Burden of disease and costs of exposure to endocrine disrupting chemicals in the European Union: an updated analysis.

A previous report documented that endocrine disrupting chemicals contribute substantially to certain forms of disease and disability. In the present analysis, our main objective was to update a range of health and economic costs that can be reasonably attributed to endocrine disrupting chemical exposures in the European Union, leveraging new burden and disease cost estimates of female reproductive conditions from accompanying report. Expert panels evaluated the epidemiologic evidence, using adapted criteria from the WHO Grading of Recommendations Assessment, Development and Evaluation Working Group, and evaluated laboratory and animal evidence of endocrine disruption using definitions recently promulgated by the Danish Environmental Protection Agency. The Delphi method was used to make decisions on the strength of the data. Expert panels consensus was achieved for probable (>20%) endocrine disrupting chemical causation for IQ loss and associated intellectual disability; autism; attention deficit hyperactivity disorder; endometriosis; fibroids; childhood obesity; adult obesity; adult diabetes; cryptorchidism; male infertility, and mortality associated with reduced testosterone. Accounting for probability of causation, and using the midpoint of each range for probability of causation, Monte Carlo simulations produced a median annual cost of €163 billion (1.28% of EU Gross Domestic Product) across 1000 simulations. We conclude that endocrine disrupting chemical exposures in the EU are likely to contribute substantially to disease and dysfunction across the life course with costs in the hundreds of billions of Euros per year. These estimates represent only those endocrine disrupting chemicals with the highest probability of causation; a broader analysis would have produced greater estimates of burden of disease and costs.

EDC-2: The Endocrine Society's Second Scientific Statement on Endocrine-Disrupting Chemicals.

The Endocrine Society's first Scientific Statement in 2009 provided a wake-up call to the scientific community about how environmental endocrine-disrupting chemicals (EDCs) affect health and disease. Five years later, a substantially larger body of literature has solidified our understanding of plausible mechanisms underlying EDC actions and how exposures in animals and humans-especially during development-may lay the foundations for disease later in life. At this point in history, we have much stronger knowledge about how EDCs alter gene-environment interactions via physiological, cellular, molecular, and epigenetic changes, thereby producing effects in exposed individuals as well as their descendants. Causal links between exposure and manifestation of disease are substantiated by experimental animal models and are consistent with correlative epidemiological data in humans. There are several caveats because differences in how experimental animal work is conducted can lead to difficulties in drawing broad conclusions, and we must continue to be cautious about inferring causality in humans. In this second Scientific Statement, we reviewed the literature on a subset of topics for which the translational evidence is strongest: 1) obesity and diabetes; 2) female reproduction; 3) male reproduction; 4) hormone-sensitive cancers in females; 5) prostate; 6) thyroid; and 7) neurodevelopment and neuroendocrine systems. Our inclusion criteria for studies were those conducted predominantly in the past 5 years deemed to be of high quality based on appropriate negative and positive control groups or populations, adequate sample size and experimental design, and mammalian animal studies with exposure levels in a range that was relevant to humans. We also focused on studies using the developmental origins of health and disease model. No report was excluded based on a positive or negative effect of the EDC exposure. The bulk of the results across the board strengthen the evidence for endocrine health-related actions of EDCs. Based on this much more complete understanding of the endocrine principles by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability, these findings can be much better translated to human health. Armed with this information, researchers, physicians, and other healthcare providers can guide regulators and policymakers as they make responsible decisions.

Executive Summary to EDC-2: The Endocrine Society's Second Scientific Statement on Endocrine-Disrupting Chemicals.

