, and the invertebrate (mysid) life cycle toxicity study.
LOW-DOSE ISSUE AND MIXTURES
Concern over endocrine disruptors has been further heightened because of reports that such compounds can elicit unique low-dose effects and exhibit nonmonotonic doseresponse relationships that may not be detected in regulatory toxicity studies, which are typically conducted at higher dose levels. The low-dose hypothesis has been a contentious issue
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among some toxicologists. The National Toxicology Program (NTP), with support from the U.S. EPA, organized a panel to evaluate the scientic evidence on the low-dose effects of endocrine disruptors (Melnick et al., 2002), dening low-dose effects as biologic changes that occur in the range of human exposures or at doses lower than those typically used in the standard testing paradigm of the U.S. EPA for evaluating reproductive and developmental toxicity. The panel concluded that (1) there was evidence for low-dose effects in laboratory animals, although in some cases, these effects have not been replicated; (2) the shape of the dose-response curve depended on the endpoint and dosing regimen used; and (3) the current reproductive/developmental toxicity testing paradigm should be reviewed and possibly revised with respect to dose selection, animal models used, ages evaluated, and endpoints measured. Some reasons identied for the inability to reproduce low-dose effects across studies included intrauterine position, strain and substrain differences in response, dietary levels of phytoestrogens or differences in caloric intake, differences in housing conditions (caging, bedding, etc.), and seasonal variation. The statistics subgroup (Haseman et al., 2001) also identied a number of important factors that should be considered in the design, analysis, and interpretation of experimental studies to help resolve low-dose issues, including study sensitivity (power), reproducibility, control for litter effect, quality control in data collection and management, procedures for data analysis, interpretation of the signicance of biological changes, and data selectivity. Ultimately, the U.S. EPA concluded, Until there is an improved scientic understanding of the low-dose hypothesis, EPA believes that it would be premature to require routine testing of substances for low-dose effects in the Endocrine Disruptor Screening Program (U.S. EPA, 2002). Following the NTP low-dose report, a number of key studies by Naciff et al. (2002, 2009, 2010) have examined changes in expression of up to 38,500 genes in various cell lines or uterine tissues exposed to very low doses of EACs, including BPA at concentrations down to 1nM. These studies collectively provide support for monotonic dose-responses for these compounds and showed negligible changes in gene expression at very low-dose levels. Furthermore, several comprehensive weight-of-evidence reviews for BPA have concluded that nonmonotonic dose-response curves and lowdose effects from BPA are not well supported by available data (Goodman et al., 2009; Kamrin, 2004, 2007). The Center for the Evaluation of Risks to Human Reproduction (Chapin et al., 2008) cited many of the issues related to using low-dose study data when evaluating BPA. Despite these reports, the Endocrine Society issued a scientic statement on endocrinedisrupting chemicals that included support of the low-dose hypothesis (Diamanti-Kandarakis et al., 2009); thus, the debate continues. The low-dose controversy has evolved to consider issues related to exposures to mixtures of EACs at low, environmentally
relevant dose levels. Although risk assessment approaches for cumulative and aggregate risk have been in place for many years (reviewed in Lipscomb et al., 2010), interest in cumulative exposures to multiple EACs has received increased attention in recent years (Carney et al., 2010; Hotchkiss et al., 2008; Kortenkamp, 2008). Mixtures have been reported to have additive effects (e.g., Gennings et al., 2004), less than additive effects (e.g., Charles et al., 2007), greater than additive effects (e.g., Laetz et al., 2009), or even counteracting effects (e.g., Tanida et al., 2009). Overall, data indicate that the interactions of chemicals in mixtures are dose dependent. Using mixture data in the context of environmental risk assessment is even more complex when one considers natural exposures to EACs in food and pharmaceuticals (Gaido et al., 1998; Safe et al., 1997). Exposures to environmental chemicals typically involve low doses of each chemical within a mixture and, given the safety factors used in risk assessment, exposures generally below the threshold for toxicity. Thus, mixtures research requires a focus on the behavior of mixtures at environmentally relevant exposures because results obtained at high-dose levels may not apply in real-world scenarios. This concept also was espoused by the Expert Working Group on Mixtures Toxicology, which was convened by the Society of Toxicology and the Society for Environmental Toxicology and Chemistry (Teuschler et al., 2002).
HISTORICAL DEVELOPMENTS IN THE BASIC SCIENCE OF ENDOCRINE SIGNALING
When the history of endocrinology is written, it is likely that the 20th century will be neatly divided into two epochs with the rst half being the era of biochemistry when the hormones were discovered and the emphasis was on proteins and the second half of the 20th century, the era of genomics when the genes for the hormone and nuclear receptors (NRs) were identied. The word hormone was rst coined in 1905, but it was not until 1926 that the rst hormones were characterized in detail when Kendeall and Reichstein isolated and determined the structures of cortisone and thyroxine (The Nobel Assembly at Karolinska Institutet, 1950). Soon thereafter, in 1929, Adolf Butenandt and Edward Adelbert Doisy independently isolated and determined the structure of estrogen (Tata, 2005). These discoveries along with other experimental observations allowed Clark to propose his model for receptor-mediated processes in his publication of the theory of occupancy. The receptor theory has been rened since the 1930s along with the vocabulary used to classify chemicals that bind to NRs or ligands, which are now classied into agonist, antagonist, partial agonist, and inverse agonist based on the biological effects elicited through NRs (Table 2). Potential EACs may produce their effects through interactions with NRs. Under normal physiological conditions, the intracellular NRs are bound by their endogenous signaling molecules, or receptor ligands, such as the steroid hormones,
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TABLE 2 Properties of Chemicals that Affect NRs

