Low-dose effects of hormones and endocrine disruptors.

Endogenous hormones have effects on tissue morphology, cell physiology, and behaviors at low doses. In fact, hormones are known to circulate in the part-per-trillion and part-per-billion concentrations, making them highly effective and potent signaling molecules. Many endocrine-disrupting chemicals (EDCs) mimic hormones, yet there is strong debate over whether these chemicals can also have effects at low doses. In the 1990s, scientists proposed the "low-dose hypothesis," which postulated that EDCs affect humans and animals at environmentally relevant doses. This chapter focuses on data that support and refute the low-dose hypothesis. A case study examining the highly controversial example of bisphenol A and its low-dose effects on the prostate is examined through the lens of endocrinology. Finally, the chapter concludes with a discussion of factors that can influence the ability of a study to detect and interpret low-dose effects appropriately.

Opposing roles of corticotropin-releasing factor and neuropeptide Y within the dorsolateral bed nucleus of the stria terminalis in the negative affective component of pain in rats.

Pain is a complex experience composed of sensory and affective components. Although the neural systems of the sensory component of pain have been studied extensively, those of its affective component remain to be determined. In the present study, we examined the effects of corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) injected into the dorsolateral bed nucleus of the stria terminalis (dlBNST) on pain-induced aversion and nociceptive behaviors in rats to examine the roles of these peptides in affective and sensory components of pain, respectively. In vivo microdialysis showed that formalin-evoked pain enhanced the release of CRF in this brain region. Using a conditioned place aversion (CPA) test, we found that intra-dlBNST injection of a CRF1 or CRF2 receptor antagonist suppressed pain-induced aversion. Intra-dlBNST CRF injection induced CPA even in the absence of pain stimulation. On the other hand, intra-dlBNST NPY injection suppressed pain-induced aversion. Coadministration of NPY inhibited CRF-induced CPA. This inhibitory effect of NPY was blocked by coadministration of a Y1 or Y5 receptor antagonist. Furthermore, whole-cell patch-clamp electrophysiology in dlBNST slices revealed that CRF increased neuronal excitability specifically in type II dlBNST neurons, whereas NPY decreased it in these neurons. Excitatory effects of CRF on type II dlBNST neurons were suppressed by NPY. These results have uncovered some of the neuronal mechanisms underlying the affective component of pain by showing opposing roles of intra-dlBNST CRF and NPY in pain-induced aversion and opposing actions of these peptides on neuronal excitability converging on the same target, type II neurons, within the dlBNST.

Endocrine disrupters: a human risk?

Endocrine disrupters (EDs) alter normal hormonal regulation and may be naturally occurring or environmental contaminants. Classically, EDs act genomically, with agonistic or antagonistic effects on steroid receptors and may alter reproductive function and/or cause feminisation by binding to oestrogen or androgen receptors; their binding to the thyroid receptor may dysregulate the neuroendocrine system. Recently, it has been shown that EDs can also act by non-genomic mechanisms, altering steroid synthesis (inhibition of cytochrome P450 isoforms) or steroid metabolism. The alkylphenol and phthalate plasticisers inhibit the inactivation of oestrogens by sulphation (via SULT 1A1 and 1E1 isoforms) and so cause a rise in levels of the free active endogenous oestrogens. A range of ED effects have been shown in mammals, fish, birds, reptiles, amphibia and aquatic invertebrates but it is not yet clear whether these processes also occur in human beings. It is evident that EDs, as well as altering reproduction, can cause changes in neurosteroid levels and so have the potential to affect immune function, behaviour and memory. This may be of long-term concern since traces of EDs such as plasticisers, brominated fire retardants, sunscreen agents and cosmetic ingredients are widely distributed in the environment and in human biofluids.

Azapeptides as pharmacological agents.

Azapeptides, formed by replacing the C(alpha) of amino acid residues by nitrogen, are promising peptidomimetic compounds. Azaamino acids impart a unique conformational property to peptide structures because of the loss of chirality and reduction of flexibility of the parent linear peptide. The peculiar conformational properties make azaamino acids an attractive tool for drug design involving specific secondary structures in peptides and proteins. Additionally, since azapeptides are less susceptible to enzymatic breakdown by proteases, they may possibly lead to orally active drugs with longer duration of action. One of the advantages of azapeptides is their unproblematic synthesis allowing retention of the amino acid side chain. Azapeptides have been developed by several groups for the design of hormone analogues, protease inhibitors and active site titrants.

