Search

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases. Inhibition of high mobility group box 1 (HMGB1) and restoration of endothelial integrity are emerging as an attractive therapeutic strategies in the management of severe sepsis or septic shock. Here, we examined the effects of methylthiouracil (MTU), used as antithyroid drug, by monitoring the effects on lipopolysaccharide (LPS)- or cecal ligation and puncture (CLP)-mediated release of HMGB1, and on the modulation of HMGB1-mediated inflammatory responses. The anti-inflammatory activities of MTU were determined by measuring permeability, leukocyte adhesion and migration, and the activation of pro-inflammatory proteins in HMGB1-activated HUVECs and mice. MTU inhibited the release of HMGB1 and downregulated HMGB1-dependent inflammatory responses in human endothelial cells. MTU also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in mice. In addition, treatment with MTU reduced CLP-induced release of HMGB1 and sepsis-related mortality and pulmonary injury. Our results indicate that MTUs could be candidate therapeutic agents for various severe vascular inflammatory diseases via the inhibition of the HMGB1 signaling pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Drug repositioning is used to discover drug candidates to treat human diseases, through the application of drugs or compounds that are approved for the treatment of other diseases. This method can significantly reduce the time required and cost of discovering new drug candidates for human diseases. Previous studies have reported pro-inflammatory responses of endothelial cells to the release of polyphosphate (PolyP). In this study, we examined the anti-inflammatory responses and mechanisms of methylthiouracil (MTU), which is an antithyroid drug, and its effects on PolyP-induced septic activities in human umbilical vein endothelial cells (HUVECs) and mice. The survival rates, septic biomarker levels, behaviour of human neutrophils and vascular permeability were determined in PolyP-activated HUVECs and mice. MTU suppressed the PolyP-mediated vascular barrier permeability, up-regulation of inflammatory biomarkers, adhesion/migration of leucocytes, and activation and/or production of nuclear factor-κB, tumour necrosis factor-α and interleukin-6. Furthermore, MTU demonstrated protective effects on PolyP-mediated lethal death and the levels of the related septic biomarkers. Therefore, these results indicated the therapeutic potential of MTU on various systemic inflammatory diseases, such as sepsis or septic shock., (© 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases. Here, methylthiouracil (MTU), an antithyroid drug, was examined for its effects on lipopolysaccharide (LPS)-mediated vascular inflammatory responses. The anti-inflammatory activities of MTU were determined by measuring permeability, human neutrophil adhesion and migration, and activation of pro-inflammatory proteins in LPS-activated human umbilical vein endothelial cells and mice. We found that post-treatment with MTU inhibited LPS-induced barrier disruption, expression of cell adhesion molecules (CAMs), and adhesion/transendothelial migration of human neutrophils to human endothelial cells. MTU induced potent inhibition of LPS-induced endothelial cell protein C receptor (EPCR) shedding. It also suppressed LPS-induced hyperpermeability and neutrophil migration in vivo. Furthermore, MTU suppressed the production of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, and the activation of nuclear factor-κB (NF-κB) and extracellular regulated kinases (ERK) 1/2 by LPS. Moreover, post-treatment with MTU resulted in reduced LPS-induced lethal endotoxemia. These results suggest that MTU exerts anti-inflammatory effects by inhibiting hyperpermeability, expression of CAMs, and adhesion and migration of leukocytes, thereby endorsing its usefulness as a therapy for vascular inflammatory diseases., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Drug repositioning refers to the development of existing drugs for new indications. These drugs may have (I) failed to show efficacy in late stage clinical trials without safety issues; (II) stalled in the development for commercial reasons; (III) passed the point of patent expiry; or (IV) are being explored in new geographic markets. Over the past decade, pressure on the pharmaceutical industry caused by the 'innovation gap' owing to rising development costs and stagnant product output have become major reasons for the growing interest in drug repositioning. Companies that offer a variety of broad platforms for identifying new indications have emerged; some have been successful in building their own pipelines of candidates with reduced risks and timelines associated with further clinical development. The business models and platforms offered by these companies will be validated if they are able to generate positive proof-of-concept clinical data for their repositioned compounds. This review describes the strategy of biomarker-guided repositioning of chemotherapeutic drugs for inflammation therapy, considering the repositioning of methylthiouracil (MTU), an antithyroid drug, as a potential anti-inflammatory reagent., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The screening of bioactive compound libraries can be an effective approach for repositioning FDA-approved drugs or discovering new treatments for human diseases. Transforming growth factor β-induced protein (TGFBIp) is an extracellular matrix protein whose expression in several cell types is greatly increased by TGF-β. TGFBIp is released by human umbilical vein endothelial cells (HUVECs), and functions as a mediator of experimental sepsis. Here, we investigated the anti-septic effects and underlying mechanisms of methylthiouracil (MTU), used as antithyroid drug, against TGFBIp-mediated septic responses in HUVECs and mice. The anti-inflammatory activities of MTU were determined by measuring permeability, human neutrophils adhesion and migration, and activation of pro-inflammatory proteins in TGFBIp-activated HUVECs and mice. According to the results, MTU effectively inhibited lipopolysaccharide-induced release of TGFBIp, and suppressed TGFBIp-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. In addition, MTU suppressed CLP-induced sepsis lethality and pulmonary injury. Collectively, these results indicate that MTU could be a potential therapeutic agent for treatment of various severe vascular inflammatory diseases via inhibition of the TGFBIp signaling pathway., (Copyright © 2015 Elsevier Inc. All rights reserved.)

