Selecting the appropriate rodent diet for endocrine disruptor research and testing studies

Selecting the optimum diet for endocrine disruptor (ED) research and testing studies in rodents is critical because the diet may determine the sensitivity to detect or properly evaluate an ED compound. Dietary estrogens can profoundly influence many molecular and cellular event actions on estrogen receptors and estrogen-sensitive genes. The source, concentration, relative potency, and significance of dietary estrogens in rodent diets are reviewed, including dietary factors that focus specifically on total metabolizable energy and phytoestrogen content, which potentially affect ED studies in rodents. Research efforts to determine dietary factors in commercially available rodent diets that affect uterotrophic assays and the time of vaginal opening in immature CD-1 mice are summarized. A checklist is provided of important factors to consider when selecting diets for ED research and testing studies in rodents. Specific metabolizable energy levels are recommended for particular bioassays. Discussions include the between-batch variation in content of the phytoestrogens daidzein and genistein, the effects of total metabolizable energy and phytoestrogens on the timing (i.e., acceleration) of vaginal opening, and increased uterine weight in immature CD-1 mice. It is concluded that rodent diets differ significantly in estrogenic activity primarily due to the large variations in phytoestrogen content; therefore animal diets used in all ED studies should ideally be free of endocrine-modulating compounds.     

Selection, acclimation, training, and preparation of dogs for the research setting

Dogs have made and will continue to make valuable contributions as animal models in biomedical research. A comprehensive approach from time of breeding through completion of in-life usage is necessary to ensure that high-quality dog models are used in studies. This approach ensures good care and minimizes the impact of interanimal variability on experimental results. Guidance related to choosing and developing high-quality laboratory dogs and managing canine research colonies is provided in this article. Ensuring that dogs are healthy, well adapted, and cooperative involves good communication between vendors, veterinarians, care staff, and researchers to develop appropriate dog husbandry programs. These programs are designed to minimize animal stress and distress from the postweaning period through the transfer and acclimation period within the research facility. Canine socialization and training programs provided by skilled personnel, together with comprehensive veterinary health programs, can further enhance animal welfare and minimize interanimal and group variability in studies.     

The importance of appropriate controls, animal feed, and animal models in interpreting results from low-dose studies of bisphenol A

Interpreting results of studies that report only negative effects is problematic. A number of published studies to determine whether chemicals with estrogenic activity can cause effects at low doses have not taken into account the possibility that the commercial animal feed being used can mask effects of even potent estrogenic drugs such as diethylstilbestrol (DES). In addition, the sensitivity of the strain of animal being used for the specific category of chemical being tested has not always been described. For environmental chemicals, such as the estrogenic polycarbonate plastic monomer bisphenol A, DES is an appropriate positive control for estrogenic effects, and using an appropriate low dose of DES can eliminate the possibility of false-negative conclusions of safety when the above or other variables contribute to the negative outcome. Only when simultaneous positive effects of low doses of a positive control chemical such as DES and negative effects of environmentally relevant low doses of the test chemical are demonstrated within the same experiment are conclusions of no effect of the test chemical warranted, and this has not been reported for bisphenol A in any study. Instead, more than 90 refereed journal publications have reported effects due to exposure to low doses of bisphenol A in a wide variety of animals (for references see: http://rcp.missouri.edu/endocrinedisruptors/vomsaal/vomsaal.html). However, due to lack of attention to the importance of appropriate positive controls, a small number of studies reporting negative effects of bisphenol A have created a false sense of controversy regarding low-dose effects of bisphenol A.     

Laboratory animal science issues in the design and conduct of studies with endocrine-active compounds

The use of rodent models for research and testing on endocrine-active compounds necessitates an awareness of a number of laboratory animal science issues to standardize bioassay methods and facilitate reproducibility of results between laboratories. These issues are not unique to endocrine research but are particularly important in this field due to the complexities and interdependencies of the endocrine system, coupled with the inherently sensitive and variable nature of physiological endpoints. Standardization of animal models and the control of animal environments depend on the establishment of strong scientific partnerships between research investigators and laboratory animal scientists. Laboratory animal care and use programs are becoming increasingly complex and are constantly changing, fueled in part by technological advances, changes in regulations concerning animal care and use, and economic pressures. Since the early 1980s, many institutions have moved to centralization of animal facility operations concomitant with numerous changes in housing systems, barrier concepts, equipment, and engineering controls of the macro- and microenvironment. These and other changes can have an impact on animals and the conduct of endocrine experiments. Despite the potential impact of animal care and use procedures on research endpoints, many investigators are surprisingly naive to the animal facility conditions that can affect in vivo studies. Several key animal care and use issues that are important to consider in endocrine experiments with rodent models are described.     

