[Thyroid hormone action beyond classical concepts. The priority programme "Thyroid Trans Act" (SPP 1629) of the German Research Foundation]. / Schilddrüsenhormonwirkung--jenseits klassischer Konzepte. Das Schwerpunktprogramm "Thyroid Trans Act" (SPP 1629) der Deutschen Forschungsgemeinschaft.

Thyroid hormones are of crucial importance for the function of nearly all organ systems. In case of dysfunction of thyroid hormone production and function many organ systems may be affected. The estimation of normal thyroid function is based on determination of TSH and the thyroid hormones T3 and T4. However, international conventions about the normal TSH range are still lacking which bears consequences for patient`s treatment. Hence not unexpected, many patients complain although their thyroid hormone status is in the normal range by clinical estimation. Here, more precise parameters are needed for a better definition of the healthy thyroid status of an individual. Recently, new key players in the system of thyroid hormone action were detected, like specific transporters for uptake of thyroid hormones and thyroid hormone derivatives. DFG, the German Research Foundation supports the priority program Thyroid Trans Act to find answers to the main question: what defines the healthy thyroid status of an individual. The overall aim of this interdisciplinary research consortium is to specify physiological and pathophysiological functions of thyroid hormone transporters and thyroid hormone derivative as new players in thyroid regulation in order to better evaluate, treat, and prevent thyroid-related disease.

Evaluation of pre- and post-zygotic mating barriers, hybrid fitness and phylogenetic relationship between Cyprinodon variegatus variegatus and Cyprinodon variegatus hubbsi (Cyprinodontiformes, Teleostei).

The euryhaline fish Cyprinodon variegatus variegatus (Cvv) is capable of tolerating ambient salinities ranging from 0.3 to 167 g l(-1) , but incapable of long-term survival in freshwater (< 2 mM Na(+) ). However, a population of this species, now designated as a subspecies (Cyprinodon variegatus hubbsi; Cvh), has been isolated in several freshwater (0.4-1 mM Na(+) ) lakes in central Florida for the past ~150 ky. We previously demonstrated that Cvh has a significantly higher affinity for Na(+) uptake suggesting that it has adapted to its dilute freshwater environment. We here evaluate whether Cvh should be considered a separate species by characterizing pre- and post-zygotic isolation, Na(+) transport characteristics of the two populations and their hybrids, and developing a molecular phylogeny of Cvv and Cvh populations in Florida using mtDNA sequence data. We found evidence of partial prezygotic isolation with Cvv females mating almost exclusively (89%) with con-specific males in choice mating experiments. Partial post-zygotic isolation was also observed with significant (59-89%) reductions in hatching success of hybrid embryos compared with con-specific embryos. Na(+) uptake kinetics in hybrids (both Cvv x Cvh and Cvh x Cvv) bred and raised under common garden conditions were intermediate to Cvh (high affinity) and Cvv (low affinity) indicating that observed differences are genetically based. Similar observations were made with respect to short-term (96 h) survival of juveniles acutely transferred from 7 mM Na(+) to a range of more dilute (0.1-2 mM Na(+) ) freshwater. Finally, although phylogenetic analysis of Cvv and Cvh populations using mtDNA sequence for ND2 were unable to fully resolve a polytomy between Cvh and Cvv populations from northeastern Florida, these data do not falsify the hypothesis that Cvh is of monophyletic origin. Overall, the available data suggest that Cvh should be considered a separate species or at a minimum an evolutionarily significant unit.

Implications of pH manipulation methods for metal toxicity: not all acidic environments are created equal.

The toxicity of many metals is impacted by environmental pH, through both competition and complexation by hydroxide and carbonate ions. To establish safe environmental regulation it is important to properly define the relationship between pH and metal toxicity, a process that involves manipulating the pH of test water in the lab. The current study compares the effects of the three most common pH manipulation methods (carbon dioxide, acid-base addition, and chemical buffers) on acute Pb toxicity of a model fish species, Pimephales promelas. Acidification of test water revealed that the Pb and Pb(2+) LC50 values were impacted by the pH manipulation method, with the following order of effects: HCl

Multi-linear regression analysis, preliminary biotic ligand modeling, and cross species comparison of the effects of water chemistry on chronic lead toxicity in invertebrates.

