Reviews in Environmental Health, 1998
Environmental Health Perspectives 106, Supplement 1, February 1998

[Citation in PubMed] [Related Articles]

Environmental Endocrine Disruption: An Effects Assessment and Analysis*

Thomas M. Crisp,^1,2 Eric D. Clegg,^1,2 Ralph L. Cooper,² William P. Wood,² David G. Anderson,³ Karl P. Baetcke,³ Jennifer L. Hoffmann,³ Melba S. Morrow,³ Donald J. Rodier,³ John E. Schaeffer,³ Leslie W. Touart,³ Maurice G. Zeeman,³ and Yogendra M. Patel⁴

Part 3: Introduction

This document provides an overview of the current state of the science relative to environmental endocrine disruption. Particular attention is paid to peer-reviewed published reports of adverse health and ecological effects attributed to specific environmental agents and to information in the Agency's pesticide registration and toxic substances databases. The document identifies gaps in our understanding of mechanisms of action for agents that disrupt the endocrine and endocrine-supported systems. It analyzes and interprets current hypotheses and specifies some of the uncertainties in our knowledge. Finally, some general research needs are recommended. This overview is not intended to address all components of the endocrine system that might be disrupted by environmental insult. Rather, it emphasizes those reports of adverse human and ecological reproductive, carcinogenic, neural, and immune effects in which a common theme of endocrine disruption has been hypothesized.

General Background

Investigators began expressing their concern for estrogenic effects of environmental xenobiotic chemicals more than 25 years ago (3-9). Within the past 5 years, this concern has become focused and intensified (1,2,10-14). Attention has been called to the potential hazards that some chemicals may pose for human health and ecological well-being (breast and reproductive tract cancers, reduced male fertility, abnormality in sexual development, etc.) (11,15-22). There has been considerable controversy over the report (23) that human sperm counts have decreased over the past 50 years.

Clear evidence exists that in utero exposure to certain potent synthetic estrogens such as DES has an adverse reproductive effect in the sons (24) and daughters of women treated with DES during their pregnancies and that a rare adenocarcinoma of the vagina was seen some 20 years later in the daughters (25). In female rats of the AEI strain, which has a low incidence of spontaneous mammary tumors, both prenatal and postnatal exposure to DES increased the numbers of mammary tumors (26). Male rats treated from gestational day 14 to postnatal day 3 with the antiandrogenic fungicide vinclozolin exhibited varied reproductive dysfunction as adults (27).

Caged male rainbow trout exposed to effluent from 15 different sewage treatment facilities in the United Kingdom expressed elevated concentrations of vitellogenin, an estrogen-induced yolk protein precursor (12). Furthermore, there is ample evidence that the pesticide DDT, now banned in this country, and its metabolites cause a dwindling bird population due to testicular feminization of male embryos leading to abnormal sex ratios of adult Western gulls in Southern California in the 1960s (28,29). More recently, declines in birthrate and increasing male reproductive tract anomalies among alligators in Florida's Lake Apopka have been reported (30).

For the past 25 years, the U.S. EPA has been committed to the protection of human health and the environment and has ongoing research programs in these areas. The Agency has followed closely the recent reports dealing with environmental endocrine disruptors on human health and ecological well-being. The U.S. EPA is particularly concerned with the possible role that xenobiotics, including endocrine disruptors, may have in the etiology of human cancers and adverse developmental, reproductive, immune, and neurological effects on human health. The Agency also is concerned with what possible adverse role these endocrine disruptors may have on growth and survival of animal species. Evidence for this concern is documented by ongoing research of the Office of Research and Development (ORD), a Risk Assessment Forum colloquium on environmental hormones held in April 1994, and two endocrine disruptor research needs workshops held in April and June 1995. Two reports titled Research Needs for the Assessment of Health and Environmental Effects of Endocrine Disruptors: A Report of the U.S. EPA-Sponsored Workshop (1) and Developing of a Research Strategy for Assessing the Ecological Risk of Endocrine Disruptors (2) have resulted from these meetings. In addition, an ORD Research Plan for Endocrine Disruptors has been written. Other Agency initiatives include a workshop on Leydig cell hyperplasia in the fall of 1995 (31), the Office of Prevention, Pesticides, and Toxic Substances' revision of the developmental and two-generation reproductive toxicity test guidelines (for mammalian species), the U.S. EPA guidelines for reproductive toxicity risk assessment, the dioxin risk assessment document, the draft proposed guidelines for ecological risk assessment, and the new proposed carcinogenesis risk assessment guidelines.