This paper describes the regiospecific synthesis of 5-halo-2-methylthiouracil nucleosides by direct glycosylation followed by halogenation by an electrophilic halogen reagent and sodium azide under mild conditions. The compounds were suggested to be effective as antiviral agents.

The postnatal development of rat microglia is marked by an important increase in the number of microglial cells and the growth of their ramified processes. We studied the role of thyroid hormone in microglial development. The distribution and morphology of microglial cells stained with isolectin B4 or monoclonal antibody ED1 were analyzed in cortical and subcortical forebrain regions of developing rats rendered hypothyroid by prenatal and postnatal treatment with methyl-thiouracil. Microglial processes were markedly less abundant in hypothyroid pups than in age-matched normal animals, from postnatal day 4 up to the end of the third postnatal week of life. A delay in process extension and a decrease in the density of microglial cell bodies, as shown by cell counts in the developing cingulate cortex of normal and hypothyroid animals, were responsible for these differences. Conversely, neonatal rat hyperthyroidism, induced by daily injections of 3,5,3'-triiodothyronine (T3), accelerated the extension of microglial processes and increased the density of cortical microglial cell bodies above physiological levels during the first postnatal week of life. Reverse transcription-PCR and immunological analyses indicated that cultured cortical ameboid microglial cells expressed the alpha1 and beta1 isoforms of nuclear thyroid hormone receptors. Consistent with the trophic and morphogenetic effects of thyroid hormone observed in situ, T3 favored the survival of cultured purified microglial cells and the growth of their processes. These results demonstrate that thyroid hormone promotes the growth and morphological differentiation of microglia during development.

During muscle development, an isozymic transition of the glycolytic enzyme enolase occurs from the embryonic and ubiquitous alphaalpha-isoform to the muscle-specific betabeta-isoform. Here, we demonstrate a stimulatory role of thyroid hormones on these two enolase genes during rat development in hindlimb muscles and an inhibitory effect on the muscle-specific enolase gene in cardiac muscle. In hindlimb muscles the ubiquitous alpha-transcript level is diminished by hypothyroidism, starting at birth. On the contrary, the more abundant muscle-specific beta-transcript is insensitive to hypothyroidism before establishment of the functional diversification of fibers and is greatly decreased thereafter. Our data support the hypothesis of a role of thyroid hormones in coordinating the expressions of contractile proteins and metabolic enzymes during muscle development. The subcellular localization of isoenolases, established here, is not modified by hypothyroidism. Our results underline the specificity of action of thyroid hormones, which modulate differentially two isozymes in the same muscle and regulate, in opposite directions, the expression of the same gene in two different muscles.