Ligand Afnity Agonists Antagonists Chemical that binds reversibly to an NR A ligands capacity to bind to an NR Ligand that produce an NR conformation that is transcriptionally active Ligands that prevent agonist binding or induce an NR conformation resulting in an action distinct from that produced by an agonist Ligands that possess weak agonist activity Ligands that induce an NR conformation that results in transcriptional repression A measure of the capacity of the ligand to activate, prevent activation, or inactivate an NR A measure of the concentration or amount of ligand needed to produce an effect through an NR
Partial agonists Inverse agonists Efcacy Potency
thyroid hormones, retinoids, vitamin D, and other as yet identied endogenous ligands. These receptor ligands either diffuse directly across the plasma membrane of cells or are sometimes produced in the same cells and bind to the NR. The commonly understood mechanism of action for NRs involves the initial ligand binding that results in receptor activation, binding at specic DNA response elements in nuclear chromatin, followed by recruitment of a variety of cofactors that modify the chromosomal organization and interaction with the transcription complex, thereby altering the rates of transcription of genes. So, binding of a ligand to an NR is only the rst step in a complex cascade of events leading to changes in gene expression and subsequent cell signaling. With the completion of the human genome, it is now clear that there are some 48 NRs expressed in humans, of which only half have identied ligands such as steroid hormone receptors that constitute only a minority of this large superfamily of receptors (Gronemeyer et al., 2004). Like the NR superfamily, coregulators are themselves a large and mixed family of proteins and possess a varied array of enzymatic activities necessary for chromosomal maintenance and gene regulation (Roeder, 2005). They can be categorized based upon their wide-ranging functional properties including acetyltransferases, ubiquitin ligases, ATP-coupled chromatin remodeling complexes, protein methylases, cell cycle regulators, RNA helicases, and bridging proteins that facilitate direct contact with components of the basal transcription machinery (McKenna and OMalley, 2003). There are currently 285 coregulators identied and many are expressed differentially by cell type, tissue type, gender, developmental stage, and species (Lonard et al., 2007; Smith and OMalley, 2004). Moreover, coregulators can be differentially recruited to the same receptor by ligand-induced conformational changes in the NR. Thus, with 48 NRs and 285 plus coregulators, the complexity of these endocrine signaling pathways is apparent. It is important to note that all these concepts of agonists, antagonists, partial agonists/antagonists, or inverse agonists are
conditional and specic to a given gene, in a specic cell and tissue under specic conditions. For example, a given ligand may be an agonist for a gene in a cell and at the same time be an antagonist for a different gene in the same cell; or a ligand can be an agonist for a gene in one cell type and an antagonist for the same gene in a different cell type or even the same cell type but under different conditions. Potency is a conditional measure that depends on parameters of afnity and efcacy, as well as tissue receptor numbers and ligand bioavailability. Thus, the intrinsic efcacy of an EAC, whether agonist, antagonist, partial agonist/antagonist, or inverse agonist for a given NR, is dependent upon both chemical properties of the EAC and the biological environment of the NR (Jordan, 2003). These differences in the properties of ligands are often lost in the discussion of EACs. An EACs potency is dependent on its afnity for an NR, and some putative EACs have ER binding afnities that are 10,000100,000 lower than ER binding afnities of potent, natural, or contraceptive estrogens (Gutendorf and Westendorf, 2001). For example, the afnity of BPA for ERs is approximately 10,000-fold weaker than that of estradiol (Kuiper et al., 1998). What this means in practical terms is that a cell would need to be exposed to 10,000 molecules of BPA to produce the same effect through the ER that one molecule of estradiol would produce through that same receptor. Furthermore, the amount of BPA that one would need to consume, breath, or absorb through the skin would be even greater because the absorption, distribution metabolism, and elimination of the BPA would limit the amount of BPA available to the cells. Even if an EAC was highly potent in one cell type, it does not mean it will have the same efcacy in another cell type or the same cell type under different conditions as efcacy is independent of ligand afnity and instead reects a ligands ability to induce a stable coactivator binding site on the NR (discussed further below). Thus, two ligands can have similar potencies for inducing a response through an NR, but the efcacy for these two ligands inducing the response can vary greatly and this can have a profound impact on the ability of these ligands to produce the same apical effects through the NR. The lower potencies and different efcacies of EACs relative to the natural hormones need to be understood and considered in the discussions of the potential impact of EACs on human health and the environment. It took another three decades after the elucidation of the structure of estrogen before the ER was isolated in 1961 by Elwood Jensen (Tata, 2005) and another two decades passed before the new tools of molecular biology allowed Pierre Chambons group to clone the gene for the human ER (Green et al., 1986). The 1980s ushered in an era of rapid discovery of the receptor genes for most of the major hormones, which was followed in the 1990s by the cloning of novel nuclear orphan receptors for which there are no known ligands. It was also ke Gustaffsons group during this period, in 1996, that Jan-A cloned a second ER, now called ERb (Kuiper et al., 1996), requiring the observations of the previous decades of estrogen
