Concern over endocrine disruptors has been further heightened because of reports that such compounds can elicit unique low-dose effects and exhibit nonmonotonic dose-response relationships that may not be detected in regulatory toxicity studies, which are typically conducted at higher dose levels. The low-dose hypothesis has been a contentious issue among some toxicologists. EPA, organized a panel to evaluate the scientific evidence on the low-dose effects of endocrine disruptors Melnick et al.
EPA for evaluating reproductive and developmental toxicity. Some reasons identified for the inability to reproduce low-dose effects across studies included intrauterine position, strain and substrain differences in response, dietary levels of phytoestrogens or differences in caloric intake, differences in housing conditions caging, bedding, etc.
The statistics subgroup Haseman et al. Ultimately, the U. Following the NTP low-dose report, a number of key studies by Naciff et al. These studies collectively provide support for monotonic dose-responses for these compounds and showed negligible changes in gene expression at very low-dose levels. Furthermore, several comprehensive weight-of-evidence reviews for BPA have concluded that nonmonotonic dose-response curves and low-dose effects from BPA are not well supported by available data Goodman et al. Despite these reports, the Endocrine Society issued a scientific statement on endocrine-disrupting chemicals that included support of the low-dose hypothesis Diamanti-Kandarakis et al.
The low-dose controversy has evolved to consider issues related to exposures to mixtures of EACs at low, environmentally relevant dose levels. Although risk assessment approaches for cumulative and aggregate risk have been in place for many years reviewed in Lipscomb et al. Mixtures have been reported to have additive effects e. Overall, data indicate that the interactions of chemicals in mixtures are dose dependent. Using mixture data in the context of environmental risk assessment is even more complex when one considers natural exposures to EACs in food and pharmaceuticals Gaido et al.
Exposures to environmental chemicals typically involve low doses of each chemical within a mixture and, given the safety factors used in risk assessment, exposures generally below the threshold for toxicity. Thus, mixtures research requires a focus on the behavior of mixtures at environmentally relevant exposures because results obtained at high-dose levels may not apply in real-world scenarios. When the history of endocrinology is written, it is likely that the 20th century will be neatly divided into two epochs with the first half being the era of biochemistry when the hormones were discovered and the emphasis was on proteins and the second half of the 20th century, the era of genomics when the genes for the hormone and nuclear receptors NRs were identified.
The word hormone was first coined in , but it was not until that the first hormones were characterized in detail when Kendeall and Reichstein isolated and determined the structures of cortisone and thyroxine The Nobel Assembly at Karolinska Institutet, Soon thereafter, in , Adolf Butenandt and Edward Adelbert Doisy independently isolated and determined the structure of estrogen Tata, These discoveries along with other experimental observations allowed Clark to propose his model for receptor-mediated processes in his publication of the theory of occupancy. Under normal physiological conditions, the intracellular NRs are bound by their endogenous signaling molecules, or receptor ligands, such as the steroid hormones, thyroid hormones, retinoids, vitamin D, and other as yet identified endogenous ligands.
These receptor ligands either diffuse directly across the plasma membrane of cells or are sometimes produced in the same cells and bind to the NR. The commonly understood mechanism of action for NRs involves the initial ligand binding that results in receptor activation, binding at specific DNA response elements in nuclear chromatin, followed by recruitment of a variety of cofactors that modify the chromosomal organization and interaction with the transcription complex, thereby altering the rates of transcription of genes.
So, binding of a ligand to an NR is only the first step in a complex cascade of events leading to changes in gene expression and subsequent cell signaling. With the completion of the human genome, it is now clear that there are some 48 NRs expressed in humans, of which only half have identified ligands such as steroid hormone receptors that constitute only a minority of this large superfamily of receptors Gronemeyer et al. Like the NR superfamily, coregulators are themselves a large and mixed family of proteins and possess a varied array of enzymatic activities necessary for chromosomal maintenance and gene regulation Roeder, They can be categorized based upon their wide-ranging functional properties including acetyltransferases, ubiquitin ligases, ATP-coupled chromatin remodeling complexes, protein methylases, cell cycle regulators, RNA helicases, and bridging proteins that facilitate direct contact with components of the basal transcription machinery McKenna and O'Malley, There are currently coregulators identified and many are expressed differentially by cell type, tissue type, gender, developmental stage, and species Lonard et al.
Moreover, coregulators can be differentially recruited to the same receptor by ligand-induced conformational changes in the NR. Thus, with 48 NRs and plus coregulators, the complexity of these endocrine signaling pathways is apparent.
