Abstract
A well-managed zoo represents a closed community with controlled animal movements. The variety of Taxa gathered in one place provide a unique opportunity to study the effects of environmental agents on a great number of species. Environmental agents with adverse effects on humans also have similar adverse effects in a variety of other species. Endocrine disrupting chemicals are environmental agents that work by mimicking or blocking the hormone signals within the endocrine system. Serum samples obtained from the Audubon Zoo's reptiles, birds, and mammals have been assayed utilizing a strain of Sacharomyces cerevisiae yeast that, in tum, has been transformed to express wild type human estrogen receptor (hER) and a reporter plasmid with two estrogen response elements (ERE) linked to a lacZ gene. Incubation of the transformed yeast with a positive control, 17P-Estradiol and appropriate P-galactosidase substrate (ONPG) has resulted in a chromogenic change that is quantified via spectrophotometry at A420. This system has been utilized to detect changes in lacZ reporter activity by incubating the transformed yeast with 17P-Estradiol in the presence of the zoo serum. Further studies are required to more clearly elucidate the degree and pattern of these changes.
Introduction
Modem open plan zoos with naturalistic environments, water systems, grasslands, and abundant flora provide extremely genetically diverse, compact, and complex ecosystems that can be comprehensively monitored.1 A well-managed zoo represents a closed community with controlled animal movements.1 The variety of species represented in a large zoological collection run the gamut of major Orders observed in numerous and diverse habitats. The variety of Taxa gathered in one place provide a unique opportunity to study the effects of environmental agents on a great number of species. A uniform effect in several species, many of which have short life cycles, would have considerable implications for the study of these agents.
Methods
Serum samples taken from apparently healthy mammals, birds, and reptiles during annual physical exams are stored at −70°C. Volumes of samples are varied, but generally consist of a minimum volume of 0.25 ml. Frozen samples collected from 1997 to date are currently being analyzed. Each sample is tested for the presence of endocrine disrupting chemicals.
Serum samples from various species at the Audubon Zoo have been assayed utilizing a strain of Sacharomyces cerevisiae yeast that, in tum, have been transformed to express wild type human estrogen receptor (hER) and a reporter plasmid with two estrogen response elements (ERE) linked to a lacZ gene. Incubation of the transformed yeast with a positive control, 17P-Estradiol and appropriate P-galactosidase substrate (ONPG) has resulted in a chromogenic change that is quantified via spectrophotometry at A420. This system has been utilized to detect changes in lacZ reporter activity by incubating the transformed yeast with 17P-Estradiol in the presence of the zoo serum.
Results
Preliminary results suggest that measurable estrogenic activity in the YES system was decreased due to competition by serum proteins for available ligand (estradiol) in the system. Incubation of estradiol in the presence of zoo animal serum variably reduced the amount of estrogen available to bind to the estrogen receptors. There also appears to be a correlation between the diets consumed by these species and the degree of decrease in reporter activity in this system. These data revealed decreases in lacZ reporter activity anywhere from 5–60% of normal activity levels. Further studies are required to more clearly elucidate the degree and pattern of these changes.
Discussion
The study of endocrine disrupting chemicals is a relatively recent but rapidly growing field. For many years the subject of environmental health has been focused on the health of human beings. More and more we are finding that environmental agents with adverse effects on humans also have similar adverse effects in a variety of other species. Endocrine disrupting chemicals are environmental agents that work by mimicking or blocking the hormone signals within the endocrine system. Recent publications provide extensive reviews on the matter.5
Experiments in the laboratory, and in both the field and clinic, have shown that numerous compounds of diverse chemical structure can function as estrogens or antiestrogens.5 The molecule estradiol-17P is the natural steroidal estrogen secreted by the ovaries of most vertebrates studied, and interacts with a conserved protein called the estrogen receptor (ER). In addition, plants such as soybeans also produce estrogenic molecules or isoflavones, which can interact with the vertebrate estrogen receptor and induce an estrogenic response in wildlife, livestock and humans.5 Another class of estrogenic environmental chemicals of natural origin are aflatoxins secreted by Fusarium sp. These aflatoxins are responsible for the "moldy com syndrome," or hyperestrogenization of pigs. There are, therefore, at least three natural sources of estrogenic chemicals: ovarian steroids, phytoestrogens, and mycotoxin estrogens.5
Other sources of estrogenic environmental chemicals include industrial pollutants such as bis-phenol A, polychlorinated biphenyls (PCBs), and dichlorodiphenyldichloroethylene (DDT); a plasticizer, a coolant, and a pesticide, respectively. These ubiquitous and high-volume contaminants are found in most ecosystems and have been measured in various tissues of humans,5 seals,5 and even alligator eggs2,4 throughout the world. Thus, most species exist in contact with a variety of estrogenic sources in addition to their own.
In laboratory experiments, fish, bird and reptile eggs have been treated with estrogenic chemicals with the resultant appearance of sex reversal in genetically male individuals.4 Likewise, laboratory rodents have been feminized by in utero treatment with estrogenic environmental chemicals.5 In acute studies in adult animals, experiments detect direct estrogenic effects in a variety of animals such as uterine growth in mammals and vitellogenin production in male nonmammalian vertebrates. Thus, the entire vertebrate kingdom responds to a number of estrogens in the environment with both acute and long-term effects.5
At another level of organization, reproductive anomalies associated with possible environmental estrogen exposure have been reported in many wildlife species including cases of intersexed fish,6 malformations of external genitalia in alligators,2,3 reproductive impairment in fish-eating birds,6 reproductive failure in seals,6 and pseudohermaphroditism in polar bears.7 Because many environmental estrogens enter the environment through agricultural runoff and industrial effluent, it is not surprising that most examples of affected wildlife either live in the water or eat aquatic organisms.5,6
A critical missing link between experiments with laboratory species and observations in the wild is the ability to pose hypotheses regarding the environment and reproduction in multiple species of wildlife in relatively controlled conditions. That missing link can be found, in part, in the experimental zoo. It is here that the academic laboratory/field work can be applied in conditions that permit replication and sampling that are not possible or relevant in the lab or field. For example, species differences in processing phytoestrogens are likely to be related to the dietary exposure to such compounds in a natural setting; carnivores are least likely and herbivores most likely to have evolved physiologic mechanisms for dealing with hormonally active compounds in plants.2 If so, exposure to these estrogens may result in different, yet predictable outcomes among such classes of animals. This would be a topic for which the experimental zoo would be an important resource.
Conclusion
Zoos can and should provide an extremely biologically diverse, well-managed, and well-monitored study population for the effects of endocrine disrupting chemicals. Long-term studies of zoo animals may provide clues and answers to the overall health of an environment, and the effect of endocrine disruptors on local, regional, and migratory wildlife. Extrapolation of relevant data may eventually influence similar studies on the human population of the region.
Literature Cited
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