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studies to be viewed through a new lens. No doubt there will be additional chemicals identied as ligands for some of the 48 NRs identied in humans, thereby necessitating an understanding of their potential for behaving as EACs.
ESTROGENS: FACTORS AFFECTING AFFINITY, POTENCY, AND EFFICACY
One of the best understood examples of an endocrine signaling system that brings all these concepts together is that of estrogens. A half-century has passed since the initial reports of a cognate ER, and it is now known that ERa and ERb exist, as well as less well-understood additional subtypes. The distribution of these two receptors is variable, with tissues such as bone having predominantly the b isoform, uterus containing mainly a, but many tissues expressing both a and b. Furthermore, a and b often regulate opposing functions providing a feedback loop for nite control of estrogen signaling. The biological effects elicited by specic ER ligands are highly cell and tissue specic. One of the best known examples of this phenomenon is the drug tamoxifen, which has been used for treatment of early stage breast cancer and as a chemoprevention agent for women at high risk for this disease (Jordan, 2003). Tamoxifen is an ER antagonist in breast cancer but an ER agonist in the bone and uterus (Fong et al., 2007). Another example is the phytoestrogen resveratrol, which shows more activity in cells that uniquely express ERb or that express higher levels of ERb than ERa and also appears to exhibit estrogen antagonist activity for ERa in certain promoter contexts (Bowers et al., 2000). In addition, only a small percentage of ER ligands are pure estrogen agonists or antagonists. Most phytoestrogens and xenoestrogens are partial agonists, meaning that they are unable to induce a maximal response matching that of the endogenous ligand, even at very high concentrations (Zhu, 2005). The latter compounds tend to act as agonists under some conditions but act as antagonists when their concentration is very high relative to more potent ligands. The research from numerous laboratories over the last ve decades has revealed the explanations for many of these observations. Differences in the tissue-specic receptor agonist and antagonist activities have long been recognized for the steroid hormone receptors and form the basis for the development of drugs for treatment of hormone-dependent diseases (Ai et al., 2009; Nettles and Greene, 2005). Tamoxifens tissue-specic activity is now thought to be because of recruitment of different coregulators to the ER by tamoxifen in breast tissue versus uterine and bone tissue (Gronemeyer et al., 2004). Tamoxifen was the rst of what are now called selective ER modulators or SERMs where the cellular response to SERMs is determined by cell type and promoter-specic differences in coregulator recruitment by the ER. Thus, coregulators extend the actions of the same hormone receptor by producing cell typespecic
FIG. 2. The actions of nuclear receptors are ligand dependent and cell context dependent. Nuclear receptors (R) form a different tertiary structure depending on the ligand that activates them (EAC-1 or EAC-2). This in turn causes the receptors to recruit different coregulator complexes, e.g., coactivator complexes or corepressor complexes that lead to gene induction or repression, respectively. In Cell A, the two different ligands (EAC-1 and EAC-2) bind to the same receptor but induce two different tertiary structures, which in turn cause the receptor to recruit a coactivator complex in the case of EAC-1 bound receptor or a corepressor complex in the case of EAC-2 bound receptor. Whereas in Cell B, the opposite occurs with EAC-1 producing a corepressor complex recruitment through the receptor, but EAC-2 produces a coactivator complex recruitment through the receptor. Thus, an individual EAC can act as an agonist, antagonist, partial agonist/antagonist, or inverse agonist depending on the coregulators present in the specic cell. Although not shown, the gene architecture inuences nuclear receptor-coregulator recruitment as well. Coactivator (coactivator complex); corepressor (corepressor complex); TBP, A, B, F, pol II (RNA polymerase II holoenzyme: RNA polymerase II and general transcription factors); TATA (TATA box promoter site); transcription (RNA transcript).
positive or negative stimuli from the same hormone ligand or different receptor ligands depending on the cell-specic coregulator availability and/or ligand-specic induced receptor conformations with differential coregulator afnities (Fig. 2). Selective receptor modulators (SRMs) have been observed for other NRs including peroxisome proliferatoractivated receptors, ARs, progesterone receptors, as well as the AhR (Gronemeyer et al., 2004; Zhang et al., 2008). Thus, SRM appears to be a common feature of ligand-activated transcription factors that explains the observed selectivity that different ligands confer to NRs on modulation of gene expression and the resultant tissue changes both within and across tissue and cell types (Ai et al., 2009; Zhang et al., 2008). No doubt, EACs will share similar characteristics with SRMs. All the discoveries discussed above and more have been harnessed to develop in vitro and in vivo assays to identify chemicals with endocrine-modulating activities. The very