In order to study the morphology and morphometry and to characterize and quantify the nucleolar organizer regions (NORs) of bovine thyroids containing methylthiouracil (MTU) residues, five animals were orally treated with a suspension of MTU (5 g/animal/day) for 20 days (group A). This treatment protocol was interrupted 5 days before the the animals were slaughtered. Six animals receiving placebos composed group B. A third group (group C) was composed of normal thyroids obtained from a slaughterhouse. All glands were previously assessed for detection of antithyroid residues by chromatography, and only those glands from MTU-treated animals were positive. Follicles of glands from group A showed wide variation in size and shape. There was a predominance of small follicles covered by multiple layers of columnar cells, sometimes forming papillary projections into the lumen, characterizing severe interfollicular and intrafollicular adenomatosis. Many follicles had vacuolated cells with nuclei showing karyolysis or pyknosis and reduced amounts of a low-density and very excavated colloid. They also showed higher follicular epithelia and larger proportions of their structural components when compared with glands of groups B and C. In the thyroids from group A, the argyrophilic nucleolar organizer region-associated proteins (AgNORs) were greater in number, with small ones scattered all over the nucleus. Although the size of AgNORs in thyroids from groups B and C was variable, these AgNORs were fewer and larger than were those in glands from group A. In conclusion, the MTU induces proliferation and regressive changes in follicular cells, and the AgNOR technique is efficient to distinguish different degrees of thyroid hyperplasia.

N3-beta-D-glucopyranosyl, galactopyranosyl and xylopyranosyl 6-methyl-2-methylthiouracil and their 5-bromo derivatives have been synthesized by coupling an alpha-acetobromosugar with the corresponding thiouracil. The new modified thiouridine analogues were evaluated for their inhibitory activity against Human Immunodeficiency Virus (HIV) replication in MT-4 cells as well as for their cytotoxicity.

This study investigates the influence of low dosages of beta-agonists combined with other growth promoters on screening and confirmation methods in male veal calves. Five groups of four calves were treated for 6 weeks with combinations of low dosages of beta-agonists (beta AG, clenbuterol, mabuterol and mapenterol, 0.4 microgram/kg each twice daily) in combination with 17 beta-estradiol (E2, 5 mg per animal per 14 days), methylthiouracil (MTU, 2.857 mg/kg bw twice daily) and dexamethasone (DEX, 4 mg per animal per 10 days during the last 20 days of treatment). Another group of four untreated animals served as controls. The weight and size of the thymus was reduced in the DEX group, the MTU group showed enlarged thyroids. Histologically the prostates showed vacuolar degeneration in the beta AG animals and metaplasia in the E2 group. Some vacuolization was also observed in the controls. The testis showed impaired development in all treatment groups, E2 leading to the most severe changes. DEX led to cortical atrophy of the thymus. MTU induced hyperplastic changes in the thyroid. These results indicate that comedication does not markedly affect the histological changes induced in the hormonal target tissues. For screening purposes histology of the prostate can be used as a marker for oestrogens, whereas the weight of the thymus and thyroid can be used as an indication for the use of corticosteroids and thyreostatics, respectively. Vacuolization in the prostate appeared no reliable indication for beta-agonists. Samples of urine, faeces, liver and eye (retina/choroid) were analysed for beta-agonists. E2 significantly increased the levels of all beta-agonists in the liver and faeces, whereas DEX significantly reduced these levels. These observations show that additional medication with different groups of growth promoters can markedly alter the excretion of beta-agonists and thus influence (regulatory) control.

The effect of hypothyroidism on adrenals and gonads in adult female rats was investigated throughout the estrous cycle. Hypothyroidism was induced by administration of 4-Methyl-2-Thiouracil (Thiouracil) in the drinking water. The weight of ovaries and adrenals, and the plasma levels of corticosterone decreased in hypothyroid rats as compared with euthyroid rats throughout the estrous cycle. Hypothyroidism resulted in decreased concentrations of plasma LH on the day of diestrus and proestrus, whereas the plasma concentrations of prolactin and progesterone increased as compared with euthyroid rats. The weight of uteri and plasma concentrations of estradiol decreased during the day of diestrus and proestrus in hypothyroid rats as compared with euthyroid rats. To further clarify the dysfunction of hypothalamo-hypophysial-adrenal axis in hypothyroid rats, animals were stressed by immobilization for 3 hr. In hypothyroid rats, a marked increase in plasma levels of ACTH in response to immobilization stress was observed compared to euthyroid control, whereas increases in plasma concentrations of corticosterone were much smaller in hypothyroid than euthyroid rats. These results clearly indicate that hypothyroidism causes both gonadal and adrenal disturbances in adult female rats. The increased concentrations of plasma progesterone may be due to hypersecretion of prolactin during the day of proestrus and estrus, which in turn result in disruption of the estrous cycle.