This Executive Summary to the Endocrine Society's second Scientific Statement on environmental endocrine-disrupting chemicals (EDCs) provides a synthesis of the key points of the complete statement. The full Scientific Statement represents a comprehensive review of the literature on seven topics for which there is strong mechanistic, experimental, animal, and epidemiological evidence for endocrine disruption, namely: obesity and diabetes, female reproduction, male reproduction, hormone-sensitive cancers in females, prostate cancer, thyroid, and neurodevelopment and neuroendocrine systems. EDCs such as bisphenol A, phthalates, pesticides, persistent organic pollutants such as polychlorinated biphenyls, polybrominated diethyl ethers, and dioxins were emphasized because these chemicals had the greatest depth and breadth of available information. The Statement also included thorough coverage of studies of developmental exposures to EDCs, especially in the fetus and infant, because these are critical life stages during which perturbations of hormones can increase the probability of a disease or dysfunction later in life. A conclusion of the Statement is that publications over the past 5 years have led to a much fuller understanding of the endocrine principles by which EDCs act, including nonmonotonic dose-responses, low-dose effects, and developmental vulnerability. These findings will prove useful to researchers, physicians, and other healthcare providers in translating the science of endocrine disruption to improved public health.

Designing Endocrine Disruption Out of the Next Generation of Chemicals.

A central goal of green chemistry is to avoid hazard in the design of new chemicals. This objective is best achieved when information about a chemical's potential hazardous effects is obtained as early in the design process as feasible. Endocrine disruption is a type of hazard that to date has been inadequately addressed by both industrial and regulatory science. To aid chemists in avoiding this hazard, we propose an endocrine disruption testing protocol for use by chemists in the design of new chemicals. The Tiered Protocol for Endocrine Disruption (TiPED) has been created under the oversight of a scientific advisory committee composed of leading representatives from both green chemistry and the environmental health sciences. TiPED is conceived as a tool for new chemical design, thus it starts with a chemist theoretically at "the drawing board." It consists of five testing tiers ranging from broad in silico evaluation up through specific cell- and whole organism-based assays. To be effective at detecting endocrine disruption, a testing protocol must be able to measure potential hormone-like or hormone-inhibiting effects of chemicals, as well as the many possible interactions and signaling sequellae such chemicals may have with cell-based receptors. Accordingly, we have designed this protocol to broadly interrogate the endocrine system. The proposed protocol will not detect all possible mechanisms of endocrine disruption, because scientific understanding of these phenomena is advancing rapidly. To ensure that the protocol remains current, we have established a plan for incorporating new assays into the protocol as the science advances. In this paper we present the principles that should guide the science of testing new chemicals for endocrine disruption, as well as principles by which to evaluate individual assays for applicability, and laboratories for reliability. In a 'proof-of-principle' test, we ran 6 endocrine disrupting chemicals (EDCs) that act via different endocrinological mechanisms through the protocol using published literature. Each was identified as endocrine active by one or more tiers. We believe that this voluntary testing protocol will be a dynamic tool to facilitate efficient and early identification of potentially problematic chemicals, while ultimately reducing the risks to public health.

The nature of the compensatory response to low thyroid hormone in the developing brain.

Thyroid hormone is essential for normal brain development, although the degree to which the developing brain is sensitive to small perturbations in serum thyroxin is not clear. An important concept related to this is that the developing brain possesses potent mechanisms to compensate for low serum thyroid hormone, and this concept is routinely employed in discussions concerning clinical treatments or public health. However, experimental studies have not directly tested whether (or the degree to which) putative compensatory mechanisms can ameliorate the consequences of small reductions in serum thyroxin (T(4)). To formally test this concept, we employed a model of graded T(4) reductions using doses of propylthiouracil (PTU) that were 200- to 67-fold lower than the dose traditionally used to produce hypothyroidism in rats. PTU produced a stepwise decrease in serum total T(4), and a stepwise increase in serum thyroid-stimulating hormone (TSH), in type 2 deiodinase mRNA expression and enzyme activity in the brain, and in the expression of the mRNA encoding the tri-iodothyronine (T(3)) transporter MCT8 in the postnatal day (P) 15 cortex. However, the mRNA encoding RC3/neurogranin, a direct target of T(3) action, exhibited a strong negative linear correlation with serum total T(4) despite these adaptive responses. In addition, single-cell analysis of RC3 mRNA levels in cortical neurones demonstrated that the co-expression of MCT8 did not alter the relationship between RC3 mRNA and serum T(4). These findings do not support the currently envisioned concept of the developing brain being capable of compensating for low T(4).

Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings.

Abstract The original concept of the critical period of thyroid hormone (TH) action on brain development was proposed to identify the postnatal period during which TH supplement must be provided to a child with congenital hypothyroidism to prevent mental retardation. As neuropsychological tools have become more sensitive, it has become apparent that even mild TH insufficiency in humans can produce measurable deficits in very specific neuropsychological functions, and that the specific consequences of TH deficiency depends on the precise developmental timing of the deficiency. Models of maternal hypothyroidism, hypothyroxinaemia and congenital hyperthyroidism have provided these insights. If the TH deficiency occurs early in pregnancy, the offspring display problems in visual attention, visual processing (i.e. acuity and strabismus) and gross motor skills. If it occurs later in pregnancy, children are at additional risk of subnormal visual (i.e. contrast sensitivity) and visuospatial skills, as well as slower response speeds and fine motor deficits. Finally, if TH insufficiency occurs after birth, language and memory skills are most predominantly affected. Although the experimental literature lags behind clinical studies in providing a mechanistic explanation for each of these observations, recent studies confirm that the specific action of TH on brain development depends upon developmental timing, and studies informing us about molecular mechanisms of TH action are generating hypotheses concerning possible mechanisms to account for these pleiotropic actions.

Differential responses of thyrotropin-releasing hormone (TRH) neurons to cold exposure or suckling indicate functional heterogeneity of the TRH system in the paraventricular nucleus of the rat hypothalamus.

Thyrotropin-releasing hormone (TRH) is released from the median eminence upon neural stimulation such as cold or suckling exposure. Concomitant with the cold- or suckling-induced release of TRH is a rapid and transient increase in the expression of proTRH mRNA in the paraventricular nucleus (PVN) of the hypothalamus. We employed two strategies to determine whether TRH neurons responding to cold exposure are different from those responding to suckling. First, we attempted to identify a marker of cellular activation in TRH neurons of the PVN. Cold induced c-fos expression in about 25% of TRH neurons of the PVN, but no induction was observed by suckling. Moreover, we explored the expression of a variety of immediate early genes including NGFI-A, fra-1 and c-jun, or CREB phosphorylation but found none to be induced by suckling. The number of cells expressing high levels of proTRH mRNA was counted and compared to total expressing cells. An increased number of cells expressing high levels of proTRH mRNA was observed when both stimuli were applied to the same animal, suggesting that different cells respond separately to each stimulus. We therefore analyzed the distribution of responsive TRH neurons as defined by the cellular level of proTRH mRNA. The proTRH mRNA signal was analyzed within three rostrocaudal zones of the PVN and within six mediolateral columns. Results showed that in response to cold, all areas of the PVN of the lactating rat present increased proTRH mRNA levels, including the anterior zone where few hypophysiotropic TRHergic cells are believed to reside. The distribution of the proTRH mRNA expressing cells in response to cold was quite comparable in female and in male rats. In contrast, the response after suckling was confined to the middle and caudal zones. Our results provide evidence of a functional specialization of TRH cells in the PVN.

Developmental and functional evidence of a role for Zfhep in neural cell development.