For example, a given ligand may be an agonist for a gene in a cell and at the same time be an antagonist for a different gene in the same cell; or a ligand can be an agonist for a gene in one cell type and an antagonist for the same gene in a different cell type or even the same cell type but under different conditions. Potency is a conditional measure that depends on parameters of affinity and efficacy, as well as tissue receptor numbers and ligand bioavailability.
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These differences in the properties of ligands are often lost in the discussion of EACs. What this means in practical terms is that a cell would need to be exposed to 10, molecules of BPA to produce the same effect through the ER that one molecule of estradiol would produce through that same receptor.
Furthermore, the amount of BPA that one would need to consume, breath, or absorb through the skin would be even greater because the absorption, distribution metabolism, and elimination of the BPA would limit the amount of BPA available to the cells. Even if an EAC was highly potent in one cell type, it does not mean it will have the same efficacy in another cell type or the same cell type under different conditions as efficacy is independent of ligand affinity and instead reflects a ligand's ability to induce a stable coactivator binding site on the NR discussed further below.
Thus, two ligands can have similar potencies for inducing a response through an NR, but the efficacy for these two ligands inducing the response can vary greatly and this can have a profound impact on the ability of these ligands to produce the same apical effects through the NR. The lower potencies and different efficacies of EACs relative to the natural hormones need to be understood and considered in the discussions of the potential impact of EACs on human health and the environment. It took another three decades after the elucidation of the structure of estrogen before the ER was isolated in by Elwood Jensen Tata, and another two decades passed before the new tools of molecular biology allowed Pierre Chambon's group to clone the gene for the human ER Green et al.
No doubt there will be additional chemicals identified as ligands for some of the 48 NRs identified in humans, thereby necessitating an understanding of their potential for behaving as EACs. One of the best understood examples of an endocrine signaling system that brings all these concepts together is that of estrogens. The biological effects elicited by specific ER ligands are highly cell and tissue specific. One of the best known examples of this phenomenon is the drug tamoxifen, which has been used for treatment of early stage breast cancer and as a chemoprevention agent for women at high risk for this disease Jordan, In addition, only a small percentage of ER ligands are pure estrogen agonists or antagonists.
Most phytoestrogens and xenoestrogens are partial agonists, meaning that they are unable to induce a maximal response matching that of the endogenous ligand, even at very high concentrations Zhu, The latter compounds tend to act as agonists under some conditions but act as antagonists when their concentration is very high relative to more potent ligands.
The research from numerous laboratories over the last five decades has revealed the explanations for many of these observations. Differences in the tissue-specific receptor agonist and antagonist activities have long been recognized for the steroid hormone receptors and form the basis for the development of drugs for treatment of hormone-dependent diseases Ai et al. Tamoxifen's tissue-specific activity is now thought to be because of recruitment of different coregulators to the ER by tamoxifen in breast tissue versus uterine and bone tissue Gronemeyer et al.
Selective receptor modulators SRMs have been observed for other NRs including peroxisome proliferator—activated receptors, ARs, progesterone receptors, as well as the AhR Gronemeyer et al. Thus, SRM appears to be a common feature of ligand-activated transcription factors that explains the observed selectivity that different ligands confer to NRs on modulation of gene expression and the resultant tissue changes both within and across tissue and cell types Ai et al. The actions of nuclear receptors are ligand dependent and cell context dependent.
This in turn causes the receptors to recruit different coregulator complexes, e. In Cell A, the two different ligands EAC-1 and EAC-2 bind to the same receptor but induce two different tertiary structures, which in turn cause the receptor to recruit a coactivator complex in the case of EAC-1 bound receptor or a corepressor complex in the case of EAC-2 bound receptor.
Whereas in Cell B, the opposite occurs with EAC-1 producing a corepressor complex recruitment through the receptor, but EAC-2 produces a coactivator complex recruitment through the receptor. Although not shown, the gene architecture influences nuclear receptor-coregulator recruitment as well. All the discoveries discussed above and more have been harnessed to develop in vitro and in vivo assays to identify chemicals with endocrine-modulating activities.
The very nature of an NR being a trans -acting transcriptional regulator allows for their exogenous expression in numerous cell types and cell lines to study the effects of potential ligands as agonists, antagonists, etc. These studies have led to the adoption of some assays into the EDSP battery of tests and many others that are used in the ToxCast panel of assays. The cloning of these receptors has also facilitated the development of NR knockout KO mouse models that provided important confirmatory evidence for the physiological role of hormones in development, reproduction, and normal physiology as well as some unexpected observations.
With the advent of the KO models, it was possible to develop transgenic receptor knock in mouse models that express human NRs in place of their endogenous mouse receptor. The development of the ER KO mouse ERKO mouse by the National Institute of Environmental Health Sciences group led by Ken Korach demonstrated that both sexes of these mutant animals are infertile and show a variety of phenotypic changes, some of which are associated with the gonads, mammary glands, reproductive tracts, and skeletal tissues Lubahn et al.