The current study examined the chronic toxicity of lead (Pb) to three invertebrate species: the cladoceran Ceriodaphnia dubia, the snail Lymnaea stagnalis and the rotifer Philodina rapida. The test media consisted of natural waters from across North America, varying in pertinent water chemistry parameters including dissolved organic carbon (DOC), calcium, pH and total CO(2). Chronic toxicity was assessed using reproductive endpoints for C. dubia and P. rapida while growth was assessed for L. stagnalis, with chronic toxicity varying markedly according to water chemistry. A multi-linear regression (MLR) approach was used to identify the relative importance of individual water chemistry components in predicting chronic Pb toxicity for each species. DOC was an integral component of MLR models for C. dubia and L. stagnalis, but surprisingly had no predictive impact on chronic Pb toxicity for P. rapida. Furthermore, sodium and total CO(2) were also identified as important factors affecting C. dubia toxicity; no other factors were predictive for L. stagnalis. The Pb toxicity of P. rapida was predicted by calcium and pH. The predictive power of the C. dubia and L. stagnalis MLR models was generally similar to that of the current C. dubia BLM, with R(2) values of 0.55 and 0.82 for the respective MLR models, compared to 0.45 and 0.79 for the respective BLMs. In contrast the BLM poorly predicted P. rapida toxicity (R(2)=0.19), as compared to the MLR (R(2)=0.92). The cross species variability in the effects of water chemistry, especially with respect to rotifers, suggests that cross species modeling of invertebrate chronic Pb toxicity using a C. dubia model may not always be appropriate.

Multi-linear regression models predict the effects of water chemistry on acute lead toxicity to Ceriodaphnia dubia and Pimephales promelas.

The current study examined the acute toxicity of lead (Pb) to Ceriodaphnia dubia and Pimephales promelas in a variety of natural waters. The natural waters were selected to range in pertinent water chemistry parameters such as calcium, pH, total CO(2) and dissolved organic carbon (DOC). Acute toxicity was determined for C. dubia and P. promelas using standard 48h and 96h protocols, respectively. For both organisms acute toxicity varied markedly according to water chemistry, with C. dubia LC50s ranging from 29 to 180µg/L and P. promelas LC50s ranging from 41 to 3598µg/L. Additionally, no Pb toxicity was observed for P. promelas in three alkaline natural waters. With respect to water chemistry parameters, DOC had the strongest protective impact for both organisms. A multi-linear regression (MLR) approach combining previous lab data and the current data was used to identify the relative importance of individual water chemistry components in predicting acute Pb toxicity for both species. As anticipated, the P. promelas best-fit MLR model combined DOC, calcium and pH. Unexpectedly, in the C. dubiaMLR model the importance of pH, TCO(2) and calcium was minimal while DOC and ionic strength were the controlling water quality variables. Adjusted R(2) values of 0.82 and 0.64 for the P. promelas and C. dubia models, respectively, are comparable to previously developed biotic ligand models for other metals.

Is Cl- protection against silver toxicity due to chemical speciation?