The Agency is aware of three recent reports (two of them published) by European governments (United Kingdom, Denmark, and Germany) dealing with environmental endocrine disruption (32,33). An extensive exploration of environmental endocrine disruption is the subject of an NAS project supported by the U.S. EPA, the Centers for Disease Control and Prevention, and the Department of the Interior (34).

Hormones

Hormones are natural secretory products of endocrine glands and travel via the bloodstream to exert their effects at distant target tissues or organs. Chemically, hormones are glycoproteins, polypeptides, peptides, steroids, modified amino acids, catecholamines, prostaglandins, and retinoic acid. They are transported in blood at very low concentrations (ng or pg/ml, i.e., 10^-9 or 10^-12 g/ml) in the free state or attached to carrier proteins. They bind to specific cell surfaces or nuclear receptors and exert important regulatory, growth, or homeostatic effects. Steroid and thyroid hormones, bound to their protein receptors, regulate gene activity (expression) as DNA transcription factors; protein and peptide hormones function by transmitting a signal (intracellular second messenger) to regulate ion channels or enzymes. Some of the major endocrine glands include the hypothalamus, pituitary, thyroid, parathyroid, pancreas, adrenal, ovary, and testis. Other endocrine tissues include the placenta, liver, kidney, and cells throughout the gastrointestinal tract. The secreted hormones help regulate general body growth and metabolism, other endocrine organs, and reproductive function. Some target organs and tissues under endocrine control include the mammary glands, bone, muscle, the nervous system, and the male and female reproductive organs.

In addition to the classical hormones found in higher vertebrates, including humans, there are hormones in invertebrates (e.g., ecdysone) and plants (e.g., auxins). Consequently, when environmental endocrine disruptors mimic or interfere with the action of endogenous hormones, they have the potential of influencing human health and exerting significant ecological effects globally.

Paracrine (secretions stimulating adjacent tissues) and autocrine (secretions targeted to the cell that synthesized the secretion) factors will not be considered in this paper because little information is available about their disruption by environmental agents.

Endocrine/Hormone Disruptors

An environmental endocrine or hormone disruptor may be defined as an exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body that are responsible for the maintenance of homeostasis, reproduction, development, and/or behavior. In this document the term endocrine disruptor will be used synonymously with hormone disruptor. Of importance here is the concept that endocrine disruptors encompass more than just environmental estrogens and include any agent that adversely affects any aspect of the entire endocrine system. Endocrine disruptors are usually either natural products or synthetic chemicals that mimic, enhance (an agonist), or inhibit (an antagonist) the action of hormones. Under some circumstances, they may act as hypertrophic (stimulatory) agents and tumor promoters. Dose, body burden, timing, and duration of exposure at critical periods of life are important considerations for assessing adverse effects of an endocrine disruptor. Effects may be reversible or irreversible, immediate (acute) or latent (not expressed for a period of time).

The endocrine system includes a number of central nervous system (CNS)-pituitary-target organ feedback pathways involved in regulating a multitude of bodily functions and maintaining homeostasis. As such, there are potentially several target organ sites at which a given environmental agent could disrupt endocrine function. Furthermore, because of the complexity of the cellular processes involved in hormonal communication, any of these loci could be involved mechanistically in a toxicant's endocrine-related effect. Thus, impaired hormonal control could occur as a consequence of altered hormone: synthesis, storage/release, transport/clearance, receptor recognition/binding, or postreceptor responses.