Dietary phytoestrogens accelerate the time of vaginal opening in immature CD-1 mice

The purpose of the study reported here was to determine the effects of dietary phytoestrogens on the time of vaginal opening (VO) in immature CD-1 mice, and to correlate it with phytoestrogen and total metabolizable energy (ME) contents of the diet in an effort to determine the most appropriate diets(s) for comparing or evaluating the estrogenic or antiestrogenic activity of endocrine disruptor compounds (EDC). Mice were weaned at postnatal day (PND) 15 and fed the test diets from PND 15 to 30. Vaginal opening was recorded from PND 20 to 30. The phytoestrogen content of the diet was highly predictive (P < 0.0001) of the proportion of mice with VO at PND 24. Total ME content also was significantly (P < 0.01) correlated with time of VO, although this variable was somewhat less predictive than was phytoestrogen content. Time of VO in mice was significantly (P < 0.05) accelerated in mice fed diets high in phytoestrogens, compared with those containing low phytoestrogen content. It was concluded that: dietary daidzein and genistein can significantly (P < 0.01) accelerate the time of VO in CD-1 mice; the advancement in time of VO is more highly correlated with daidzein and genistein contents of the diets than with total ME content; advancement in the time of VO is a sensitive end point for evaluating the estrogenic activity of EDCs, and should be part of the standard protocol for evaluating EDCs. Phytoestrogen-free diet(s) containing the same amount of ME should be used in bioassays that compare the time of VO, or increases in uterine weight as end points for evaluating the estrogenic activity of an EDC.     

Chemical safety in animal care,use, and research

Chemical safety is an essential element of an effective occupational health and safety program. Controlling exposures to chemical agents requires a careful process of hazard recognition, risk assessment, development of control measures, communication of the risks and control measures, and training to ensure that the indicated controls will be utilized. Managing chemical safety in animal care and use presents a unique challenge, in part because research is frequently conducted in two very different environments--the research laboratory and the animal care facility. The chemical agents specific to each of these environments are typically well understood by the employees working there; however, the extent of understanding may not be adequate when these individuals, or chemicals, cross over into the other environment. In addition, many chemicals utilized in animal research are not typically used in the research laboratory, and therefore the level of employee knowledge and proficiency may be less compared with more routinely used materials. Finally, the research protocol may involve the exposure of laboratory animals to either toxic chemicals or chemicals with unknown hazards. Such animal protocols require careful review to minimize the potential for unanticipated exposures of the research staff or animal care personnel. Numerous guidelines and regulations are cited, which define the standard of practice for the safe use of chemicals. Key chemical safety issues relevant to personnel involved in the care and use of research animals are discussed.     

Developmental exposure to low-dose estrogenic endocrine disruptors alters sex differences in exploration and emotional responses in mice

Estrogenic endocrine disruptors (EEDs) are naturally occurring or man-made compounds present in the environment that are able to bind to estrogen receptors and interfere with normal cellular development in target organs and tissues. There is mounting evidence that EEDs can interfere with the processes of sexual differentiation of brain and behavior in different animal models. We investigated the effects of maternal exposure to EEDs, at concentrations within the range of human exposure and not patently teratogenic, on behavioral responses of male and female house mice (Mus musculus domesticus) before and after puberty. Pregnant dams were trained to spontaneously drink daily doses of corn oil with or without the estrogenic plastic derivative, bisphenol A (BPA 10 microg/kg), or the estrogenic insecticide methoxychlor (MXC 20 microg/kg) from gestation day 11 to postpartum day 8. Their male and female offspring were examined at different ages to examine several components of explorative and emotional behaviors in 3 experimental paradigms: a novelty test before puberty and, as adults, a free-exploratory open-field test and the elevated plus maze test. The main results are sex differences in control mice on a number of behavioral responses at both ages and in all experimental paradigms, while perinatal exposure to BPA or MXC decreased or eliminated such sex differences. The present findings are evidence of long-term consequences of developmental exposure to BPA and MXC on neurobehavioral development and suggest a differential effect of low-dose exposure to these estrogenic chemicals in males and females.     