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nature of an NR being a trans-acting transcriptional regulator allows for their exogenous expression in numerous cell types and cell lines to study the effects of potential ligands as agonists, antagonists, etc. These studies have led to the adoption of some assays into the EDSP battery of tests and many others that are used in the ToxCast panel of assays. The cloning of these receptors has also facilitated the development of NR knockout (KO) mouse models that provided important conrmatory evidence for the physiological role of hormones in development, reproduction, and normal physiology as well as some unexpected observations. With the advent of the KO models, it was possible to develop transgenic receptor knock in mouse models that express human NRs in place of their endogenous mouse receptor. The development of the ER KO mouse (ERKO mouse) by the National Institute of Environmental Health Sciences group led by Ken Korach demonstrated that both sexes of these mutant animals are infertile and show a variety of phenotypic changes, some of which are associated with the gonads, mammary glands, reproductive tracts, and skeletal tissues (Lubahn et al., 1993). The ERKO mice and other hormone receptor KO mouse models can be used to conrm the endocrine effects of putative EACs and demonstrate important differences in the effects produced by endocrine chemicals compared with endogenous hormones. By knocking in the human gene for an NR into the appropriate receptor KO mouse, new transgenic mouse models are made that allow comparison of putative EACs acting through the human receptor or mouse receptor. The observations from such studies are important to consider in the weight of evidence for predicting the effects of EACs to humans. Future mouse models are being developed that express the human proteins for multiple NRs and accessory proteins, and these will allow further renement of the effects of EACs on human pathways. In addition, the technology is now available to make KO rats and other species, and this will be followed by the ability to humanize these models as well, thereby furthering our ability to understand the human relevance for EACs identied in wildtype laboratory models and in vitro assays.
FUTURE DIRECTIONS IN ENDOCRINE DISRUPTOR SCREENING AND TESTING
Endocrinology has come far since the term hormones were rst introduced in 1905. What started as a simple concept has evolved into a very complex array of hormones, receptors, and cofactors. Because a chemical ligand for an NR can have multiple intrinsic efcacies (i.e., induce several NR responses) depending upon the cellular and tissue environment of the NR, relying upon in vitro cell and biochemical assays to dene EACs has important implications. With suspect EACs possessing the potential for multiple induced NR responses, the validity for transferring inferences about the efcacy of the EAC-induced
effect obtained for one response to another response is questionable. Moreover, the problem of confounding is even more problematic when considering that measured parameters of in vitro high-throughput screening systems are by nature different from those found for receptor systems in vivo. Therefore, the results from these in vitro screening systems may seldom accurately reect the intrinsic efcacies of NR ligands in vivo and challenge the validity for extrapolating observations to humans. Even if a single in vitro system was reective of a human response under certain conditions, the fact that the intrinsic efcacy of an EAC is dependent upon the cellular context of the NR means that multiple systems and or conditions would need to be employed to ensure that all the possible conditions in vitro reect similar conditions in humans in vivo. Although this review only touched briey on some of the key developments to date concerning endocrine-mediated toxicity and safety assessment, it should be readily apparent that the issue has raised more questions and challenges than have been answered. Most likely, we are only at the very beginning of a long journey toward improved understanding of the basic mechanisms of endocrine-mediated toxicity and the extent to which relevant exposures to hormonally active agents impact human and environmental health. As we forge ahead, this active and often controversial topic has already accelerated the science of toxicology and risk assessment and has redirected it into some fundamentally different directions. One such direction is down. This is not meant in a negative sense but refers to the examination of much lower, environmentally relevant dose levels than have been examined in the traditional high-dose toxicology paradigm. This new direction was spurred by research, albeit controversial, that hormonally active agents might not adhere to the monotonic dose-response behavior, which has been a fundamental principle of toxicology since its inception as a eld of science. However, further progress in this area is possible thanks to modern technology, such as toxicogenomics, which allows one to examine for precursor changes on the cellular and molecular level (Naciff et al., 2009) and thus help to better understand the fundamental biology underlying dose-response for environmentally relevant exposures. In addition, the increased utilization of human, rather than animal, cells holds promise for enhancing human relevance even further. Another research direction points upward, namely, toward the investigation of higher numbers of chemicals and other stressors and their potential cumulative effects. For years, toxicologists have tended to avoid studying cumulative effects because it was considered an intractable problem. This was especially so for hormonally active agents because of the vast array of ligands and multiplicity of sources, which includes not only man-made chemicals but also compounds in plants, endogenous hormones, and even nonchemical stressors. However, because of new high-throughput tools, rened statistical methods, and economical experimental designs for mixture studies (Gennings, 1996), questions about combined