In freshwater teleosts, the primary mechanism of acute silver toxicity is inhibition of Na(+)/K(+) ATPase and carbonic anhydrase at the gill, leading to net Na(+) and Cl(-) loss due to the continued diffusion of these ions into the hypoosmotic external environment. External Cl(-) has been shown to protect rainbow trout (Oncorhychus mykiss) against silver toxicity presumably by complexation to form AgCl. However, Cl(-) does not appear to greatly influence silver toxicity to at least two other species, the European eel (Anguilla Anguilla) and the fathead minnow (Pimephales promelas). We hypothesized that differences in protective effects of Cl(-) at the gill were due to differing requirements or mechanisms for Cl(-) uptake among fish species. To test this hypothesis, we exposed Fundulus heteroclitus, which does not take up Cl(-) across the gills, and Danio rerio and P. promelas, which do rely on Cl(-) uptake across the gills, to Ag(+) in waters of varying Cl(-) concentration. The 96-h LC50s of F. heteroclitus exposed to Ag(+) in soft water with 10 microM Cl(-), 1mM KCl, and 0.5mM MgCl(2) were 3.88, 1.20, and 3.20 microg/L, respectively, and not significantly different. The 96-h LC50s for D. rerio exposed to Ag(+) in soft water with 10 microM Cl(-) and 1mM KCl were 10.3 and 11.3 microg/L, respectively and P. promelas exposed under the same conditions were 2.32 and 2.67 microg/L, respectively. Based on these results, increasing external Cl(-) concentration by as much as 1mM (35.5mg/L) did not offer protection against Ag(+) toxicity to any fish species tested. Although previous results in our laboratory have demonstrated that P. promelas do take up Cl(-) at the gill, a mechanism of uptake has not been identified. Additional experiments, investigating the mechanisms of Na(+) and Cl(-) influx at the gill of P. promelas and the influence of silver, demonstrated that Cl(-) uptake in P. promelas acclimated to soft water occurs through both a Na(+):K(+):2Cl(-) co-transporter and a Cl(-)/HCO(3)(-) exchanger, but is not dependent on carbonic anhydrase. Further, acclimation water chemistry was found to greatly influence subsequent branchial silver accumulation, but Cl(-) uptake was not sensitive to 10 microg/L Ag(+).

Physiology is pivotal for interactions between salinity and acute copper toxicity to fish and invertebrates.

The present paper presents original data and a review of the copper (Cu) toxicity literature for estuarine and marine environments. For the first time, acute Cu toxicity across the full salinity range was determined. Killifish, Fundulus heteroclitus, eggs were hatched in freshwater (FW), 2.5, 5, 10, 15, 22 and 35 ppt (seawater, SW) and juveniles were allowed to acclimate for 7 days prior to acute toxicity testing. Sensitivity was highest in FW (96 h LC50: 18 microg/l), followed by SW (96 h LC50: 294 microg/l) with fish at intermediate salinities being the most tolerant (96 h LC50 > 963 microg/l at 10 ppt). This approximately 50-fold, non-linear variation in sensitivity could not be accounted for by Cu speciation or competition among cations but can be explained by physiology. The relative Na(+) gradient from the blood plasma to the water is greatest in FW followed by SW and is smallest at 10 ppt. Regression of Cu toxicity versus the equilibrium potential for Na(+), which reflects the relative Na(+) gradient, revealed that 93% of the variation can be attributed to Na(+) gradients and thus osmoregulatory physiology. Examination of the existing literature on acute Cu toxicity in SW (defined as >25 ppt) confirmed that early life stages generally are most sensitive but this pattern may be attributable to size rather than developmental stage. Regardless of developmental stage and phylogeny, size clearly matters for Cu sensitivity. The existing literature on the influence of salinity on acute Cu toxicity as well as studies of mechanisms of Cu toxicity in fish and invertebrates are reviewed.

Effects of copper, cadmium, and zinc on the hatching success of brine shrimp (Artemia franciscana).

Previous studies indicate that the hatching success of brine shrimp (Artemia franciscana) cysts is surprisingly sensitive to ambient metal concentrations. These studies estimated median effective concentrations (EC50s) of 7, 5, and 28 microg l-1 for Cd, Cu, and Zn, suggesting that the hatching end point for A. franciscana is the most sensitive tested to date for Cd and Zn in saline environments and comparable in sensitivity with the most sensitive tested to date for Cu. Furthermore, these data suggest that brine shrimp are at significant risk from Cu and Zn in Great Salt Lake (GSL), UT, where ambient concentrations as high as 10 and 14 microg l-1, respectively, have been measured. Given that brine shrimp appear to be successfully reproducing in GSL, we hypothesized that these toxicity values were either biased low as a result of an artifact of the test method used or that site-specific water-quality conditions in the lake had decreased metal bioavailability such that brine shrimp could successfully reproduce. To test these hypotheses, we initiated a step-wise series of experiments. First we investigated the effects of pretreatment of brine shrimp cysts with antibiotics on brine shrimp sensitivity to metals because previous investigators as part of their test methods have used antibiotics. Next we considered the effect of ionic composition of the artificial test media on sensitivity. Finally, we evaluated the effects of the site-specific water quality of the GSL on metal bioavailability and toxicity. Results indicate that pretreatment of cysts with antibiotics had no effect on sensitivity. However, we were unable to repeat the previous values for Cd and Zn, obtaining EC50s of 11,859 and 289 microg l-1 for Cd and Zn, respectively. For Cu, however, we estimated an EC50 of 12 microg l-1, so we conducted further testing on the artificial media, adjusting the media composition to better reflect the Ca2+ and HCO3- concentration of normal seawater. This increased the EC50 to 28 microg l-1. Finally we evaluated the toxicity of Cu in GSL water and obtained an EC50 of 68 microg l-1, suggesting that the increased dissolved organic carbon in GSL has a significant protective effect. Overall, the results of this study suggest that brine shrimp hatching success is not particularly sensitive to Cd and Zn, but it is sensitive to Cu. However, site-specific water-quality conditions ensure that brine shrimp cyst hatching success is not significantly affected by any of these metals at the normal background concentrations that occur in GSL (<15 microg l-1).

