Last year, increases in the numbers of adult eagles and nest counts in certain regions of the United States led to a widely-hailed conservation "success"the lowering or down-listing of the bald eagle's legal status from "endangered" to "threatened". The bald eagle's partial comeback is a product of numerous factors including the outlawing of most uses of DDT, which was associated in the 1960s with the widespread occurrence of eggshell thinning in populations of bald eagles and other species. But the bald eagle has not made a complete recovery everywhere in the United States (Bowerman et al. 1995; Colborn 1991; Wiemeyer et al. 1993). Eggshell thinning and outright mortality are no longer visible and the absence of these conspicuous endpoints has led to the assumption that bald eagles and their populations are healthy. However, bald eagles nesting along the shores of the Great Lakes and feeding primarily on contaminated Great Lakes fish have far lower reproductive success than bald eagles nesting farther inland.
About the same time that the down-listing was reported, the popular press and electronic media reported an increasing number of disturbing findings about other wildlife species. These reports arose from research that described developmental effects associated with endocrine disruption in alligators (Guillette et al. 1994, 1995; Guillette and Crain 1996), shifts in sex ratios in turtles (Bergeron et al. 1994), altered bird behavior (Fry 1996), egg yolk proteins (vitellogenin) production in male fish (Tyler et al. 1996), female-female pairing in roseate terns (Nisbet and Hatch, personal communication), and feminization and demasculinization of male birds (Nisbet et al. 1996). Many of these animals appear normal and healthy. However, their reproductivity and survivorship are compromised and several of the populations appear to be in jeopardy. Given these findings and the growing evidence of endocrine disruption, it is apparent that more subtle measures of wildlife health must be established.
What is the endocrine system?
The endocrine system operates through a complex series of events triggered by chemical messengers that choreograph development and function. The chemical messengers (1) are involved in sexual differentiation; (2) prime the rates of cell division leading to the construction of tissues and organs that eventually determine future function, such as sperm production and ovulation; (3) control the development of the populations of cells comprising the immune system, thereby affecting future ability to combat disease; and (4) influence neural development, such as that required for bird behavior, vocalization, and parental care.
Components of the endocrine system that control development and function include the ovary, testis, and thyroid glands and their respective hormones, estrogen, testosterone, and thyroxine. In each case, the response depends upon the binding together of natural messengers with specific receptors (proteins) that are located throughout the body. Together, they initiate specific responses in the cell through genes coded on the DNA, yielding enzymes that mediate specific biochemical pathways, changes in rates of cellular activity, shifts within reproductive cycles, or increases in rates of cell divisions. Cells in different tissues respond differently to the same hormone. For example, estrogen in brain tissue can alter behavior, while the same hormone and receptors in cells lining the reproductive tract can initiate changes preparing the animal for new phases in a reproductive cycle. Likewise, cells of the same tissue may respond differently at different stages of development. Most importantly, signals occurring early in development frequently lead to a cascading of developmental events that are irreversible (Colborn and Clement 1992). Because of the critical role of the endocrine system in directing development and maintaining physiological homeostasis of animals throughout life, it is highly conserved. In other words, molecular biologists currently sequencing genes for receptors find little variation in genes among species, which is not surprising since many of these specific hormone messengers are shared from fish through humans (Gerhart and Kirschner 1997). From this, we may infer wider concern for all wildlife based on the observed effects of disruption of the endocrine system reported in alligators, turtles, birds, and mammals.
What is endocrine disruption?
That man-made chemicals can perturb the endocrine system of animals is not a new and startling revelation. Such perturbations have been reported in numerous laboratory studies using high dose testing, and in field studies involving exposure of wildlife to chemicals released into the environment. The well documented story of diethylstilbestrol (DES), a pharmaceutical administered to pregnant women to increase the probability of successful births, provides a model for estrogen-like compounds released into the environment (Bern 1992). This drug is a confirmed estrogen mimic. The effects to the mother were minimal and their babies were born "healthy". However, many of the exposed children developed a variety of anomalies later in life. Subsequent research linked the effects to disruption in utero by the estrogen mimic, DES.
Endocrine disrupters work by a variety of mechanisms. First, they can impersonate natural hormones by binding to receptors and initiating a new cellular response. Second, an endocrine disrupting chemical may bind and block the receptor, thereby making these regulatory switches unavailable to signals from the body's naturally produced hormone messengers. Third, concentrations of the natural hormone can also be affected when man-made chemicals promote or interfere with the breakdown of the hormone by the liver's enzyme system. Fourth, during development, endocrine disrupting chemicals can alter the number of receptors in developing tissue types, thereby predisposing these tissues to abnormal responses later in life. The net result is a perturbation to systems that are critical for the creation and maintenance of the body plan which has been molded by natural selection over countless generationsperturbations resulting not from a genetic mutation but from confused chemical messenger systems and thus alter how genes are expressed. These changes in expression can lead to "functional deficits"changes in how well an organism's immune, reproductive, and other systems perform.
What are the links to wildlife?
