Animal Models and Experimental Design Considerations for Endocrine Disruptor Research and Testing

Volume 45(4) of the Institute for Laboratory Animal Research (ILAR) Journal reviews and discusses important laboratory animal experimental design issues for endocrine disruptor research and identifies the main experimental and environmental factors that can confound endocrine disruptor research findings. The articles provide valuable insight regarding the role of different models for endocrine disruptor chemicals (EDCs) and research in the Endocrine Disruptor Screening Program (EDSP) which is designed to identify EDCs that may adversely affect humans and ecologically important animal species.

Experimental Design Considerations for Endocrine Disruptor Studies

Scientific evidence suggests that certain chemicals may bind to endogenous hormone receptors and disturb normal endocrine functioning, thereby increasing the risk of reproductive problems and cancer in humans. This has led to international efforts to screen chemicals for endocrine activity and potential health effects. However, recent studies have shown that the use of traditional laboratory animal models, "classical" procedures and toxicity testing methods, has not previously identified many of the endocrine-related adverse effects of some chemicals, especially weakly adverse effect on the fetus. Inter-laboratory variability was also identified as a significant problem when attempting to replicate and interpret results, especially at low-dose levels. Among the factors that likely contributed to the variation in results between laboratories is the standard laboratory animal diet traditionally used for endocrine research. The standard diet contains high and variable levels of phytoestrogens (soy-derived isoflavones) which can modulate physiologic and behavior responses similar to both endogenous estrogen and exogenous estrogenic chemicals. In short- and long-term studies, it was found that a diet with phytoestrogens had specific influence on food and water intake, adipose deposition, serum leptin and insulin levels, as well as on learning process, memory and anxiety-related behavior. Further study is required to examine the influence of dietary isoflavones on basic regulatory behaviors which cascade into other physiological and neuroendocrine hormonal mechanisms of normal and different pathological conditions. The findings suggest the importance of controlling and characterizing dietary phytoestrogen content not only for endocrine disruptor studies, but also for all types of research and testing using animals to minimize potentially confounding effects and to maximize the possibility for replication.

Another important consideration in endocrine disruptor studies (EDS) is the selection of the appropriate species and strain/stock of animal models. Experimental data have shown that some commonly used outbred mice and rats are less responsive to estrogenic substances than certain inbred mouse and rat strains. The authors emphasize the importance of considering the strain/stock sensitivity for the specific endpoints of interest in endocrine disruptor studies, and the importance of selecting a sensitive strain/stock that will minimize the likelihood of false-negative results.

Although differences in diet and strain/stock sensitivity are well-known sources of variation in EDS, there are numerous possible variations in the environment in which experimental animals are housed or tested that may also confound research and testing results. Potential confounding factors that should be selected, standardized, and controlled to provide scientifically sound and reproducible research findings include genetic management, microbial and pathogen status, breeding schemes, housing and husbandry methods, diet, physical environment (lighting, room temperature, humidity, and macro- and microenvironment), and caging systems.

The effect of exposure to potential EDCs during in utero development are often evaluated in EDS. However, recent studies have demonstrated the heightened sensitivity of the fetus to endogenous hormones during this critical period of organ and system development. For example, the intrauterine proximity of female fetuses to adjacent male fetuses affects a number of sexually dimorphic anatomical, physiological, and behavioral traits. Accordingly, for some EDS studies, it may be important to observe or estimate intrauterine position for each fetus or pup, respectively, to provide optimal sensitivity and specificity to accomplish the research and testing objectives.

Animal Models for Endocrine Disruptor Studies

During the last 5-10 years, the number of chemicals known to have endocrine-disrupting potential has increased exponentially. Indeed, the need for the development of a rapid prioritization testing program becomes obvious when one considers that more than 100,000 already existing and new chemicals are listed for evaluation. According to this concern the Endocrine Disruptor Screening and Testing Advisory Committee (EDSTAC), convened by the Environmental Protection Agency (EPA), recommended a tiered screening and testing approach for the evaluation of estrogen, androgen, and thyroid -related effects of existing and new commercial chemicals and environmental contaminants. The Tier I Screening battery was designed to identify substances with endocrine-disruption potential for further testing, to be quick and inexpensive, and more sensitive than specific. The Tier II Testing battery was designed to identify, characterize, and quantify EDC adverse effects and establish dose-response relationships for hazard assessment.

Invertebrates are essential components of every ecosystem on earth and have been used effectively in biological screening and testing assays for many years. Unique hormone systems which are found in all vertebra phyla, including arthropods, mollusks, nematodes, and many others, offer a real opportunity for the study and identification of EDCs that may affect ecologically and economically important invertebrate species of ecosystems. The EPA plans to include an opossum shrimp (Mysidacea) and/or other invertebrate life cycle toxicity assays as part of its EDSP Tier II analysis. However, further comparative endocrinology research is needed to understand the usefulness and limitations of this approach.

Fish tests are an important component of EDC screening and testing programs. Because of their genetic uniformity these strains can minimize the experimental variability normally associated with other species. Partial and full-life cycle tests with fish that are focused on key aspects of reproduction and development are effective models for quantitative predictions of ecological risks of EDCs to fish populations and screening EDCs for generalized effects on vertebrate endocrine systems. The EPA proposed the inclusion of fish tests in both Tier I Screening and Tier II Testing studies.