Hormonal changes with cholesterol reduction: a double-blind pilot study.

BACKGROUND: The lowering of high serum cholesterol levels may be associated with increased non-cardiac mortality due to behavioral changes, although such endpoints are likely rare. OBJECTIVE: This current study sought to determine if hormonal changes accompany pharmacologically induced decreases in serum cholesterol levels. METHOD: Cholesterol, dopamine, homovanillic acid (HVA), serotonin, 5-HIAA, testosterone, cortisol and pregnenolone were measured at baseline and after 4 weeks of treatment. RESULTS: Subjects' cholesterol levels significantly declined within 4 weeks. Concomitant significant increase in dopamine and HVA were noted. CONCLUSION: Although this study is limited in size, it raises the possibility that cholesterol-lowering drug treatment is associated with hormonal perturbations.

Is exposure to endocrine disrupting compounds during fetal/post-natal development affecting the reproductive potential of farm animals?

Concerns have been raised about the potential adverse effects on reproductive health and immune status of farm animals following exposure to a range of natural and synthetic environmental compounds that disrupt normal hormonal actions. These compounds range from natural plant oestrogens (e.g. genistein, coumesterol) and mycoestrogens (e.g. Aflatoxins, zearalenone) to growth promoting pharmaceuticals (e.g. trenbolone acetate, melengastrol acetate) to chemicals spread in water, sewage sludge or the atmosphere such as detergents and surfactants (e.g. octylphenol, nonylphenol), plastics (e.g. bisphenol-A, phthalates), pesticides (e.g. methoxychlor, dieldrin, DDT) and industrial chemicals (e.g. PCB, TCDD). These compounds are commonly termed 'endocrine disrupting compounds' (EDCs) or 'endocrine disruptors' due to their ability to act as either hormone agonists or antagonists or the ability to disrupt hormone synthesis, storage or metabolism. A similar group of compounds are called 'immunotoxicants' and are thought to affect the immune system mainly by disrupting B and T cell homeostasis. As more studies are performed it is becoming clear that many compounds can directly or indirectly affect both the endocrine and immune systems. The susceptibility of target tissues is related to the stage of development, the cumulative exposure dose and the immune status of the individual. While some of the effects of the EDCs on the endocrine and immune systems are quite distinct, many are subtle and identifying the causative agent from the vast array of environmental challenges including EDCs, nutrition, temperature, etc. can be problematic. Identifying the causative agent is confounded by the possibility that effects that are observed in the adult may be due to exposure to EDCs during fetal life. This has major implications for the determination of universal end-point measurements to assess exposure to EDCs in farm animals.

Amphibians as a model for the study of endocrine disruptors.

Evidence shows that environmental compounds can interfere with the endocrine systems of wildlife and humans. The main sink of such substances, called endocrine disruptors (EDs), which are mainly of anthropogenic origin, is surface water; thus, aquatic vertebrates such as fishes and amphibians are most endangered. Despite numerous reports on EDs in fishes, information about EDs in amphibians is scarce, and this paucity of information is of particular concern in view of the worldwide decline of amphibians. EDs could contribute to changes of amphibian populations via adverse effects on reproduction and the thyroid system. In amphibians, EDs can affect reproduction by (anti)estrogenic and (anti)androgenic modes of action that produce severe effects including abnormal sexual differentiation. ED actions on the thyroid system cause acceleration or retardation of metamorphosis, which may also affect population levels. Our broad knowledge of amphibian biology and endocrinology indicates that amphibians are very suitable models for the study of EDs. In particular, effects of EDs on the thyroid system triggering metamorphosis can be determined easily and most sensitively in amphibians compared to other vertebrates. A new classification of EDs according to their biological modes of action is proposed because EDs have quite heterogeneous chemical structures, which do not allow prediction of their biological effects. Methods and strategies are proposed for identification and risk assessment of EDs, whether as pure test substances or as mixtures from environmental samples. Effects of EDs on the thyroid system of amphibians can be assessed by a single animal model (Xenopus laevis), whereas the various types of reproduction need comparative studies to investigate whether general endocrine principles do exist among several species of anurans and urodeles. Thus, at least one anuran and one urodelean model are needed to determine ED interference with reproduction.

How can chemical compounds alter human fertility?