Endocrine disruptors in bottled mineral water: estrogenic activity in the E-Screen

Human exposure to endocrine disruptors is well documented by biomonitoring data. However, this information is limited to few chemicals like bisphenol A or phthalate plasticizers. To account for so-far unidentified endocrine disruptors and potential mixture effects we employ bioassays to detect endocrine activity in foodstuff and consequently characterize the integrated exposure to endocrine active compounds. Recently, we reported a broad contamination of commercially available bottled water with estrogenic activity and presented evidence for the plastic packaging being a source of this contamination. In continuation of that work, we here compare different sample preparation methods to extract estrogen-like compounds from bottled water. These data demonstrate that inappropriate extraction methods and sample treatment may lead to false-negative results when testing water extracts in bioassays. Using an optimized sample preparation strategy, we furthermore present data on the estrogenic activity of bottled water from France, Germany, and Italy: eleven of the 18 analyzed water samples (61.1%) induced a significant estrogenic response in a bioassay employing a human carcinoma cell line (MCF7, E-Screen). The relative proliferative effects ranged from 19.8 to 50.2% corresponding to an estrogenic activity of 1.9-12.2 pg estradiol equivalents per liter bottled water. When comparing water of the same spring that is packed in glass or plastic bottles made of polyethylene terephthalate (PET), estrogenic activity is three times higher in water from plastic bottles. These data support the hypothesis that PET packaging materials are a source of estrogen-like compounds. Furthermore, the findings presented here conform to previous studies and indicate that the contamination of bottled water with endocrine disruptors is a transnational phenomenon.     

Survey of estrogenic activity in fish feed by yeast estrogen-screen assay

Fishes have been used as laboratory animal for research of estrogenic endocrine disrupters by many researchers. However, much less attention was paid to the possibility that compounds with estrogenic activity are present in fish diets. In order to examine this possibility, we measured the estrogenic activity in commercial fish feed by in vitro yeast estrogen-screen (YES) assay based on the binding ability of tested compounds to estrogen receptors. Estrogenic activity was detected in all the commercial fish feed examined (0.2-6.2 ng estradiol equivalent/g fish feed), some phytoestrogens (genistein, formononetin, equol and coumestrol; relative activity to estradiol, 8.6 x 10(-6)-1.1 x 10(-4) by giving a value of 1.0 to estradiol) and some androgens (testosterone, 11-ketotestosterone and 5 alpha-dihydrotestosterone; relative activity to estradiol, 3.0 x 10(-6)-1.2 x 10(-4)). Therefore, it is possible that these compounds could affect the results of in vivo estrogen assay, such as vitellogenin production in male fish, especially when fish are fed commercial feed.     

Alternatives to animal experimentation for hormonal compounds research

Alternatives to animal testing and the identification of reliable methods that may decrease the need for animals are currently the subject of intense investigation worldwide. Alternative testing procedures are particularly important for synthetic and natural chemicals that exert their biological actions through binding nuclear receptors, called nuclear receptors-interacting compounds (NR-ICs), for which research is increasingly emphasizing the limits of several models in the accurate estimation of the physiological consequences of exposure to these compounds. In particular, estrogen receptor interacting compounds (ER-ICs) have a great impact on human health from the therapeutic, nutritional, and toxicological point of view due to the highly permissive nature of the estrogen receptors towards a large number of natural and synthetic compounds. Similar to in vitro systems, recently generated animal models (e.g., animal models generated for the study of estrogen receptor ligands) may fulfill the 3R principles: refine, reduce, and replace. If used correctly, NR-regulated models, such as reporter mice, xenopus, or zebrafish, and models obtained by somatic gene transfer in reporter systems, combined with imaging technologies, may contribute to strongly decreasing the overall number of animals required for NR-IC testing and research. With these models, flexible and highly standardized parameters and reporter marker quantification can be obtained. Here, we highlight the need for the substitution of currently used testing models with more appropriate ones that can reproduce the features and reactivity of specific mammalian target tissue/organs. We consider the promotion of this advancement a research priority bearing scientific, economic, social, and ethical relevance.     