Altered Hormone Synthesis. A number of compounds possess the ability to inhibit synthesis of various hormones. Some compounds inhibit specific enzymatic steps in the biosynthetic pathway of steroidogenesis (e.g., aminoglutethimide, cyanoketone, ketoconazole). Estrogen biosynthesis can be inhibited by exposure to aromatase inhibitors such as the fungicide fenarimol (35).

Alterations in protein hormone synthesis can be induced by gonadal steroids and potentially by environmental estrogens and antiandrogens. Both estrogen and testosterone have been shown to affect pituitary hormone synthesis directly or by changes in the glycosylation of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) (36). A decrease in glycosylation of these glycoproteins reduces the biological activity of the hormones. Any environmental compound that mimics or antagonizes the action of these steroid hormones could presumably alter glycosylation. The biopotency of pituitary hormones also may be altered by changes in glycosylation in response to treatment with biogenic amines (i.e., dopamine) and gonadotropin-releasing hormone (GnRH) [for review, see Wilson et al. (36)] .

Synthesis of nonpeptide, nonsteroidal hormones such as epinephrine and melatonin, which also serve as CNS neurotransmitters, can be altered by environmental agents. Changes in the synthesis of norepinephrine and epinephrine have been observed following exposure to a number of dithiocarbamates, metam sodium, and carbon disulfide (37-39). Exposure to these copper chelating compounds suppresses the activity of dopamine-ß-hydroxylase, thereby inhibiting the conversion of dopamine to norepinephrine and subsequently to epinephrine.

Altered Hormone Storage and/or Release. Catecholamine hormones are stored in granular vesicles of chromaffin cells within the adrenal medulla and within presynaptic terminals in the CNS. This mechanism for storage is important to the maintenance of normal concentrations of the hormone so that they can be released quickly on demand. Without such a storage mechanism, the hormones are subject to deamination by monoamine oxidase. Reserpine and amphetamine are well-known examples of compounds that can affect this storage process. In contrast, steroid hormones do not appear to be stored intracellularly within membranous secretory granules. For example, testosterone is synthesized by Leydig cells of the testis and released on activation of the LH receptor. Thus, compounds that block the LH receptor or the activation of the 3´,5´-cyclic AMP (cAMP)-dependent cascade involved in testosterone synthesis can rapidly alter the secretion of this hormone.

The release of many protein hormones depends on activation of second messenger pathways such as cAMP, phosphatidylinositol 4,5-bisphosphate, inositol 1,4,5-trisphosphate, tyrosine kinase, and Ca²⁺. Interference with these processes consequently will alter the serum levels (availability) of many hormones. Several metal cations have been shown to disrupt pituitary hormone release presumably by interfering with Ca²⁺ flux (40).

Altered Hormone Transport and Clearance. Hormones are transported in blood in the free or bound state. Lipid-soluble hormones are transported in the blood by specialized transport (carrier) proteins synthesized in the liver. The same binding globulin, known as sex/steroid hormone-binding globulin (SHBG) or testosterone-estrogen-binding globulin (TEBG), can associate with either testosterone or estrogen. Glucocorticoids are bound to corticosteroid-binding globulin (CBG) or transcortin in the circulation. Similarly, the thyroid hormones, triiodothyronine (T₃) and thyroxine (T₄), are transported in the blood on thyroxine-binding globulin, prealbumin, and albumin. Regulation of the concentration of these binding globulins in the blood is of some practical significance because there may be either increases or decreases that could affect steroid hormone availability. The levels of both TEBG and transcortin have been shown to be modified by gonadectomy and gonadal steroid hormone replacement. Salicylates and diphenylhydantoin may modify the circulating levels of T₄ because of changes in thyroxine-binding globulin. Estrogens increase the TEBG concentration in plasma, whereas androgens and pharmacologic doses of glucocorticoids decrease TEBG (41).