Alterations in the superoxide dismutase (SOD) content of thyroid tissues occurring in association with thyroid dysfunction have been reported. In this study, the Mn-SOD content was found to increase in thyroid tissues of rats administered thyroid stimulating hormone (TSH) and in thyrocytes cultured in medium supplemented with TSH. Furthermore, in the thyroid glands of rats whose serum TSH level was elevated by inhibiting the synthesis of T3 and T4 by 6-methyl-2-thiouracil, the Mn-SOD increased as the TSH concentration increased. In the cultured thyrocytes, the increase in Mn-SOD induced by TSH was inhibited by the C-kinase inhibitor H7. These findings suggest the induction of Mn-SOD by TSH in thyroid cells and point to a role of C-kinase in this process, thereby indicating that a close relationship exists between the serum TSH level and the change in Mn-SOD content in thyrocytes with thyroid dysfunction.

Adult female turtles (Lissemys punctata punctata) were treated with pineal indoleamine melatonin (100 micrograms/100 g) or the antithyroid agent, methylthiouracil (100 micrograms/100 g) or melatonin together with methylthiouracil (100 micrograms of each drug/100 g) for 12 days. Melatonin alone inhibited thyroid activity as evidenced by reduction in the gland weight, follicular epithelial cell height, thyroid peroxidase, and plasma thyroxine levels. Methylthiouracil caused hyperplasia of the gland, although it inhibited thyroid activity and reduced thyroid peroxidase and plasma thyroxine levels. Melatonin together with methylthiouracil produced changes similar to those obtained with melatonin alone. The results indicate that melatonin probably exerts inhibitory effects influences on both thyrotropin release from the pituitary and the activity of the thyroid itself in turtles.

Regulation of annexin concentration and localization were investigated in thyroid tissues of hypothyroid [methylthiouracil (MeSur) treatment], euthyroid (control) and hyperthyroid [thyroxine (T4) treatment] rats. A low level of circulating thyroid hormones induces a decrease of total thyroid calcium-binding protein concentration when compared with the concentration in unstimulated animals. Conversely, concentrations of annexins I, II and V increase. The accumulation of these proteins in two subcellular compartments (cytosolic and particulate fractions) can be reversed by addition of thyroid hormones. The finding of a specific increase in annexins concentration in thyroid-hormone-deficient rats, with a general decrease of the total calcium-binding protein content points to a very important role of these proteins in the cells. Furthermore, hyperthyroidisnt gives opposite results. To investigate the transduction pathway of annexins I-, II- and V-induced biosynthesis by thyroid hormones in thyroid glands, we used cultured pig thyroid cells as in vitro model system. In previous work [16], we have shown that annexin concentrations and localization are under TSH control via the adenylate cyclase pathway. In the presence of MeSur (in the culture medium), the protein-binding iodine remains low, indicative of weak thyroid hormone synthesis (data not shown) and that the annexins content is unchanged. These results suggest that, in thyroid tissue, an indirect mechanism links thyroid hormones to annexin expressions via the TSH feed-back loop, and excludes autocrine regulation.