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exposures to hormonally active agents can be addressed in a scientically rigorous and efcient manner. This movement toward evaluating more chemicals is also being combined with a fundamental change toward mechanismbased testing (inside out) versus the traditional adverse effectsbased system (outside in) as described in the landmark report by the U.S. NRC Committee on Toxicity Testing and 1365 Assessment of Environmental Agents, Board of Environmental Studies and Toxicology, Institute for Laboratory Animal Research (2007) report entitled Toxicity Testing in the 21st Century: A Vision and a Strategy. Within this report, endocrine screening is highlighted as an area of toxicological concern that could benet from the use of molecular-based screening methods. Progress has been made toward implementing this approach. EPAs ToxCast program contains numerous endocrine-related screening assays, including ER and AR receptor binding assays, ER, AR, and thyroid receptor reporter gene assays, an assay for thyrotropin-releasing hormone receptor, and an aromatase enzymatic activity assay (Houck, 2009). These assays are conducted in high-throughput mode, so results are generated quickly. However, there are concerns about relying on these in vitro and alternative assays for regulatory decisions in determining potential endocrine activity (e.g., Bus and Becker, 2009) because of the lack of toxicokinetic considerations (absorption, distribution, metabolism, and excretion), the imperfect concordance of ToxCast assays with in vivo data, and achieving agreement on a mechanismbased denition of adverse. In the short run, these data may contribute to the weight of evidence for some test compounds in determining potential endocrine activity. Furthermore, if the vision of Toxicology in the 21st Century is realized, such an approach could prove to be far more efcient and cost effective than the existing EDSP assays, some of which are resource intensive and use large numbers of animals to screen for a limited number of MOAs. Finally, future research in this area needs to look outward, meaning that information on specic components of the endocrine system (e.g., specic cell culture responses) needs to be interpreted in the context of whole-body physiology, preferably that of humans. In the case of environmental species effects, integrative frameworks such as the adverse outcomes pathway concept have recently been developed to address links between events at the molecular level all the way up through adverse outcomes at the population level (Ankley et al., 2010). Because the endocrine system integrates responses across distant cells, tissues, and organs, a reductionist approach that solely focuses on isolated components of an endocrine circuit can generate incomplete answers and may even guide public health professionals in the wrong direction. In this same vein, basic principles of toxicology and in particular, toxicokinetics, should not be forgotten and need to be part of any research program if it is to be meaningful for risk assessment. For example, recent research on the toxicokinetics of BPA and genistein has
highlighted the tremendous impact of hepatic rst pass metabolism in converting these compounds to their biologically inactive glucuronide forms (Teeguarden and Barton, 2004; Teeguarden et al., 2005). Therefore, route of exposure can have a dramatic effect on the percentage of free, biologically active compound and consequently the effects produced. The key point is that as we delve deeper into the details of individual mechanisms, it is essential that these new ndings be interpreted in a holistic, physiological framework. Fortunately, new tools in exposure modeling, physiologically based pharmacokinetic modeling, and systems biology are increasingly available to assist with experimental design, data integration, and data interpretation to achieve this goal.
CONCLUSION
Concerns about EACs have come to the forefront of toxicology over the past 15 years. The precise impact of EACs is difcult to discern as EACs can be benecial or adverse, producing different effects in different tissues at different life stages. To complicate this issue further, exposure to low-level environmental EACs occurs in an environment in which natural EACs (e.g., phytoestrogens) and/or exposures to pharmaceuticals contribute to the background hormonal mileau. The focus on endocrine disruption represents a new paradigm in toxicology, wherein the MOA, rather than toxicological outcome, is the focus of chemical screening. Development of the EDSP has been more complex than originally envisioned, and the difculty in identifying assays, efciently conducting those assays, and data interpretation have become apparent. As endocrine issues have increased in scope, increased research has led to a better understanding of receptor biology. To take full advantage of this new knowledge and efciently meet the needs to screen compounds for endocrine activity, the EDSP should be iterative and allow for reevaluation of earlier assumptions based on the state of the science. Clearly, the past 15 years have demonstrated that legislative mandates for specic types of toxicity testing that precede scientic understanding are difcult to implement and limit the latitude to adapt to evolving science. Even so, the signicant resources dedicated to developing and validating the panel of EDSP assays have led to an improved understanding of the challenges involved with implementing and interpreting EDSP assays, and these lessons will be benecial to eventual implementation of the EPAs ToxCast assays and human primary cell assays as proposed in the National Research Council report Toxicity Testing in the 21st Century.
ACKNOWLEDGMENTS
The authors would like to thank Drs James Bus, Matt Lebaron, and Katie Coady for their thoughtful reviews of the manuscript and Dr David Geter for contributing Figure 1.
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TOXICOLOGICAL SCIENCES 120(S1), S93S108 (2011) doi:10.