The clearance of hormones is influenced by compounds that alter liver enzymes involved in hormone clearance. For example, DDT analogs are potent inducers of hepatic microsomal monooxygenase activity in vivo (42). Induction of this activity by treatment with DDT analogs could possibly cause a decrease in testicular androgen as a result of enhanced degradation of endogenous androgens by the monooxygenase system. Similarly, treatment with lindane (-hexachlorocyclohexane) has been reported to increase the clearance of estrogen (43). However, no evidence for enhanced clearance was noted in a study by Laws et al. (44) in which serum estradiol was measured at multiple time points after estrogen administration via subcutaneous silastic implants in doses aimed at producing physiological levels of the steroid hormone. It should be pointed out that pan-fried meat and cruciferous vegetables can induce cytochrome P4501A2 in humans (45). Recently, a mechanistic, dosimetric model of TCDD effects on increasing hepatic UDP-gluconosyltransferase for removal of T₄ has been reported (46).

Altered Hormone Receptor Recognition/Binding. Hormones elicit responses from their respective target tissues through direct interactions with either intracellular receptors or membrane-bound receptors. Specific binding of the natural ligand to its receptor is a critical step in hormone function. Intracellular (nuclear) receptors such as those for sex steroids, adrenal steroids, thyroid hormones, vitamin D, and retinoic acid regulate gene transcription in a ligand-dependent manner through their interaction with specific DNA sequences (response elements). New messenger RNAs are synthesized, processed, and translated to produce new proteins.

A number of environmental agents may alter this process by mimicking the natural ligand and acting as an agonist or by inhibiting binding and acting as an antagonist. The best known examples are methoxychlor, chlordecone (Kepone), DDT, some PCBs, and alkylphenols (e.g., nonylphenols and octylphenols), which can disrupt estrogen receptor function (47,48). The antiandrogenic action of the dicarboximide fungicide vinclozolin (49) is the result of an affinity of this compound's metabolites for the androgen receptor (17). Interestingly, the DDT metabolite p,p´-DDE has been found to bind also to the androgen receptor and block testosterone-induced cellular responses in vitro (50,51).

Many of the chemicals classified as environmental estrogens can actually inhibit binding to more than one type of intracellular receptor. For example, o,p´-DDT and chlordecone can inhibit endogenous ligand binding to the estrogen and progesterone receptors, with each compound having IC₅₀ values that are nearly identical for the two receptors. Other compounds such as nonylphenol and the metabolite of methoxychlor, 2,2-bis(hydroxyphenyl)-1,1,1-trichloroethane, have the ability to inhibit binding to the estrogen, progesterone, and androgen receptors with similar affinities (52).

Receptors for protein hormones are located on and in the cell membrane. When these hormones bind to their receptors, transduction of a signal across the membrane is mediated by the activation of second-messenger systems. These may include alterations in G-protein-cAMP-dependent protein kinase A (e.g., after LH stimulation of the Leydig cell), phosphatidylinositol regulation of protein kinase C and inositol triphosphate (e.g., after GnRH stimulation of gonadotrophs; thyrotropin-releasing hormone stimulation of thyrotrophs), tyrosine kinase (e.g., after insulin binding to the membrane receptor), and calcium ion flux. Xenobiotics thus can disrupt signal transduction of peptide hormones if they interfere with one or more of these processes.