The rat Zfhep gene encodes a member of the Zfh family of transcription factors having a homeodomain-like sequence and multiple zinc fingers. We examined expression of Zfhep in the rat forebrain during embryonic and postnatal development. Zfhep mRNA was strongly expressed in the progenitor cells of the ventricular zone around the lateral ventricles on E14 and E16, but showed little expression in cells that had migrated to form the developing cortex. Dual labeling with PCNA demonstrated expression of Zfhep mRNA in proliferating cells. Expression of Zfhep in the ventricular zone decreases during late development as the population of progenitor cells decreases. This pattern is distinctly different from other members of the Zfh family. We also examined the expression of Zfhep protein during retinoic acid-induced neurogenesis of P19 embryonal carcinoma cells. Zfhep is highly expressed in P19 neuroblasts, and expression decreases by the time of morphological neurogenesis. Hence, both P19 cells and embryonic brain demonstrate a loss of Zfhep expression during the transition from proliferating precursor to differentiated neural cells. We investigated a possible link between Zfhep and proliferation by treating human glial cell lines with Zfhep antisense phosphorothioate oligodeoxynucleotides. Two Zfhep antisense oligonucleotides repressed proliferation of either U-138 or U-343 glioblastoma cells more than control oligonucleotides. Based on the expression patterns of Zfhep in vivo and in the P19 cell model of neurogenesis, we suggest that Zfhep may play a role in proliferation or differentiation of neural cells.

Maternal hypothyroidism selectively affects the expression of neuroendocrine-specific protein A messenger ribonucleic acid in the proliferative zone of the fetal rat brain cortex.

Thyroid hormone is essential for mammalian brain development, but the mechanisms by which thyroid hormone exerts its effects, the developmental processes affected, and the timing of thyroid hormone effects are poorly understood. An important question is whether thyroid hormone of maternal origin is essential in guiding fetal brain development. In both humans and rats, thyroid hormone of maternal origin reaches the fetus before the onset of fetal thyroid function. Moreover, receptors for thyroid hormone (TRs) are present in the fetal brain and are occupied by thyroid hormone. Finally, a recent report strongly indicates that transient undiagnosed maternal hypothyroidism can lead to measurable neurological deficits in the offspring despite the lack of neonatal hypothyroidism. Considering that TRs are ligand-activated transcription factors, we recently initiated a project to identify thyroid hormone-responsive genes in the fetal cortex before the onset of fetal thyroid function. One of the thyroid hormone-responsive genes we identified, neuroendocrine-specific protein (NSP), is expressed as two separate transcripts, NSP-A and NSP-C. Only NSP-A is affected by maternal thyroid hormone. We now demonstrate that the messenger RNA encoding NSP-A is expressed exclusively in the proliferative zone of the fetal cortex, and that its expression is affected by maternal hypothyroidism. Moreover, as development proceeds, NSP-A becomes selectively expressed in Purkinje cells of the cerebellum, a well known thyroid hormone-responsive cell. These findings strongly support the concept that thyroid hormone of maternal origin exerts specific receptor-mediated effects on fetal brain development.

Thyroid hormone of maternal origin regulates the expression of RC3/neurogranin mRNA in the fetal rat brain.

Recent clinical studies indicate that thyroid hormone plays essential roles in fetal brain development. However, the mechanism by which thyroid hormone affects fetal brain development is poorly studied. We recently identified several genes expressed in the fetal cortex whose abundance is affected by thyroid hormone of maternal origin. However, it is unclear whether these genes are directly regulated by thyroid hormone. Because these are the first genes known to be regulated by thyroid hormone during fetal development, we sought to expand our investigation to genes known to be regulated directly by thyroid hormone. We now report that the well-known thyroid hormone-responsive gene RC3/neurogranin is expressed in the fetal brain and is regulated by thyroid hormone of maternal origin. These findings support the concept that maternal thyroid hormone exerts a direct action on the expression of genes in the fetal brain that are important for normal neurological development.

Acute changes in maternal thyroid hormone induce rapid and transient changes in gene expression in fetal rat brain.

Despite clinical evidence that thyroid hormone is essential for brain development before birth, effects of thyroid hormone on the fetal brain have been largely unexplored. One mechanism of thyroid hormone action is regulation of gene expression, because thyroid hormone receptors (TRs) are ligand-activated transcription factors. We used differential display to identify genes affected by acute T(4) administration to the dam before the onset of fetal thyroid function. Eight of the 11 genes that we identified were selectively expressed in brain areas known to contain TRs, indicating that these genes were directly regulated by thyroid hormone. Using in situ hybridization, we confirmed that the cortical expression of both neuroendocrine-specific protein (NSP) and Oct-1 was affected by changes in maternal thyroid status. Additionally, we demonstrated that both NSP and Oct-1 were expressed in the adult brain and that their responsiveness to thyroid hormone was retained. These data are the first to identify thyroid hormone-responsive genes in the fetal brain.