Birds were one of the first classes of wildlife in which endocrine disruption, thought to be due to the pesticide DDT (dichlorodiphenyltrichloroethane), was first reported. Consequently, avian tests to assess reproductive capabilities are a usual requirement for pesticide registration. The EPAs EDSP includes an avian two-generation toxicity test as part of the Tier II Testing battery. The test is intended to expose all four critical life stages (in ovo, juvenile, subadults, and adults) to assess any adverse effects associated with a putative EDC quantitatively. The rationale for including birds in the EDSP was reviewed and the experimental design parameters and important aspects of test methodology to ensure appropriate exposure during life stage were described. The author outlined some general research needs including improvements in the husbandry and experimental handling of birds to reduce both the occurrence of confounding behaviors and unintended morbidity and mortality.

The biological effects of estrogen in mammalian target tissues are important for the function of multiple organ systems including the male and female reproductive tract, the neuro-endocrine system, the skeletal system, and the cardiovascular system. Adverse health effects from exogenous EDCs with estrogenic and/or antiestrogenic activity are of significant concern to both humans and animals. The degree of confidence with which hazard may be estimated depends on a thorough understanding of the types, distribution, and function of estrogen receptors. Recently generated genetically modified mouse models, that lack estrogen receptor genes, provide a unique and valuable tool for investigators and scientists to assess the role each receptor may play in various tissues and the specific functions of different estrogen receptors in normal and pathological physiology.

Current in vitro screening assays that are part of the Tier I test batteries consist of the estrogen/androgen receptor binding, estrogen/androgen gene transactivation, and minced testis; and alternate (placental aromatase) in vitro screening assays. The receptor binding and transactivation assays evaluate the potential of exogenous agents to mimic or block the action of the natural estrogen receptor and androgen receptor ligands with their receptors and alter hormonal regulation through altered gene expression. The minced (sliced) testis assays was designed to identify the potential of compounds to disrupt any of the intracellular pathways involved in the gonadal biosynthesis of testosterone. The placental aromatase assay was designed to identify specific inhibitors of aromatase enzymes, which catalyze the conversion of androgen to estrogens. The aromatase assay has been used in the assessment of the activity of different substances including pesticides like atrazine, diuron, fenarimol, organochlorines, polychlorinated biphenyls, TCDD, organotin, and phytoestrogens. However, these assays do not cover all of the possible ways by which the synthesis and metabolism of steroid hormones can be disrupted and require the evaluation of the negative impact of cytotoxicity on gonadal biosynthesis and aromatase activity. The advantages of in vitro tests, in general, include cost effectiveness, speed of the test, reproducibility (consistency), capability of handling large number of samples and indication of the mechanism of action. However, in vitro assays alone cannot account for adsorbtion, distribution, metabolism, and excretion of chemicals, and they should be not be used directly in the risk assessment paradigm.

Due to limitations of the in vitro assays, it was necessary to include in vivo assays in the screening and testing batteries. The laboratory rat has served as the traditional in vivo animal model for regulatory toxicological and endocrinology research, and developmental and reproductive toxicity testing conducted to reveal the potential adverse health effects on humans and animals, supporting human health risk assessment. Currently, three short-term rat assays are undergoing standardization and validation for the EDSP Tier I Screening Battery: The first is the 3-day rat uterotrophic assay, which is designed to detect estrogen agonists based on the induction or inhibition of increased weight changes in the uterus. The second is the 10-day rat Hershberger assay, which detects androgen and anti-androgen activity by measuring changes in the weight of male reproductive tissues. The third is the 21-day pubertal female rat assay where the age of the vaginal opening is monitored, serum thyroid hormones and uterine and ovarian weight are measured, and histology is evaluated. This assay was found highly reproducible and very sensitive regarding to potential EDCs with anti-thyroid and estrogenic activity, as well those that may inhibit steroidogenesis. Other assays undergoing evaluation for possible inclusion in the Tier I Screening battery include the pubertal male rat assay and an in utero-lactational assay with exposure during both gestation and lactation periods. The rat was also selected as the animal model for the EDSP Tier II mammalian multi-generation tests. These tests expose animals during all critical stages of development, and evaluate the reproductive function of animals that were exposed in utero. Furthermore, the development of methods to detect estrogen, androgen, and thyroid hormone perturbations is needed.

The EPA is developing requirements for the screening and testing of thousands of pesticides, commercial chemicals, and environmental contaminants for their potential to disrupt the endocrine system. However, the endocrine system can be readily modulated by many experimental factors. Selection of appropriate animal models and experimental design parameters for endocrine disruptor research and testing will minimize confounding experimental variables, increase the likelihood of replicable experimental results, and contribute to more reliable and relevant test system. Alternative methods, such as in vitro and computer technologies, and the use of nonmammalian organisms, are playing an increasingly important role in this process. Furthermore, scientists continue to to identify, develop, and validate new screening and testing methods which will improve the ability to predict the harmful effects of new and existing chemicals, animal well-being and further minimize the number of animals needed for testing.