Use and role of invertebrate models in endocrine disruptor research and testing

Historically, invertebrates have been excellent models for studying endocrine systems and for testing toxic chemicals. Some invertebrate endocrine systems are well suited for testing chemicals and environmental media because of the ease of using certain species, their sensitivity to toxic chemicals, and the broad choice of models from which to choose. Such assays will be useful in identifying endocrine disruptors to protect invertebrate populations and as screening systems for vertebrates. Hormone systems are found in all animal phyla, although the most simple animals may have only rudimentary endocrine systems. Invertebrate endocrine systems use a variety of types of hormones, including steroids, peptides, simple amides, and terpenes. The most well-studied hormone systems are the molting and juvenile hormones in insects, the molting hormones in crustaceans, and several of the neurohormones in molluscs and arthropods. These groups offer several options for assays that may be useful for predicting endocrine disruption in invertebrates. A few invertebrate phyla offer predictive capabilities for understanding vertebrate endocrine-disrupting chemicals. The echinoderms, and to a lesser extent molluscs, have closer evolutionary relationships with the vertebrates than the arthropods and these phyla. The recently identified estrogen receptor structure within the genome of the marine gastropod, Aplysia, indicates that the estrogens, and probably the basic steroid receptor, are quite old evolutionarily. This review of the recent literature confirms the effects of some endocrine-disrupting chemicals on invertebrates--tributyltin on snails, pesticides on insects and crustaceans, and industrial compounds on marine animals.     

A treatise on hazards of endocrine disruptors and tool to evaluate them

Hormones mediate a major part of our essential physiological functions. Both endogenous and exogenous compounds and their metabolites are known to act through hormone receptors leading to regulation of endocrine function. The endogenous ligands that control reproductive functions are generally steroids such as 17beta-estradiol, androgens, progesterone, pregnenolone and glucocorticoids. However, exogenous compounds that are structurally and functionally similar gain entry into animals including humans through the diet or by occupational exposures, causing endocrine disruption. In the recent decade, there is a lot of apprehension about the so-called "endocrine disruptors" which are wide spread in the environment, mainly due to unrestricted human activity. These compounds of anthropogenic or natural origin mimic the action of sex hormones and can interfere with the endocrine system. It has been hypothesized that environmental exposure to synthetic estrogenic chemicals and related endocrine active compounds may be responsible for malformations in the male reproductive tract, crytorchidism, hypospadias, decrease in sperm counts, decreased male reproductive capacity and even testicular cancers. The increasing concern in both public and scientific communities about these abnormalities have prompted the initiation of epidemiological studies to not only identify, but to also analyze the short and long term effects of endocrine disruptors. As a result, a number of assays have been developed and are undergoing validation aimed at high throughput screening of chemical agents that disrupt endocrine activity. This review consolidates the findings of epidemiological studies, particularly in relation to male reproductive disorders and brings to light the various types of in vitro and in vivo models that are available for tiered testing of suspected compounds.     

缺血性中风动物模型研究现状

利用实验动物或细胞模型完全模拟人类的中风十分困难,动物模型与临床的拟合具有重要意义。本文对目前缺血性中风动物模型研究中的实验动物选择、模型评价标准及造模方法 ,以及主要局灶缺血模型的优缺点进行述评,为缺血性中风的基础和应用研究选择合适的实验动物模型提供参考。     

Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes

Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria".     

Determining Indicators of Exposure and Effects for Endocrine Disrupting Chemicals (EDCs): An Introduction

Endocrine disruptors are characterized by their influence on animal endocrine systems resulting in reproductive, developmental, neurological, and immune dysfunction. The purpose of this overview is to provide the reader with a sense of the activities within the U.S. Environmental Protection Agency (USEPA), in particular NHEERL, that address the many facets of research on endocrine disrupting chemicals (EDCs) and to highlight the approach being taken at the different organizational levels within the USEPA, including screening, testing and evaluating endocrine disrupting chemicals. As a part of this endeavor, the USEPA continues to evaluate the current research activities in order to better understand and refine the process of risk characterization of EDCs. Thus, the participants in this session were asked to review their research within the framework of a better identification of EDC effects, better characterization of those compounds that have endocrine disrupting activity and how to incorporate this information into the risk assessment paradigm. Specifically, the goals of the ensuing papers were to compare individual vs. population indicators of endocrine disrupting effects, examine comparable and multiple mechanisms of toxicity, and describe the use of effects as indicators to identify toxicants and their sources. Mammalian and fish reproductive endpoints served as models to emphasize commonalities between human and wildlife risks.     