Developmental exposure to polychlorinated biphenyls exerts thyroid hormone-like effects on the expression of RC3/neurogranin and myelin basic protein messenger ribonucleic acids in the developing rat brain.

Polychlorinated biphenyls (PCBs) are a class of industrial compounds consisting of paired phenyl rings with various degrees of chlorination. They are now ubiquitous, persistent environmental contaminants that are routinely found in samples of human and animal tissues and are known to affect brain development. The effects of PCBs on brain development may be attributable, at least in part, to their ability to reduce circulating levels of thyroid hormone. However, the developmental effects of PCB exposure are not fully consistent with hypothyroidism. Because some individual PCB congeners interact strongly with various thyroid hormone binding proteins, several investigators have speculated that these congeners may be producing thyroid hormone-like effects on brain development. Therefore, we tested whether a mixture of PCBs, Aroclor 1254 (A1254), would produce an antithyroid or thyromimetic effect on the expression of known thyroid hormone-responsive genes in the developing brain. Pregnant female rats were fed various doses of A1254 (0, 1, 4, and 8 mg/kg) from gestational day 6 to weaning on postnatal day (P) 21. Pups derived from these dams were sampled on P5, P15, and P30. Total T4 was reduced by A1254 in a dose-dependent manner, but body weight of the pups or dams was not affected. The expression of RC3/Neurogranin and myelin basic protein was not affected by A1254 on P5 or P30. However, on P15, RC3/Neurogranin was elevated by A1254 in a dose-dependent manner, and myelin basic protein expression followed this general pattern. These data clearly demonstrate that the developmental effects of PCB exposure are not simply a function of PCB-induced hypothyroidism.

Thyroid hormone action in fetal brain development and potential for disruption by environmental chemicals.

Thyroid hormone is well-known to play essential roles in brain development. Therefore, environmental factors that interfere with thyroid function or thyroid hormone action may produce deleterious effects on brain development by interfering with thyroid hormone action in the developing brain. The purpose of this review is to identify in broad terms the gaps in our knowledge of thyroid hormone action in brain development, to relate these gaps to present information on thyroid disruption, and to review briefly our recent research that is germane to these issues. The endocrinology of the thyroid system is first reviewed briefly with an emphasis on the neuroendocrine and extrathyroidal mechanisms controlling circulating levels of thyroid hormones. The second section reviews the evidence that thyroid hormone is important for fetal, as well as neonatal, brain development. We review the mechanism of thyroid hormone action in the third section and briefly relate this information to information about the mechanism of thyroid hormone action on brain development. In the final section, we review the endocrinology of thyroid disruption with an emphasis on disruption of thyroid hormone action.

Evaluating the effects of endocrine disruptors on endocrine function during development.

The major concerns with endocrine disruptors in the environment are based mostly on effects that have been observed on the developing embryo and fetus. The focus of the present manuscript is on disruption of three hormonal systems: estrogens, androgens, and thyroid hormones. These three hormonal systems have been well characterized with regard to their roles in normal development, and their actions during development are known to be perturbed by endocrine-disrupting chemicals. During development, organs are especially sensitive to low concentrations of the sex steroids and thyroid hormones. Changes induced by exposure to these hormones during development are often irreversible, in contrast with the reversible changes induced by transient hormone exposure in the adult. Although it is known that there are differences in embryonic/fetal/neonatal versus adult endocrine responses, minimal experimental information is available to aid in characterizing the risk of endocrine disruptors with regard to a number of issues. Issues discussed here include the hypothesis of greater sensitivity of embryos/fetuses to endocrine disruptors, irreversible consequences of exposure before maturation of homeostatic systems and during periods of genetic imprinting, and quantitative information related to the shape of the dose-response curve for specific developmental phenomena.