Influence of Ca, humic acid and pH on lead accumulation and toxicity in the fathead minnow during prolonged water-borne lead exposure.

The present study examines the influence of Ca2+ as (CaSO4), dissolved organic carbon (DOC) and pH on chronic water-borne lead (Pb) toxicity to the larval fathead minnow (Pimephales promelas) under flow-through conditions. The 30 day LC50 for low hardness basic test water (19 mg CaCO3 L(-1)) was 39 (range: 27-51) microg dissolved Pb L(-1) and was greatly increased by increasing concentrations of CaSO4 and DOC to as much as 1903 (range: 1812-1992) mug dissolved Pb L(-1). Both reduced and increased pH (6.7 and 8.1, respectively) compared to control pH of 7.4 appeared to increase Pb toxicity substantially. Whole body Pb accumulation did not reflect water chemistry and thus exhibited no correlation with Pb induced mortality. One possible explanation for this lack of correlation is that mortality occurred predominantly during the first 4-6 days of exposure, whereas Pb accumulation was determined in surviving fish at the end of 30 days of exposure. Chronic Pb exposure resulted in a general iono-regulatory disturbance affecting K+, Na+ and Ca2+ homeostasis. However, recovery of Na+ and K+ levels and reversal of effects on Ca2+ homeostasis during continued exposure strongly suggest fathead minnow can acclimate to Pb. The gills accumulate the highest Pb concentrations during chronic exposure but the skeleton contains the largest mass of Pb by contributing up to approximately 80% of whole body Pb. In conclusion, water chemistry characteristics like Ca2+ and DOC should be considered for chronic water quality criteria.

The effects of metals on embryo-larval and adult life stages of the sea urchin, Diadema antillarum.

Since the massive population decline of the long-spined sea urchin, Diadema antillarum, in the early 1980s, the dynamics of coral reef ecosystems in the Caribbean have changed tremendously. The absence of D. antillarum, once a keystone herbivore, has led to macroalgal dominance in many of these reef communities. D. antillarum is not only important ecologically, but may also be a sensitive bioindicator species for toxicant exposure. Echinoderm larval development tests were conducted with D. antillarum exposed to elevated levels of aqueous copper (Cu), silver (Ag), nickel (Ni), or selenium (Se). All metals significantly affected larval development, based on normal development to the pluteus stage. The EC50s based on dissolved metal concentrations were 11 microg/L Cu, 6 microg/L Ag, 15 microg/L Ni, and 26 microg/L Se. Adult sea urchins were exposed to aqueous copper under flow through conditions for 96 h. The 96-h LC50 for this exposure was 25 microg/L dissolved Cu. Additionally, behavioral and physiological disturbance was observed. The physiological responses included both acid-base balance disturbance, as evidenced by reduced coelomic fluid pH and apparent ionoregulatory effects. In addition, behavioral effects included spatial orientation within the exposure tank, spine closure, and loss of spines. The high sensitivity of both adult and larval D. antillarum to these metals supports the use of this organism as an important biological indicator for metal exposure in marine environments.

Assessing acute and chronic copper risks to freshwater aquatic life using species sensitivity distributions for different taxonomic groups.