The effects of environmental toxins, such as pesticides, solvents and industrial waste, on human and animal health have caused much public fear. The suggested mechanism of action for these xenobiotics is their capacity to interact with steroid hormones receptors, in particular those for estrogens and androgens. Concern was reinforced by the "historical" example of diethylstilbestrol, an estradiol mimetic causing genital cancer in girls exposed in utero. The real harm of these environmental xenobiotics is controversial. Some authors estimate that they do not reach sufficiently high concentrations to do damage and much experimental work has been done. In this review, we summarise the latest findings on the molecular mechanisms of action of three environmental toxicants, xenohormones, dioxin and glycol ethers and compare animal and cell experimental model data with epidemiological studies.

Assessment of environmental endocrine disruptors in bald eagles of the Great Lakes.

Environmental endocrine disruption in wildlife has primarily focused on estrogenic/androgenic end points and their antagonists. We describe here the work that has occurred within the Great Lakes of North America that has used the bald eagle (Haliaeetus leucocephalus) as a sentinel species of the effects of environmental toxicants, including endocrine disruption. Our data suggests that population level effects of hormone disrupting chemicals, not necessarily estrogen/androgen mimics and their antagonists, have been associated with reproductive and teratogenic effects observed in the bald eagle population within the Great Lakes Basin. Additional laboratory and field studies are necessary to further clarify the role of environmental endocrine disruptors on reproduction in avian populations. The use of sea eagles (Haliaeetus spp.) as biosentinels of pollution in other regions of the world is also discussed.

Rational discovery of novel nuclear hormone receptor antagonists.

Nuclear hormone receptors (NRs) are potential targets for therapeutic approaches to many clinical conditions, including cancer, diabetes, and neurological diseases. The crystal structure of the ligand binding domain of agonist-bound NRs enables the design of compounds with agonist activity. However, with the exception of the human estrogen receptor-alpha, the lack of antagonist-bound "inactive" receptor structures hinders the rational design of receptor antagonists. In this study, we present a strategy for designing such antagonists. We constructed a model of the inactive conformation of human retinoic acid receptor-alpha by using information derived from antagonist-bound estrogen receptor-alpha and applied a computer-based virtual screening algorithm to identify retinoic acid receptor antagonists. Thus, the currently available crystal structures of NRs may be used for the rational design of antagonists, which could lead to the development of novel drugs for a variety of diseases.

Current status of anabolic hormone administration in human burn injury.

Survival after massive burns has increased due to advances in critical care and wound closure techniques. Because of the ravages of hypermetabolism that is so prevalent in these patients, survivors are left with significant lean body mass losses that correspond to decreased strength with which to begin the rehabilitation phase. Efforts to decrease lean body mass catabolism by environmental regulation, early wound closure, and sufficient caloric provision modify the hypermetabolic response to an extent; however, further manipulations are required to optimize recovery fully. Pharmacologic intervention with hormone agonists and antagonists holds this promise. This article reviews some of the current investigations in this area and points out the future work that needs to be done to elucidate the field of anabolic hormones after severe injury.

Biological regulation of receptor-hormone complex concentrations in relation to dose-response assessments for endocrine-active compounds.

Some endocrine-active compounds (EACs) act as agonists or antagonists of specific hormones and may interfere with cellular control processes that regulate gene transcription. Many mechanisms controlling gene expression are universal to organisms ranging from unicellular bacteria to more complex plants and animals. One mechanism, coordinated control of batteries of gene products, is critical in adaptation of bacteria to new environments and for development and tissue differentiation in multi-cellular organisms. To coordinately activate sets of genes, all living organisms have devised molecular modules to permit transitions, or switching, between different functional states over a small range of hormone concentration, and other modules to stabilize the new state through homeostatic interactions. Both switching and homeostasis are regulated by controlling concentrations of hormone-receptor complexes. Molecular control processes for switching and homeostasis are inherently nonlinear and often utilize autoregulatory feedback loops. Among the biological processes contributing to switching phenomena are receptor autoinduction, induction of enzymes for ligand synthesis, mRNA stabilization/activation, and receptor polymerization. This paper discusses a variety of molecular switches found in animal species, devises simple quantitative models illustrating roles of specific molecular interactions in creating switching modules, and outlines the impact of these switching processes and other feedback loops for risk assessments with EACs. Quantitative simulation modeling of these switching mechanisms made it apparent that highly nonlinear dose-response curves for hormones and EACs readily arise from interactions of several linear processes acting in concert on a common control point. These nonlinear mechanisms involve amplification of response, rather than multimeric molecular interactions as in conventional Hill relationships.