Effects of prenatal ethanol exposure on hypothalamic-pituitary-adrenal responses to chronic cold stress in rats.

Animals prenatally exposed to ethanol typically exhibit hypothalamic-pituitary-adrenal (HPA) hyperresponsiveness to stressors. In contrast to previous studies that have investigated effects of prenatal ethanol exposure on HPA responses to acute or intermittent stressors, our study investigated HPA responses to a chronic continuous stressor, cold stress (4 degrees C for 0, 1, or 3 days). We tested the hypothesis that prenatal ethanol exposure would result in increased plasma corticosterone (CORT) and adrenocorticotropin (ACTH) responses and increased peptide [corticotropin-releasing factor and vasopressin] mRNA levels in the paraventricular nucleus (PVN) of the hypothalamus compared to that in control animals. In addition, CORT and ACTH responses were measured after exposure to an acute stressor (i.p. isotonic saline injection), superimposed during chronic cold exposure, to examine possible sensitization of the HPA response to the acute stress. Thus, blood samples were collected at the end of each of the three periods of cold exposure, either before (0 min) or 15 min after acute stress. The subjects were adult male and female Sprague-Dawley rat offspring from prenatal ethanol (E), pair-fed (PF), and ad libitum-fed control (C) treatment groups. Exposure to cold stress resulted in significant body weight loss in E males at 1 day and in both males and females of all prenatal treatment groups by 3 days of cold stress. Males in all prenatal groups also exhibited significant increases in adrenal weight:body weight ratios. Cold stress alone (0 min condition) increased CORT levels in E males and overall ACTH levels in E males and females compared to controls. ACTH levels were also higher overall in E compared to control males after acute stress (15 min condition). Sensitization of the CORT response to acute stress was observed in males but not females across all prenatal treatment groups. Corticotropin-releasing factor and vasopressin mRNA levels in the PVN were not significantly affected by prenatal treatment or chronic cold stress in either males or females. In contrast, both males and females displayed increases in PVN thyrotropin-releasing hormone (TRH) mRNA levels after cold stress. These data support and extend previous work demonstrating differential effects of prenatal ethanol exposure on HPA responsiveness of male and female offspring, and suggest that E males may be more vulnerable to the effects of chronic cold stress than E females.

Prenatal ethanol exposure selectively reduces the mRNA encoding alpha-1 thyroid hormone receptor in fetal rat brain.

Some of the developmental defects characteristic of congenital or experimental hypothyroidism are also observed in children or experimental animals prenatally exposed to ethanol, suggesting that a subset of neurological defects attributable to ethanol exposure are produced by interfering with thyroid hormone action. In this article, we tested whether an ethanol treatment regimen known to produce neurological damage in rats can alter the expression of the mRNAs encoding the thyroid hormone receptor isoforms (TR alpha-1, TR alpha-2, and TR beta-1) in the fetal rat brain neocortex and hippocampus. Rats were fed an ethanol-containing diet beginning on gestational day (G) 6 and continuing until sacrifice on G15, G17, or G21; controls included animals pair-fed a liquid control diet or fed lab chow. Ethanol selectively reduced the expression of TR alpha-1 mRNA in the neocortex and hippocampus on G21, compared with pair-fed and control fetuses. In contrast, pair-feeding selectively reduced TR alpha-2 mRNA in both neocortex and hippocampus on G21, and increased TR beta-1 mRNA on G17. These data support the hypothesis that ethanol may interfere with thyroid hormone action during fetal brain development. In addition, these data indicate that ethanol and pair-feeding exert independent effects on thyroid hormone receptor expression in the developing brain.

Identification of a phylogenetically conserved Sug1 CAD family member that is differentially expressed in the mouse nervous system.