Following oral administration of methionine in high doses to normotensive (NR) and spontaneously hypertensive (SHR) rats, its degradation product, homocysteine (HC), which is markedly elevated in serum, exerts an angiotoxic action directed to the aorta. This is accompanied by considerable loss of endothelium and degeneration, partly with dissolution of the media cells with formation of characteristic processes of the degenerating mitochondria, and by elevated HC and cystathion (CT) values in the aortic wall. At the arterial vessels of other organs similar alterations did not occur. There are quantitative differences between NR and SHR. In SHR, serum shows higher HC and CT concentrations than in NR, and the methionine-related aortic alterations are considerably more pronounced and develop earlier, with the additional formation of connective tissue. Here, a certain dependence on the methionine dose is noted, in contrast to NR, for which the magnitude of the reaction appears to be more related to the length of time of methionine application. Additional administration of atherogenic substances (cholestane-3 beta, 5 alpha, 6 beta-triol, cholesterol, angiotensin II, cholic acid with methylthiouracil) in SHR causes an exacerbation of the methionine-related aortic alterations. Only cholestane-triol has the same effect on the aortic wall in NR and SHR, with more accentuation in SHR. Cholestane-triol has, in NR as well as in SHR, a high coincidence with methionine-induced morphological reactions including the formation of mitochondrial processes. Simultaneous application of these two substances did not cause a potentiation of the effect. High doses of cholesterol bring about aortic alterations in SHR but not in NR. Thus, in addition to the disorder of fat and carbohydrate metabolism, disturbed protein metabolism is of decisive importance as a risk factor for coronary and other vascular diseases.

Thyroid hormones or methylthiouracil (MTU) were given to healthy rats or animals with hepatic fibrosis. Administration of thyroid hormones to rats with carbon tetrachloride-induced hepatic fibrosis caused an increase in serum activity of matrix-degrading hydrolases although the increase was lower than that in healthy animals treated with thyroid hormones only. Methylthiouracil administration had no effect on the enzyme activity in healthy rats and resulted in a decreased activity in rats with hepatic fibrosis.

Type I iodothyronine deiodinase (ID-I) is a selenoenzyme, which is important for the conversion of the prohormone thyroxine (T4) to the bioactive thyroid hormone 3,3',5-triiodothyronine (T3). 2-Thiouracil derivatives inhibit ID-I by interaction with an enzyme form generated during catalysis. We have now tested the potential inhibitory effects of the selenocompounds 6-methyl- (MSU) and 6-propyl-2-selenouracil (PSU) in comparison with their thioanalogs 6-methyl- (MTU) and 6-propyl-2-thiouracil (PTU) on rat liver ID-I activity using 3,3',5-triiodothyronine (reverse T3, rT3) as substrate and dithiothreitol (DTT) as cofactor. All compounds showed dose-dependent inhibition of ID-I with IC50 values of 1, 0.5, 0.4 and 0.2 microM for MTU, MSU, PTU and PSU, respectively. Our results further suggest that these inhibitions are uncompetitive with substrate and competitive with cofactor. The high potency of selenouracils may be due to reaction with a substrate-induced enzyme selenenyl iodide intermediate under formation of a stable enzyme-selenouracil diselenide.

1. Male Sprague-Dawley rats were made either hyper- or hypothyroid with thyroxine or 4-methyl-2-thiouracil, respectively. Bronchial smooth muscle (BSM) contractility and lung cyclic adenosine 3',5'-monophosphate (cAMP) content were measured in both conditions. 2. Bronchial smooth muscle contractility was significantly weaker in hyperthyroid rats, while the BSM contractility of hypothyroid rats was the same as controls. 3. The cAMP content of hyperthyroid rat lungs was similar to controls but was decreased in hypothyroid rats. 4. These studies demonstrated that both the hyper- and hypothyroid states affect respiration, although the mechanisms involved with different for each condition.

The immunoregulatory role of thyroid hormone was investigated in mice weighing 20-30 g. The experimental results showed that the antibody production, transformation of lymphocytes, DTH response (delayed-type hypersensitivity response) and phagocytosis of the peritoneal macrophages were all markedly enhanced when the animals were fed with a daily dose of 2 mg thyroid hormone for 14-18 days. The number of mature T cells in peripheral blood was also increased significantly by the thyroid hormone. When mice were administered with antithyroid drug (methylthiouracil), the antibody production, the transformation of lymphocytes and DTH response were inhibited. Thyroidectomy could also suppress the antibody production. The results suggest that thyroid hormone is involved in the regulation of immune function.