Commentary: setting aside tradition when dealing with endocrine disruptors

In 1996, the US Congress directed the Environmental Protection Agency to produce screens and assays to detect estrogenic and other endocrine-disrupting chemicals in food and water. To date, there are none. Years have been wasted in attempts to utilize traditional toxicological approaches to solve the problem, when in retrospect, it is now apparent that the delay in part stems from the reluctance to attack the problem with entirely new approaches. To develop new testing protocols, it is necessary to set aside much of the dogma of toxicology and to begin again with open minds. A few pertinent examples are provided concerning what has been overlooked and what needs to be done. In particular, it is necessary to give close attention to the selection of animal strain and diet, factors that were only loosely controlled historically when one takes into consideration what has been learned in the last decade. Vast numbers of animals have been sacrificed, and more will be sacrificed, in futile attempts to validate assays and to develop safety standards unless knowledge gained over the past decade concerning the sensitivity and complexity of the endocrine system is taken into consideration.     

Endocrine disrupting compounds: effect of octylphenol on reproduction over three generations

With the growing concern that environmental chemicals might impair human and animal fertility, it is important to investigate the possible influence of these substances on sexual differentiation and genital development of mammals. Many of these substances are suspected to interfere with endocrine processes, and exposure during critical periods of prenatal development might affect reproductive performance over several generations. Alkylphenols and their metabolites are lipophilic substances exerting apparent estrogenic action in in vitro and in vivo testing systems. With the widespread industrial use of alkylphenols, these are disseminated in the environment with sewage sludge, and domestic animals and humans are likely to be exposed via the food chain. Using the pig as an in vivo model, we studied the effect of intrauterine exposure to tertiary octylphenol (OP) on essential reproductive parameters over 3 generations. Sows were treated daily from D 23 to 85 of pregnancy with either 0, 10 or 1000 micrograms OP/kg body weight. Treatment with OP extended pregnancy length and induced basal cell proliferation in the cervical epithelium of the parental generation. In F1 offspring of sows treated with the low dosage of OP, onset of puberty was accelerated. Furthermore, when F1 gilts and F1 boars originating from sows treated with high dosages of OP were bred, the litter size was reduced. The results of the present study are compared with previous reports on estrogenicity of OP, and the usefulness of in vivo animal or embryo models for the evaluation of possible consequences of human exposure to endocrine disrupting compounds is discussed. Furthermore, possible consequences of exposure to endocrine disrupting compounds for the embryo transfer industry are addressed.     

Phytoestrogen content of purified, open- and closed-formula laboratory animal diets.

BACKGROUND AND PURPOSE: Phytoestrogens exert estrogenic effects on the central nervous system, induce estrus, and stimulate growth of the genital tract of female animals. Over 300 plants and plant products, including some used in laboratory animal diets, contain phytoestrogens. Therefore, the source and concentration of phytoestrogens in rodent diets were determined. METHODS: Twelve rodent diets and six major dietary ingredients were assayed for phytoestrogens (daidzein, genistein, formononetin, biochanin A, and coumestrol), using high-performance liquid chromatography. Three rodent diets recently formulated to reduce phytoestrogen content also were assayed. RESULTS: Formononetin, biochanin A, and coumestrol were not detected. Soybean meal was the major source of daidzein and genistein; their concentrations were directly correlated to the percentage of soybean meal in each diet. CONCLUSIONS: High, variable concentrations of daidzein and genistein are present in some rodent diets, and dietary phytoestrogens have the potential to alter results of studies of estrogenicity. Careful attention should be given to diet phytoestrogen content, and their concentration should be reported. A standardized, open-formula diet in which estrogenic substances have been reduced to levels that do not alter results of studies that are influenced by exogenous estrogens is recommended.     

Regulatory issues surrounding the use of companion animals in clinical investigations,trials, and studies

Laboratory animal veterinarians sometimes encounter animals with rare conditions and may subsequently become involved in the performance of related animal research outside the laboratory, in homes, in veterinary clinics, or in universities to which owners have donated their animals for study. Similarly, veterinarians may monitor animal companion vaccination studies, performed to optimize preventive health care or minimize physiological variability and research confounders associated with a preventive medicine program for dogs and cats utilized for research procedures. These nontraditional uses of dogs, cats, and other companion animals in research have spurred the establishment of regulations to ensure that the animals benefit from clinical veterinary products and techniques. Included and described are the 2002 Public Health Service Policy, the Animal Welfare Act (AWA), the Federal Food, Drug, and Cosmetic Act, and the regulations of the US Department of Agriculture in response to the AWA. The complexities of clinical research with companion animals outside standard biomedical research facilities are discussed.