Much of what is known about endocrine disruption comes from new multidisciplinary research. A gathering of scientists met in 1991 at the Wingspread Conference Center, Racine Wisconsin (Colborn and Clement 1992). After two days of discussion, the scientists, from 17 diverse disciplines such as medicine, molecular biology, pharmacology, physiology, psychology, reproductive and developmental biology, and zoology, agreed with certainty that "a large number of man-made chemicals that have been released into the environment, as well as a few natural ones, have the potential to disrupt the endocrine system of animals." The basis of the concern arose from the presentations outlining each investigator's narrowly focused research specialty, each contributing a single piece from which the picture of endocrine disruption emerged. A consensus statement issued by the participants identified observable effects seen in wildlife, including thyroid dysfunction in birds and fish; decreased fertility in birds, fish, shellfish, and mammals; decreased hatching success in birds, fish, and turtles; gross birth deformities in birds, fish, and turtles; metabolic abnormalities in birds, fish, and mammals; behavioral abnormalities in birds; demasculinization and feminization of male fish, birds, and mammals; defeminization and masculinization of female fish and birds; and compromised immune systems in birds and mammals. These effects when reported alone appeared as unique events. What the Wingspread participants came to realize is that such events share a common mechanism of action, perturbation of the endocrine system, and are more widespread than previously understood.
The list of endocrine disrupting chemicals is growing (Colborn et al. 1993). Some man-made chemicals are of special concern because they are produced in large quantities, widely used, and, when released, can travel long distances in water or through the air. Many are very persistent since they resist degradation in the environment or detoxification by enzymes in organisms. Some are altered in the body into different chemicals that are more biologically active and interfere with the function of normal endocrine systems. Many can bioaccumulate in the fat of animals and are passed up through the food web when prey is eaten by predators. Many chemicals on the list are pesticides. Preliminary studies have identified endosulfan, methoxychlor, dicofol, lindane, DDT and its metabolites, vinclozolin, chlor-decone, toxaphene, 2,4-D, 2,4,5-T, atrazine, carbaryl, dieldrin, heptachlor, mirex, malathion, synthetic pyrethroids, and chlordane as endocrine disrupting chemicals. Industrial and commercial chemicals on the list, such as polychlorinated biphenyls (PCBs), furans, dioxins, brominated biphenyls, phthalates, and phenol ethoxylates, are also found in wildlife tissue and have endocrine disrupting characteristics.
Concerns for wildlife
Much of the concern for wildlife originally emerged from the extensive research by many scientists working in the Great Lakes region (Colborn et al. 1990). Since then, there has been a steady flow of reports about developmental, reproductive, behavioral, immunological, and physiological changes in various wildlife species around the world. Die-offs in populations, such as seals and dolphins, have been linked to contaminant exposures (Lahvis et al. 1995; de Swart et al. 1996; Ross et al. 1996). Bill deformities have been described for a wide variety of birds in the Great Lakes region, including herring gulls, ring-billed gulls, common terns, Caspian terns, Forster's terns, black-crowned night herons, great blue herons, double-crested cormorants, Virginia rails, and bald eagles (Bowerman et al. 1994; Fry and Toone 1981; Fry 1996). Dioxins, furans, and PCBs are the primary suspects with regard to bill deformities. Also correlating with elevated concentrations of these chemicals are impaired reproduction and increased nestling mortality in many of these same species in the Great Lakes (Gilbertson et al. 1991; Kubiak et al. 1989). These are obvious morphological markers of development gone awry. New sophisticated, more refined methodologies are now revealing the invisible effects that can undermine a wildlife population (Giesy et al. 1994).
The developing offspring is the most sensitive target of endocrine disruption (Bern 1992). Many man-made chemicals can cross the placental barrier, thereby allowing the mother's body burden to be shared with her developing offspring. Further intake occurs as nursing animals drink fat-loving chemicals that are bound into fat-rich milk. In egg laying species, the chemicals are transported from the mother to the egg yolk where the chemicals cause irreversible damage during incubation. These concentrated doses of chemicals during embryonic, fetal, and early postnatal development can be the highest exposures encountered throughout life. Again, the timing for such exposures is of concern since much of the neural, reproductive, and immune development continues long after birth or hatching.
Concerns about the effects of DDT and its metabolites on the health of wildlife and humans have a lengthy history. DDT has long been described as an estrogen mimic, and a variety of effects in birds has been attributed to this biological activity. Besides DDT's well documented effects on eggshell thinning, a number of abnormalities were reported in male sexual development. These effects were proposed to be due in part to estrogen receptor interference, but only recently Kelce and coworkers (1995, 1997) have shown that the primary metabolite of DDT, p,p'-DDE, is a potent androgen antagonist. It binds to the androgen receptor, blocking a switch critical for the development of normal males. Prenatal exposure to p,p'-DDE leads to feminization in male mice, including the development of teats in a species which does not express them, as well as shortened penises. Exposure to p,p'-DDE is a serious concern since it dissolves in fat and resists degradation. The half-life of p,p'-DDE in animals is measured in decades and, coupled with the transfer in the food web, concentrations are frequently elevated in fish, wildlife and humans the world over. Even when exposure is low on a daily basis, the concentrations in body tissues increase over the years. By the time a female reaches reproductive age, the concentrations of chemicals such as p,p'-DDE can be substantial. Consequently, chemicals such as DDT and its metabolites can have a wide range of effects. Threats to the developing male by the anti-androgen p,p'-DDE differ from the estrogen mimicking effects of o,p'-DDT. There may be other more cryptic effects from meddling with a soup of endocrine disrupters.