The total and specific activity of thyroid homogenate of normal rat was 0.95 times 10- minus 3 GU (guaiacol unit) per rat and 0.61 times 10- minus 3 GU per mg protein. These were increased 3.4 fold and 1.6 fold by chronic administration of thyroid stimulating hormone (TSH) and 5.1 fold and 2.5 fold by treatment with methylthiouracil (MTU), respectively. However, the soluble fractions, which were obtained by centrifugation at 105,000 xg for 120 min from the homogenates, manifested only 2-3% of the activity of the original homogenates, and the specific activity was scarcely affected by the administration of TSH and MTU. Moreover, even the low activity of the soluble fraction might be due to contaminated microsomes. None of the results obtained favors the view that the peroxidase is released from the cell to the colloid lumen. The number of peroxidase molecule per cell of thyroid was roughly estimated to be 1.5 times 10- minus 5 for normal and 3.9 times 10- minus 5 for MTU-treated rat.

In homogenate supernatants of kidneys of male rats the extent of deiodination of L-diiodotyrosine (L-DJT) and L-thyroxine (L-T4) was investigated in dependence on the thyroid function (hypo- and hyperthyroidized) and also in dependence on age. In rats hypothyroidized by loading with Methylthiouracil (MTU) or Methimazol (MMI) the deiodination for L-DJT and L-T4 was significantly reduced, in rats loaded with 40 mug T4 sc. for 10 days, the deiodination was significantly enhanced compared with untreated control animals. With advancing age (6 weeks, 3 or 12 month) the deiodination activity is highly significantly reduced. The results underline relations between thyroid gland function and deiodination activity in kidney.

The hypothalamic serotonin content in the altered pituitary thyrotropic function was determined in mature male rabbits by the fluorescent method. No distinct correlation was found between the intensity of the pituitary thyrotropic function and serotonin level in the hypothalamus. Intensification of the hypophysial thyrotropic function under the effect of 6-methylthiouracil or partial thyroidectomy was accompanied by a rise in serotonin concentration, while in aseptic inflammation in the thyroid gland, or in combination of the upper cervical sympathetic ganglia ablation with chlorpromazine administration this adenohypophysial function proved to increase without any significant shifts in the hypothalamic serotonin level. The mentioned findings suggest that the role of serotonin in the hypothalamic regulation of the hypophysial thyrotropic function could not be considered as determining.

Changes in the content of cyclic nucleotides (cAMP and cGMP) and related enzyme activities were observed in the rat thyroid, pituitary and plasma during the prolonged increase of endogenous TSH produced by treatment with methylthiouracil (MTU). Experiments were performed after 4 weeks treatment with MTU. The wet weight and cAMP content per wet weight of the thyroid increased 3 and 1.4 times respectively, but cGMP showed a slight decrease. Pituitary weight increased 1.3 times, but cAMP and cGMP content did not change. The cAMP level in plasma also increased about 1.3 times, but cGMP did not increase. The cAMP-phosphodiesterase activity in the thyroid, pituitary and plasma was increased 1.9, 1.4 and 1.3 times respectively after MTU treatment, while cGMP-phosphodiesterase showed no significant change. ATPase activity in the thyroid and pituitary was also increased more than 1.5 times after MTU treatment, while 5'-nucleotidase activitity decreased remarkably. These data indicate that the metabolism of the cyclic nucleotide system in the thyroid is stimulated by TSH.

Interference microscopy was used to measure dry mass of pyramidal neurones of hippocampal CA1 and CA3 areas in control and hypothyroid rats aged 14 and 21 days and 2 months. Hypothyrosis was induced in the newborn by intraperitoneal injection of methylthiouracil to the lactating female during the lactation period (for 1 month). Hypothyrosis caused a considerable retardation of the animals' growth. A significant decrease in he concentration of dry substances was detected only in the cytoplasm of Ca1 area neurones in the group of 2-month-old hypothyroid animals. The measurements have demonstrated that hypothyrosis gives rise to pronounced retardation of the neuronal build-up and accumulation of protein products in the cytoplasma of hippocampal pyramidal cells in all the animal groups examined. The size and dry weight of neuronal nuclei of both the hyppocampal areas substantially diminish in the group of 2-month-old hypothyroid animals.