Using copper as an example, we present a method for assessing chemical risks to an aquatic community using species sensitivity distributions (SSDs) for different taxonomic groups. This method fits probability models to chemical exposure and effects data to estimate the percentage of aquatic species potentially at risk and expands on existing probabilistic risk assessment methodologies. Due to a paucity of chronic toxicity data for many chemicals, this methodology typically uses an acute-chronic ratio (ACR) to estimate the chronic effects distribution from the acute effects distribution. We expanded on existing methods in two ways. First, copper SSDs were developed for different organism groups (e.g., insects, fish) that share similar sensitivities or ecological functions. Integration of exposure and effects distributions provides an estimate of which organism groups may be at risk. These results were then compared with a site-specific food web, allowing an estimation of whether key food web components are potentially at risk and whether the overall aquatic community may be at risk from the perspective of ecosystem function. Second, chronic SSDs were estimated using the relationship between copper ACRs and acute toxicity (i.e., the less acutely sensitive a species, the larger the ACR). This correction in the ACR removes concerns previously identified with use of the ACR and allows evaluation of a significantly expanded chronic data set with the same approach as that for assessing acute risks.

Cysteine proteinases mediate extracellular prohormone processing in the thyroid.

Thyroglobulin, the precursor of thyroid hormones, is extracellularly stored in a highly condensed and covalently cross-linked form. Solublization of thyroglobulin is facilitated by cysteine proteinases like cathepsins B and K which are proteolytically active at the surface of thyroid epithelial cells. The cysteine proteinases mediate the processing of thyroglobulin by limited extracellular proteolysis at the apical plasma membrane, thereby rapidly liberating thyroxine. The trafficking of cysteine proteinases in thyroid epithelial cells includes their targeting to lysosomes where they become maturated before being transported to the apical plasma membrane and, thus, into the extracellular follicle lumen. We propose that thyroid stimulating hormone regulates extracellular proteolysis of thyroglobulin in that it enhances the rate of exocytosis of lysosomal proteins at the apical plasma membrane. Later, thyroid stimulating hormone upregulates thyroglobulin synthesis and its secretion into the follicle lumen for subsequent compaction by covalent cross-linking. Hence, cycles of thyroglobulin proteolysis and thyroglobulin deposition might result in the regulation of the size of the luminal content of thyroid follicles. We conclude that the biological significance of extracellularly acting cysteine proteinases of the thyroid is the rapid utilization of thyroglobulin for the maintenance of constant thyroid hormone levels in vertebrate organisms.

Acute toxicity of sodium selenate to two daphnids and three amphipods.

This study evaluated the acute toxicity of sodium selenate to two daphnid and three gammarid amphipod species. The daphnids, Ceriodaphnia dubia and Daphnia pulex, were evaluated in 48-hour static tests and the amphipods, Gammarus pseudolimnaeus, Gammarus lacustris, and Hyalella azteca, were evaluated in 96-hour static and flow-through tests. Tests resulted in mean LC50's of 1.92, 9.12, 1.82, 3.05, and 1.95 mg/L selenium for C. dubia, D. pulex, G. pseudolimnaeus, G. lacustris, and H. azteca, respectively. The LC50's for the G. pseudolimnaeus tests are more than 30-fold higher than previously reported LC50's for the same or similar species. The explanation for these differing results appears to be partially, but not entirely, explained by differences in ambient pH between the new studies and previous ones. Depending on how the new data are included in U.S. EPA's selenium freshwater quality criterion data set, the selenate acute water quality criterion (i.e., Criterion Maximum Concentration) increases from 12.8 to as high as 583 microg/L selenium.

Effects of sulfate on the acute toxicity of selenate to freshwater organisms.

This study evaluated the relationship between ambient sulfate concentrations and acute selenate toxicity to freshwater aquatic life. Previous studies indicated that increasing sulfate concentrations reduced selenate bioconcentration and toxicity. However, these studies generally were not conducted in a manner that was conducive to their use in deriving a water quality criterion. We compiled results from previous studies and generated additional data to help define a selenate-sulfate relationship for acute toxicity. Selenate toxicity was determined in standardized test waters with varying sulfate concentrations using Ceriodaphnia dubia, Gammarus pseudolimnaeus, Hyalella azteca, and Pimephales promelas as the test organisms. Analysis of test results indicated that a significant relationship does exist between acute selenate toxicity and ambient sulfate concentrations. Data from these tests and previous studies were combined to develop a statistical relationship sufficiently robust to derive a sulfate-dependent water quality criterion for selenate. The relationship is similar to those commonly derived between divalent metals and hardness to adjust water quality criteria.