Chaos caused by alterations to the messages sent by Mullerian Inhibiting Substance (MIS) is another example of endocrine disruption. For instance, MIS is normally released in developing male vertebrates to cause the resorption of the embryonic tissues that would produce a female reproductive system. All embryos have the potential to become either male or female, and simultaneously develop two separate kinds of tissues, one that gives rise to male and the other to female reproductive systems. Early in life, a developmental switch is thrown and the proper set of tissues is signaled to develop appropriate reproductive organs while existing tissues fated for the opposite sex are signaled to self-destruct. The sex chromosomes determine in which direction the switch is thrown, male or female, thereby setting in motion specific activities along a number of endocrine pathways. The resulting chorus of messengers directs the construction of anatomy, morphology, physiology, and behaviors necessary for that sex. It is perturbations to these hormonal ebbs and flows that confound development and cause potentially serious problems. Crossed messages signaling development of the sexes can cause varied intensities of feminization and demasculinization of males or defeminization and masculinization of females. As a result, the offspring become some intermediate design when compared to those that develop by genetic inheritance alone. Babble added to thyroid, estrogen, testosterone, and other hormone systems can contribute to reduced growth, functional abnormalities, altered behavior, lowered fertility, learning disabilities, reduced intelligence and increased susceptibility to disease.
A number of organochlorine chemicals are known to affect wildlife behavior. Increased concentrations of these chemicals are associated with increases in aberrant courtship behaviors, breeding asynchrony between mated pairs, faulty nest construction, and alterations in incubating and parental care behaviors in birds (Fry 1996; Fox et al. 1978; Kubiak et al. 1989). Some of these are also correlated with alterations in the quantity of circulating androgens, estrogens, and thyroid hormones. Such subtle changes in the behavior of birds are not easy to observe, let alone quantify, under standard field conditions. However, because of their importance they need to be monitored.
Songbirds are known for their complex behavioral repertoires in courtship, mating, and territorial displays. For example, male zebra finches sing, whereas females never sing even if given testosterone as adults. However, when this steroid hormone is administered shortly after hatching, females will sing as adults (Arnold et al. 1996; Bottjer and Arnold 1997). Steroid hormones present early in development are critical for the expression of the bird songs by which zebra finches court and defend territory, factors necessary to insure successful reproduction. The differences between males and females are in the numbers of neurons, the arrangements of the synaptic connections in the brain, and the size of nuclei, all choreographed by an interplay between steroid hormones, neuro-trophins, and their receptors. If endocrine disrupting chemicals compete for receptor sites, mimic estrogens or block androgen receptors, or inhibit aromatase activity, neurological development could be altered through cascading effects that would not be discerned even by the trained eyes of serious bird watchers. Trouble might only become evident when adult birds disappear from Christmas bird counts or other formalized surveys. Given the prevalence of endocrine disrupting chemicals in the environment, the evidence of maternal transfer between females and their offspring, and the important relationship between the presence of steroid hormones and brain and behavioral development, it is important that such subtle changes be recognized in wildlife.
Endocrine disruption has been documented in a wide variety of vertebrates, in both laboratory and field conditions. Numerous pesticides, industrial chemicals, and commercial products that have been released into the environment are endocrine disrupters. Monitoring of animal tissues has documented that some of these chemicals biomagnify in food webs, with notable concentrations reported in top predators in some communities and regions around the world. Recent findings, however, reveal that concentrations of chemicals in wildlife tissues cannot be used to assess their health. In light of what is known about the potential of synthetic chemicals to disrupt development and homeostasis, it is imperative that the animals' functional integrity be measured in hazard assessments. Merely observing an adult animal with young or seemingly healthy immature animals by themselves must not be the end product of an assessment of population health. Instead, the fate of the offspring must be followed to determine if they mature completely and have the potential to reproduce, thereby contributing to the viability of the population and the species. Besides the bald eagle, concern is growing for the viability of many other species of birds, including albatrosses, hawks, spoonbills, herons, cormorants, terns, and migratory shorebirds and songbirds. Birds are not the only species threatened by endocrine disrupting chemicals. Florida panthers, alligators, turtles, dolphins, porpoises, whales, otters, mink, and a growing list of fish species are being drawn into the web of endocrine disruption as scientists report on the status of these species. This is the nature of endocrine disruptionstealth damage caused by interference with endogenous messengers, the messengers that build and maintain the complex biochemistry that is ultimately critical for an individual's and species' survival.
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Michael Smolen and Theo Colborn work in the Wildlife and Contaminants Program of the World Wildlife Fund. Michael is a conservation scientist and Theo is the Director of the Program. Their address is: World Wildlife Fund, 1250 Twenty-Fourth St., NW, Washington, DC 20037-1175.
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