5' -Nucleotidase activity was determined in rat thyroid and some other organs employing a specific assay method. During the course of methylthiouracil (MTU) treatment, thyroid 5'-nucleotidase activity decreased significantly. This decrease was specific for this enzyme since the activity of neutral phosphatase did not change and the activity of alkaline phosphatase and Mg2+-activated adenosine triphosphatase increased markedly. The 5'-nucleotidase activity of the adenohypophysis also decreased following MTU treatment. This enzyme activity of the liver, heart and whole brain remained unchanged after the treatment. The role of this enzyme was discussed in relation to tissue growth and increased contents of RNA and DNA in the thyroid and adenohypophysis.

Effect of chronic methylthiouracil (MTU) treatment on the thyroid arginase activity and thyroidal concentration of arginine, ornithine and other amino acids was studied in the rat. The activity of thyroid arginase increased significantly at 15 days of MTU treatment and the elevated enzyme activity was reduced to normal by L-thyroxine (T4) injection. The thyroidal concentration of polyamines was increased by MTU and decreased by T4 with the exception of spermine. The thyroidal concentration of arginine and lysine, a substrate and an inhibitor for arginase respectively decreased significantly, while that of ornithine remained unchanged after MTU treatment. T4 injected to MTU-pretreated rats restored the decreased arginine concentration to normal. These results suggest that ornithine supply for polyamine biosynthesis is regulated by the level of both arginase and lysine in the thyroid.

Changes in ornithine decarboxylase (ODC) activity and in polyamine contents of the rat thyroid were studied under various experimental conditions. Methylthiouracil (MTU) treatment produced several-fold increases in the thyroid ODC activity and in the content of putrescine, spermidine and spermine within a week. While serum thyrotropin (TSH) levels increased gradually up to 3 weeks, the content of both putrescine and spermidine tended to reach a plateau after 2 weeks of the goitrogen treatment; spermine content continued to increase progressively for 3 weeks. Discontinuance of MTU at 7 days resulted in a rapid decline in the elevated thyroid ODC activity, followed by a diminution of putrescine, spermidine and RNA contents. Thyroidal putrescine, spermidine and RNA responded more sensitively to both introduction and withdrawal of TSH stimulation than thyroidal spermine and DNA. Excess iodide, having no effect on the basal level of thyroid ODC, suppressed the MTU-induced increase in this enzyme activity without affecting circulating TSH, thyroxine (T4) and triiodothyronine (T3) levels. There was a significant negative correlation between the ODC activity and intrathyroidal concentration of iodine in MTU-pretreated rats. Theophylline increased the thyroid weight and ODC activity when given to rats fed with a subeffective dose of MTU. Analyses of serum TSH, T4, T3 and of thyroidal iodine revealed that TSH-induced thyroid ODC activity was suppressed by increased circulating thyroid hormones and/or intrathyroidal iodine. Furthermore, it was suggested that thyroid hormones and excess iodide acted directly on the thyroid to alter polyamine biosynthesis, possibly by changing the responsiveness of the gland to TSH.

Experiments in 16-week old male Wistar strain rats showed that deficiency of thyroid hormones caused a decrease of cytochrome aa3, RNA, myofibrillar and sarcoplasmic protein contents in m. quadriceps femoris, Mg2+ activated actomyosin ATPase activity and physical working capacity of untrained and trained animals also reduced. Physical activity the intensity of which corresponded to metabolic power output of 190%. Vo2max stimulated thyroid function, elicited a significant elevation of blood thyroxine level, oxidative potential and intensity of protein synthesis in m. quadriceps femoris, the character of these changes depending on the type of muscle fibers. The stimulation of thyroid function caused an elevation of muscle content of cytochrome aa3 and content of RNA, especially in the glycolytic fibers of hypothyroid rats.

The effect of methylthiouracil (MTU) on the colloidopexic capacity of rat hepatosplenic macrophages was experimentally studied. MTU administration determined the depression of the colloidopexic function tested with isamine blue. Under such conditions, thyroid insufficiency may be responsible for the decrease of the hepatosplenic colloidopexic activity. X

There is a marked reduction of diuresis (revealed by water load) during the action of 6-methylthiouracil (MTU). In the course of the thyroid gland restitution (after the discontinuance of the 6-MTU action) the diuresis at first becomes intensified and then it repeatedly either becomes diminished or enhanced. Secretory activity of the cells of the supraoptic nucleus of the anterior hypothalamus also becomes alternately enhanced and diminished. Dynamics of the shifts in this activity failed to correlate with the "ricochet" enhancement of the thyrotropic function of the hypophysis, which occurred on the 11th restitution day and corresponded to the "ricochet" enhancement of iodine consumption by the thyroid gland. Thus, there was no association between the state of the cells of the supraoptic nucleus and the changes in the thyrotropic function of the hypophysis.