We have isolated a cDNA clone from mouse, m56, that encodes a member of the Conserved ATPase-containing Domain (CAD) protein family. Sequence analysis revealed that m56 is identical to mouse mSug1/FZA-B and shares high homology with human Trip1, moth 18-56, and yeast Sug1. When examined, Sug1-like CAD proteins appear to function in the regulation of the 26S proteasome, as well as associate with members of the steroid/thyroid receptor superfamily and other transcriptional activators. m56 can complement the lethal phenotype of loss of SUG1 in yeast. We have examined the tissue distribution of m56 using Northern and Western blots, in addition to immunocytochemistry and in situ hybridization. While m56 was expressed in all tissues and cells examined, several classes of neurons, most notably in the hippocampus, olfactory bulb, and cerebellum, displayed elevated levels of m56 mRNA and protein. We also examined distribution of RNA polymerase II and 26S proteasome subunit 4 (S4) within the mouse brain by in situ hybridization. While all three genes had similar patterns of expression, there were significant differences among them. In moths, the expression of the Sug1 homolog 18-56 is dramatically up-regulated during programmed cell death. In addition, it has been previously demonstrated that the proteasome plays an essential role in the regulation of apoptosis in mammals. We examined the expression of m56 in mouse during natural and induced cell death in a variety of tissues and found no significant changes in expression. Taken together, the data presented here suggest that while m56 is a highly conserved gene that presumably plays essential but complex roles in basal and developmental processes, it may not represent a rate-limiting step in these processes.

N-ethylmaleimide (NEM) can significantly improve in situ hybridization results using 35S-labeled oligodeoxynucleotide or complementary RNA probes.

We predicted that a significant source of background labeling after in situ hybridization (ISH) using 35S-labeled probes is attributable to a chemical reaction between the phosphorothioate moiety of the probe [O3P = S] and disulfides in tissue. These covalent bonds would immobilize probe in the tissue, thereby increasing background labeling. On the basis of this view, we have explored the use of N-ethylmaleimide (NEM) to irreversibly alkylate the phosphorothioate moiety of the probe and/or to alkylate free sulfhydryls in tissue to block the formation of disulfides as a method of reducing background labeling. We report that NEM can significantly decrease background labeling of 35S-labeled oligodeoxynucleotide or cRNA probes but does not affect specific labeling. We conclude that the use of NEM in ISH protocols, as outlined here, may be an additional element researchers may consider to improve the signal-to-noise ratio.

Chronic ethanol treatment reduces the responsiveness of the hypothalamic-pituitary-thyroid axis to central stimulation.

The hypothalamic-pituitary-thyroid (HPT) axis functions abnormally in man and animals chronically exposed to ethanol. The most consistent observation in humans is that the thyrotropin response to thyrotropin-releasing hormone (TRH) is blunted. We have tested the hypothesis that chronic ethanol treatment in rats leads to a diminished responsiveness of the hypothalamus to central stimulation. Animals were maintained on 1 of 3 diets for 4 weeks: (1) laboratory chow and water provided ad libitum (chow-fed), (2) Sustacal chocolate liquid diet with vitamin mixture containing 5% (w/v) ethanol provided ad libitum (ethanol), or (3) Sustacal chocolate liquid diet with vitamin mixture containing sucrose substituted isocalorically (35%) for ethanol and provided in amounts matched to a weight-paired, ethanol-treated animal (pair-fed). At the end of 4 weeks, the animals were evaluated for their response to a single injection of ethanol (3 g/kg, ip) and/or exposure to 5 degrees C. Chronic ethanol treatment produced an increase in TRH mRNA in neurons of the paraventricular nucleus and fully blocked the thyrotropic response to cold exposure. However, chronic ethanol-treated animals did not exhibit altered basal levels of triiodothyronine or thyrotropin, nor did they have an altered response to a single injection of ethanol. These data demonstrate that chronic alcohol exposure alters functioning of the hypothalamic-pituitary-thyroid axis at least in part by affecting TRH neurons of the paraventricular nucleus.