Cathepsin K in thyroid epithelial cells: sequence, localization and possible function in extracellular proteolysis of thyroglobulin.

Extracellular proteolysis of thyroglobulin at the apical surface of thyroid epithelial cells results in liberation of thyroxine, and is mediated by lysosomal cysteine proteases such as cathepsins B and L. Here, we report on the expression of the cysteine protease cathepsin K in thyroid epithelial cells. The cDNA for porcine thyroid cathepsin K showed homologies ranging from 71% to 94% to the cDNA of cathepsin K from various species and cell types. The deduced amino acid sequence of porcine thyroid cathepsin K predicted a 37 kDa preproenzyme, with the active site residues Cys-140, His-277 and Asn-297, and one potential N-glycosylation site. The localization of cathepsin K was not restricted to lysosomes. Rather, secreted cathepsin K was predominantly found within the follicular lumen and in association with the apical plasma membrane of thyroid epithelial cells. Enzyme cytochemistry showed that cell-surface associated cathepsin K was proteolytically active at neutral pH. In vitro, recombinant cathepsin K liberated thyroxine from thyroglobulin by limited proteolysis at neutral pH. We postulate that its localization enables cathepsin K to contribute to the extracellular proteolysis of thyroglobulin, i.e. thyroid hormone liberation, at the apical surface of thyroid epithelial cells in situ.

Thyroglobulin type-I-like domains in invariant chain fusion proteins mediate resistance to cathepsin L digestion.

The MHCII associated invariant chain isoform Ii41 shows homology to a repeat in thyroglobulin (TgR). We show that the Ii31 isoform, which lacks the TgR-like domain, is sensitive to cathepsin L treatment whereas Ii41 displays substantial resistance. The TgR-like sequence of Ii41 was exchanged for thyroglobulin type-IA and -IB repeats, that contain six or four cysteine residues. Resistance to cathepsin L digestion was maintained upon substitution of the Ii41 TgR for homologous sequences from TgR type-IA. Mutation of a conserved cysteine in the TgR domain of an Ii fusion protein strongly reduced resistance to cathepsin L digestion.

Covalent cross-linking of secreted bovine thyroglobulin by transglutaminase.

Extracellular storage of thyroglobulin (TG) is a prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of TG within the follicle lumen is achieved by compactation and by the formation of covalent cross-links between TG molecules. In bovine thyroids, approximately 75% of the cross-links are other than disulfide bonds (J. Cell Biol. 180, 1071-1081). We have now shown that polymeric TG contains a large number of N(epsilon)(gamma-glutamyl)lysine cross-links and that only traces of these can be found in the soluble form of TG. Because such isopeptide bridges are generated usually by the action of a transglutaminase, it is reasonable to propose that the covalent polymerization of TG in the globules is under the control of this enzyme. Soluble TG was shown to be a substrate for transglutaminase in vitro; moreover, the presence of transglutaminase was demonstrated by immunofluorescence and by immunoblotting in freshly isolated bovine thyroid globules. With immunoelectron microscopy, transglutaminase was detected in the cytoplasm of thyrocytes, but not in compartments of the secretory pathway. Only one messenger RNA for transglutaminase was found by Northern blotting. Sequencing of the cloned gene failed to reveal a secretory signal, which supports the notion that the thyroid transglutaminase is the cytosolic type. Apparently, the enzyme reaches the lumen of the follicle by an as yet unknown pathway to catalyze the covalent cross-linking of thyroid globules in this extracellular compartment.

Hyperproliferative hepatocellular alterations after intraportal transplantation of thyroid follicles.