Altered Hormone Postreceptor Activation. Once the endogenous ligand or an agonist binds to its receptor, a cascade of events is initiated indicative of the appropriate cellular response. This includes the response necessary for signal transduction across the membrane or, in the case of nuclear receptors, the initiation of or alteration in transcription and protein synthesis. A variety of environmental compounds can interfere with the membrane's second messenger systems. For example, cellular responses that depend on the flux of calcium ions through the membrane (and the initiation of the calcium/calmodulin-dependent cellular response) are altered by a variety of metal cations (i.e., lead, zinc, cadmium) (40). Disruption of G proteins and transduction of receptor-generated signals leading to a biological response (activation of protein kinase A) occur from exposure to cholera and pertussis toxins (53). Similarly, lindane, among other environmental compounds, has been demonstrated to decrease phosphatidylinositol turnover in the membrane and thus reduce protein kinase C activation. Interestingly, the well-known antiestrogen tamoxifen also inhibits protein kinase C activity (54). Alternatively, the phorbol esters are known to mimic diacylglycerol and enhance protein kinase C activity.

Steroid hormone receptor activation can be modified by indirect mechanisms such as a downregulation of the receptor (temporary decreased sensitivity to ligand), as seen after TCDD exposure (including the estrogen, progesterone, and glucocorticoid receptors) (55,56). Consequently, because of the diverse known pathways of endocrine disruption, any assessment must consider the net result of all influences on hormone receptor function and feedback regulation.

Risk Assessment Paradigm

Evaluation and analysis of reported environmental endocrine disruption phenomena should be examined from a risk assessment perspective. Generally, quantitative risk assessment includes estimation of levels of exposure to a toxic substance that leads to specified increases in lifetime incidence rates or in the probable occurrence of an undesirable consequence (57). The four components of the noncancer risk assessment paradigm for human health are hazard characterization, dose-response assessment, exposure assessment, and risk characterization (58).

The ecological risk assessment framework is conceptually similar to the approach used for human health risk assessment, with a few distinctions. Ecological risk assessment considers effects beyond individuals of a single species and may examine population, community, or ecosystem-level risks. The framework consists of three major phases: problem formulation, analysis (which includes exposure and effects assessment), and risk characterization. The end points for ecological risks most often considered are survival, growth, and reproduction of individuals of a few representative species and populations. Although not specific to endocrine disruption effects, some limited inferences about endocrine-controlled processes may be made.

Hazard characterization focuses on the qualitative evaluation of the adverse effects of an agent on human and animal health and ecological well-being. Health end points of particular concern with environmental hormones are reproductive (including developmental) effects, cancer, and neurological and immunologic effects.

For human health, relevant and adequate epidemiologic studies and case reports for the agent(s) are preferable. In the absence of this information, pertinent test animal toxicology studies should provide useful information. In vitro studies may provide useful data for elucidating mechanisms of toxicity but are not sufficient by themselves to characterize a hazard. Important factors to consider in the evaluation of a hazard include inherent toxicity, route of exposure, dose level, timing and duration of exposure, body burden, susceptible populations and interspecies differences, and all of the assumptions and uncertainties in the data.

Dose-response assessment is the process of characterizing the relationship between the dose of an agent and the incidence/degree of an adverse effect. Factors to consider in the dose-response assessment are the intensity or frequency of the response with increasing dose, the shape and slope of the dose-response curve, pharmacokinetics (uptake, distribution, metabolism/detoxification, elimination), and the methods used for extrapolation of data from surrogate or sentinel species to ecological end points or to humans.

The exposure assessment component of the paradigm attempts to measure the intensity, frequency, and duration of exposure to an agent in the environment or to estimate hypothetical exposures that might arise from the release of new chemicals. Factors to consider in the exposure assessment include the amount of the agent in the environment; reactivity; half-life; environmental fate and disposition of the agent; the magnitude, duration (acute, subchronic, lifetime), schedule (timing), and route of exposure (oral, inhalation, dermal, aquatic); the size and nature of the exposed population; and all of the uncertainties and assumptions in the estimates.

Risk characterization is the process of estimating the incidence of a health or ecological effect under various conditions of human and biotic exposure. It draws together the hazard, dose-response, and exposure assessments. It discusses the assumptions, uncertainties, and limitations of all of the data.