The ERKO mice and other hormone receptor KO mouse models can be used to confirm the endocrine effects of putative EACs and demonstrate important differences in the effects produced by endocrine chemicals compared with endogenous hormones. By knocking in the human gene for an NR into the appropriate receptor KO mouse, new transgenic mouse models are made that allow comparison of putative EACs acting through the human receptor or mouse receptor.
The observations from such studies are important to consider in the weight of evidence for predicting the effects of EACs to humans. Future mouse models are being developed that express the human proteins for multiple NRs and accessory proteins, and these will allow further refinement of the effects of EACs on human pathways. In addition, the technology is now available to make KO rats and other species, and this will be followed by the ability to humanize these models as well, thereby furthering our ability to understand the human relevance for EACs identified in wild-type laboratory models and in vitro assays.
Endocrinology has come far since the term hormones were first introduced in What started as a simple concept has evolved into a very complex array of hormones, receptors, and cofactors. Because a chemical ligand for an NR can have multiple intrinsic efficacies i. With suspect EACs possessing the potential for multiple induced NR responses, the validity for transferring inferences about the efficacy of the EAC-induced effect obtained for one response to another response is questionable.
Moreover, the problem of confounding is even more problematic when considering that measured parameters of in vitro high-throughput screening systems are by nature different from those found for receptor systems in vivo. Therefore, the results from these in vitro screening systems may seldom accurately reflect the intrinsic efficacies of NR ligands in vivo and challenge the validity for extrapolating observations to humans. Even if a single in vitro system was reflective of a human response under certain conditions, the fact that the intrinsic efficacy of an EAC is dependent upon the cellular context of the NR means that multiple systems and or conditions would need to be employed to ensure that all the possible conditions in vitro reflect similar conditions in humans in vivo.
Although this review only touched briefly on some of the key developments to date concerning endocrine-mediated toxicity and safety assessment, it should be readily apparent that the issue has raised more questions and challenges than have been answered. Most likely, we are only at the very beginning of a long journey toward improved understanding of the basic mechanisms of endocrine-mediated toxicity and the extent to which relevant exposures to hormonally active agents impact human and environmental health.
As we forge ahead, this active and often controversial topic has already accelerated the science of toxicology and risk assessment and has redirected it into some fundamentally different directions. One such direction is down. This is not meant in a negative sense but refers to the examination of much lower, environmentally relevant dose levels than have been examined in the traditional high-dose toxicology paradigm.
This new direction was spurred by research, albeit controversial, that hormonally active agents might not adhere to the monotonic dose-response behavior, which has been a fundamental principle of toxicology since its inception as a field of science. However, further progress in this area is possible thanks to modern technology, such as toxicogenomics, which allows one to examine for precursor changes on the cellular and molecular level Naciff et al. In addition, the increased utilization of human, rather than animal, cells holds promise for enhancing human relevance even further.
Another research direction points upward, namely, toward the investigation of higher numbers of chemicals and other stressors and their potential cumulative effects. For years, toxicologists have tended to avoid studying cumulative effects because it was considered an intractable problem. This was especially so for hormonally active agents because of the vast array of ligands and multiplicity of sources, which includes not only man-made chemicals but also compounds in plants, endogenous hormones, and even nonchemical stressors.
However, because of new high-throughput tools, refined statistical methods, and economical experimental designs for mixture studies Gennings, , questions about combined exposures to hormonally active agents can be addressed in a scientifically rigorous and efficient manner. This movement toward evaluating more chemicals is also being combined with a fundamental change toward mechanism-based testing inside out versus the traditional adverse effects-based system outside in as described in the landmark report by the U.
Progress has been made toward implementing this approach. EPA's ToxCast program contains numerous endocrine-related screening assays, including ER and AR receptor binding assays, ER, AR, and thyroid receptor reporter gene assays, an assay for thyrotropin-releasing hormone receptor, and an aromatase enzymatic activity assay Houck, These assays are conducted in high-throughput mode, so results are generated quickly. However, there are concerns about relying on these in vitro and alternative assays for regulatory decisions in determining potential endocrine activity e.
In the short run, these data may contribute to the weight of evidence for some test compounds in determining potential endocrine activity. Finally, future research in this area needs to look outward, meaning that information on specific components of the endocrine system e.
Because the endocrine system integrates responses across distant cells, tissues, and organs, a reductionist approach that solely focuses on isolated components of an endocrine circuit can generate incomplete answers and may even guide public health professionals in the wrong direction. In this same vein, basic principles of toxicology and in particular, toxicokinetics, should not be forgotten and need to be part of any research program if it is to be meaningful for risk assessment.