New screening methods for chemicals with hormonal activities using interaction of nuclear hormone receptor with coactivator.

The endocrine system exerts important functions in a multitude of physiological processes including embryogenesis, differentiation, and homeostasis. Xenobiotics may modify natural endocrine function and so affect human health and wildlife. It is necessary, therefore, to understand the degree to which xenobiotics can disrupt endocrine systems. The key targets of endocrine disruptors are nuclear hormone receptors, which bind to steroid hormones and regulate their gene transcription. We have developed relevant assay systems based on the ligand-dependent interaction between nuclear hormone receptor and coactivator. The coactivators used in this study contained CBP, p300, RIP140, SRC1, TIF1, and TIF2. By two hybrid assay in yeast, the interactions of estrogen receptor with RIP140, SRC1, TIF1, and TIF2 were detected and they were completely dependent on the presence of estrogen. Specificity of this assay was assessed by determining the effect of steroids, known estrogen receptor agonists, and phytoestrogens. The pattern of response to chemicals were consistent with estrogenic activity measured by other assay systems, indicating that this assay system is reliable for measuring estrogenic activity. In addition, we carried out in vitro binding studies: GST pull-down assay and surface plasmon resonance analysis. The estrogen receptor also bound to coactivator in response to chemicals depending on their estrogenic activity in vitro. These data demonstrate that the measurement of interaction between steroid hormone receptor and coactivator serves as a useful tool for identifying chemicals that interact with steroid receptors.

Molecular determinants of hormone mimicry: halogenated aromatic hydrocarbon environmental agents.

The potential of ostensibly structurally diverse environmental chemicals to modulate endocrine processes in biological systems has been recognized. Difficulty in classifying endocrine system modulators by chemical structure may in large part be due to lack of understanding of mechanisms of action. New developments in understanding nuclear receptor mechanisms of hormone action support a more complex mechanism, possibly involving dimerization/aggregation events leading to multimeric receptor complexes in agonist action. Because of the requirement for high structural specificity in agonist action, it is suggested that most environmental chemicals of concern are likely to function as imperfect hormones with partial agonist-antagonist properties, especially at environmentally realistic concentrations. In the absence of having appropriately placed molecular recognition domains to affect agonist action, partial agonism-antagonism may be associated with favorable low-energy conformational flexibility and complementary receptor protein flexibility. The halogenated aromatic hydrocarbons are of particular concern as hormone mimics since they often have (1) similar molecular recognition factors but in many cases relatively more flexible structures, (2) similar bulk physico-chemical properties controlling uptake and distribution in biological systems, and (3) are relatively more resistant to metabolism and elimination. Some important molecular reactivity properties underlying thyromimetic and estrogenic actions of some of these chemicals are identified and described in terms of structure-activity relationships (SARs). It is proposed that specificity of hormone action in the nucleus could be associated with differential interaction of ligand-bound receptor dimeric forms with other transcription factors specific to the target cell. The small-molecule ligand can be viewed as playing a central, multifunctional role in nuclear receptor action as an organic unmasking and reclustering agent for critical macromolecules. Evidence is discussed in support of a nuclear heterodimerization model for dioxin and related compound action involving a structural transition mechanism. These models with some molecular detail also have utility in understanding the different structural properties of agonists and antagonists. There would appear to be ample opportunities for environmental chemicals to act as antagonists for multiple receptor systems with little more than anchor-ring similarities in structure. The application of three-dimensional quantitative structure-activity (3D QSAR) models incorporating such structural information should be a useful adjunct for identifying endocrine system modulating chemicals. This data has implications for (1) improved drug design, (2) understanding of chemical interaction toxicity, (3) removing undesirable chemicals from our environment, and (4) reducing their chemical release.

9-cis retinoic acid induces the expression of the uncoupling protein-2 gene in brown adipocytes.

The expression of uncoupling protein-2 (UCP2) mRNA is up-regulated during the differentiation of brown adipocytes in primary culture. When differentiation of brown adipocytes is impaired, UCP2 mRNA expression is down-regulated. 9-cis Retinoic acid causes a dose-dependent induction of UCP2 mRNA levels in brown adipocytes, whereas all-trans retinoic acid has no effect. Specific agonists of retinoid X receptors (RXR) induce UCP2 mRNA expression, whereas specific activators of retinoic acid receptors do not. 9-cis Retinoic acid, acting through RXR receptors, is identified as a major regulator of the expression of the UCP2 gene in the brown fat cell.