1093/toxsci/kfq329 Advance Access publication October 25, 2010

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EARLY HISTORY OF TOXICITY TESTING FOR ENDOCRINE-MEDIATED EFFECTS

MARTY, CARNEY, AND ROWLANDS

THE BIRTH OF ENDOCRINE DISRUPTORS

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GENESIS OF ENDOCRINE SCREENING AND TESTING

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EPAS ENDOCRINE DISRUPTOR SCREENING PROGRAM TIER I ASSAYS

MARTY, CARNEY, AND ROWLANDS

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MARTY, CARNEY, AND ROWLANDS

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LOW-DOSE ISSUE AND MIXTURES

MARTY, CARNEY, AND ROWLANDS

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HISTORICAL DEVELOPMENTS IN THE BASIC SCIENCE OF ENDOCRINE SIGNALING

TABLE 2 Properties of Chemicals that Affect NRs

Partial agonists Inverse agonists Efcacy Potency

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MARTY, CARNEY, AND ROWLANDS

ESTROGENS: FACTORS AFFECTING AFFINITY, POTENCY, AND EFFICACY

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Downloaded from http://toxsci.oxfordjournals.org/ at University of Nairobi on November 16, 2013

Downloaded from http://toxsci.oxfordjournals.org/ at University of Nairobi on November 16, 2013

Downloaded from http://toxsci.oxfordjournals.org/ at University of Nairobi on November 16, 2013

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