The thyroid hormone 3,5,3'-triiodo-L-thyronine (T3) is a strong direct hepatocyte mitogen in vivo. The effects of T3 resemble those of peroxisome proliferators, which are known to induce hepatocellular tumors in rats. With the aim of studying long-term local effects of thyroid hormones on liver parenchyma, small pieces of thyroid tissue were transplanted via the portal veins into the livers of thyroidectomized male Lewis rats. At 1 week, 3 weeks, 3 months, and 18 months after transplantation, the transplants were found to proliferate, to synthesize thyroglobulin, and to release thyroxine and T3. At 3 and 18 months after transplantation, the hepatocytes of the liver acini downstream of the transplanted follicles showed an increase in cytoplasmic basophilia, a loss of glycogen, an enlargement and hyperchromasia of their nuclei, and a strong increase in cell turnover compared with unaltered liver acini. The altered hepatocytes exhibited an increase in the activities of glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, malic enzyme, mitochondrial glycerol-3-phosphate dehydrogenase, cytochrome-c-oxidase, and acid phosphatase; the activities of glycogen synthase and glycogen phosphorylase were strongly decreased. The hepatocytic alterations downstream of the transplanted follicles could be explained by effects of T3. On the other hand, they resembled alterations characteristic of amphophilic preneoplastic liver foci observed in different models of hepatocarcinogenesis.

Subcellular distribution, secretion, and posttranslational modifications of clusterin in thyrocytes.

A prominent secretory glycoprotein was detected in the culture medium of porcine thyrocytes which was identified as clusterin by microsequencing. Treatment of thyrocytes with thyroid stimulating hormone revealed a tight regulation of both synthesis and secretion of clusterin, with a distinct fraction of clusterin being always associated with the cells. At least three N-bound glycans were found on each subunit of clusterin, receiving most of the incorporated [(32)P]phosphate-label. Binding of clusterin to the immobilized cation-independent mannose 6-phosphate (M6P) receptor indicated that part of the phosphate label was contained in M6P moieties. Immunolabeling of cultured thyrocytes and of thyrocytes in situ showed clusterin on the apical cell surfaces where it colocalized with gp330/megalin, which is known to serve as a binding site for clusterin. The association with the apical plasma membrane, which, in thyrocytes, carries the iodinating system, was confirmed by biosynthetic iodination, an as yet unknown posttranslational modification of clusterin. On the basolateral plasma membranes clusterin was found within distinct, bipartite patches, suggesting that it is a constituent of cell-adhesion complexes and that it participates in cell-cell and cell-matrix interactions.

Review of health consequences from high-, intermediate- and low-level exposure to organophosphorus nerve agents.

Short and long-term health effects from exposure to organophosphorus (OP) military and insecticidal nerve agents are evaluated based on the abundant scientific literature published over five decades on health effects in humans (from human experimentation and occupational exposures) and in laboratory animals. Four distinct health effects are identified: acute cholinergic toxicity; organophosphate-induced delayed neuropathy (OPIDN); subtle long-term neuropsychological and neurophysiological effects; and a reversible muscular weakness called 'intermediate syndrome'. Some effects are subtle and difficult to differentiate from health effects caused by other diseases or occupational exposures. Each effect has data suggesting threshold exposure levels below which it is unlikely to be clinically detectable. Therefore, meaningful interpretation of human and animal studies requires rigid exposure characterization. Because precise exposure levels are often difficult to reconstruct, a system for characterizing exposure is proposed based upon observed initial acute signs and symptoms, as high-level (definitive cholinergic poisoning); intermediate-level (threshold cholinergic effects including miosis, rhinorrhea or clinically measurable depression of cholinesterase); and low-level (no immediate clinical signs or symptoms) exposure. Threshold exposure levels for known long-term effects from OP nerve agent are at or above intermediate-level exposure. Long-term health effects seen at intermediate-level exposures or in many survivors of high-level exposure are subtle, detectable in exposed populations but not individuals, and not reported in individuals experiencing low-level exposure alone. Co-exposure to other pharmaceutical agents may promote or protect against health effects from OP nerve agents, but qualitatively they are the same effects seen with OP nerve agents alone. Thus, the system for characterizing exposure based on initial acute effects is also useful for evaluating health outcomes from co-exposure to OP nerve and other agents.