With respect to recent reports of hazard (i.e., endocrine disruption causing human health or ecological effects), a critical element for risk assessment is the exposure assessment component. Without a clear understanding as to the magnitude and distribution of exposure and the potency and nature of endocrine activity, development of a credible risk assessment for specific endocrine-disrupting agents is not feasible. Another factor to consider in the evaluation of possible risk is whether testing paradigms in past or current use are capable of adequately identifying an agent as an environmental endocrine disruptor.

It should be emphasized that this special report is an interim effects and analysis document until the NAS releases its assessment report on environmental endocrine disruption. The current document focuses primarily on human health and ecological hazard effects (characterization) as found within peer-reviewed literature.

Controversy within the Scientific Community

In the wake of media coverage dealing with possible reproductive health and cancer concerns (59,60), a few toxicologists have questioned whether these adverse health effects can be attributed to environmental endocrine disruption (56,61,62). Arguments for a demonstrable link between hormone-disruptive environmental agents and human reproductive health effects are supported by the fact that many pesticides and other agents with estrogenic or antiandrogenic activity operate via hormone receptor mechanisms. However, in the few studies of suspected weak estrogens, such as the alkylphenols, some 1,000 to 10,000 times more of the weak estrogen is required to bind 50% of the estrogen receptor than estradiol itself (48). In other assays, 10⁶ times more of the agent may be required than for estradiol. Of course, crucial to risk assessment is the need to know how many receptors must be occupied before activation of a response can ensue. For some hormones such as human chorionic gonadotropin (hCG), as little as 0.5 to 5% receptor occupancy is required for full activation of response. For other hormones (those that require protein synthesis for expression of effect), higher levels of receptor occupancy are needed (63).

In general, because of the precise yet adaptable control mechanisms and the intertwined nature of the hormonal balance, modest amounts of chemical exposure seldom compromise normal physiological functions. Fluctuations of hormone concentration and receptor activities, by design, absorb some environmental and physiological challenges to maintain homeostasis in adults. Only when the equilibrium control mechanisms are overwhelmed do deleterious effects occur. An important question is whether homeostatic mechanisms are operative in the embryo and fetus. -Fetoprotein, to which endogenous sex steroids bind avidly, is thought to exert some protective function in developing fetuses to elevated estradiol that occurs during pregnancy. However, it is known that free estradiol, under experimental conditions in female rats, may have access to brain and other target organs in the fetus and neonate (64). DES is not bound to *-fetoprotein (65) and is not metabolized by the placenta as is estradiol (66). Whether other xenoestrogens behave in a similar manner is not known.

Production of any hormone in the endocrine system is the result of a chain of events involving precisely choreographed interactions of many other endocrine organs. Therefore, manifestation of an endocrine disorder may be associated with multiple changes in hormone concentrations.

Some investigators (67,68) have proposed the use of in vitro assays to screen for estrogenic or other hormonal activity. Although steroid receptors bound to their ligand act as transcription factors for gene expression in the target tissue, simple in vitro screening assays based on binding to a receptor are not sufficient in themselves for measuring hormone activity. Binding of ligand to its specific receptor must be correlated with a physiologic response. For such screening assays to be accepted as indicative of hormonal alteration, they must be thoroughly validated in a number of qualified, independent laboratories. This validation requires the correlation of receptor binding with a physiologic end point, for example, induction of the progesterone receptor (44), increase in uterine peroxidase (69), or an increase in vitellogenin in the case of the estrogen receptor. Furthermore, before screening assays can be used in a tier approach for evaluating hormone effects, in vitro assays need to be validated in vivo (in the whole animal). In the case of estrogen-mimicking agents, uterotrophic responses, progesterone receptor induction, or gonadotrophin inhibitory responses in ovariectomized rats or mice should be undertaken for validation in the whole animal. Although estrogenic effects have been cited as examples in this document, it is important to realize that any hormone has the potential of being disrupted in one way or another by an environmental agent, and considerations similar to those for estrogenic effects apply.

                       

Last Update: March 20, 1998