There exist some histometric methods for the morphological quantification of different strongly stimulating effects on the thyroid gland induced by drugs and/or other chemical substances in dependence upon dose and duration of application. But in respect of technical and temporal expense and also diagnostic statement, there are considerable differences between these recording procedures. Therefore we examined the 3 mostly used methods synchronously (i.e. determination of thyroid epithelial cell height, nuclear volume in thyrocytes, and estimation of relative volume parts in the thyroid gland by the point counting method) by investigating the thyroid glands of methylthiouracil-(MTU)-stimulated rats and corresponding controls in order to compare the diagnostic value and temporal expense. The largest temporal expense was required in the nuclear volume determination, the smallest in the point-counting method. On principle, all 3 procedures allow the determination of hypertrophic alterations but only by help of the point-counting method, also hyperplastic changes are recognizable. By nuclear volume determination, we found significant differences between central and peripheral parts of the thyroid gland. Therefore, to avoid the subjective error, it will be necessary to measure a large number of nuclei in many planes of the gland. Also the determination of epithelial cell high reinforces the subjective error because of the heterological structure especially in unstimulated thyroid gland. If the number of counting points is exactly determined and, full of sense, limited, the point-counting method allows a nearly complete measuring of the whole object to be tested within an acceptable investigation time. In this way, the heterological structure of thyroid gland will be regarded, and comparability and reproducibility are guaranteed on an high level.

The development of the vegetative muscular-gastric plexus ganglia has been studied in 17-day-old chick embryos at administration of thyroxin and the inhibitor of its production - methylthiouracil. The substances in question are injected into the air chamber of the egg on the 3d days of incubation. In histological preparations, stained after Nissl, with hematoxylin - eosin and silver impregnated, the total number of cells of the neuroblastic line and that of differentiated neurons are calculated in 50 ganglia. Under thyroxin effect the number of the differentiated neurons in the ganglia increases, while the total number of the neuroblastic cells (comparing to the control) remains constant. Under methylthiouracil effect the number of the differentiated neurons decreases and so does the total number of the neuroblastic cells. Thyroxin and methylthiouracil differently affect the differentiation rate of the neurons at various stages of this process. The effect of the drugs applied is poorly manifested in the duodenum, where (in the control) the total portion of the differentiated cells is higher than in other segments of the digestive tube, and the number of the ganglia without such cells is lower, while the portion of the ganglia with numerous differentiated neurons is more significant.

In the control animals of thyroid peroxidase is localized within the membrane of rough endoplasmic reticulum, perinuclear cisternae, microvilli, lamellar structures of the GOLGI apparatus and dispersed through the cytoplasm small vesicles. 3 weeks treatment of the animals with MTU leads to disappearance of the peroxidase activity from the follicular cells. However, a prolongation of MTU administration until the 6th month and latter causes a reappearance of the peroxidase activity within the same structures of the proliferating cells as in the control animals. In the epithelial cells of follicular and papillary carcinomas the reaction product is observed predominantly within the membrane of the rough endoplasmic reticulum, perinuclear space and outher membrane of the microvilli. The changes in the inhibitory effect of MTU on the peroxidase activity during thyroid carcinogenesis are discussed.

The influence of thyroid status modifications on the metastasis of 256 Walker carcinosarcoma intratesticularly grafted to rats was examined. Inhibition of metastasization and of the secondary tumoral growth was noticed in animals chronically treated with thyroxine, while hypothyroidization realized either by surgery or by administration of synthetical antithyroid drugs enhanced metastasization and growth of the secondary tumors. The primary tumoral development was not significantly influenced by the thyroid status modifications. The variations of the thyroid hormonal level influenced nonspecifically the proliferation of the in vitro cultivated tumoral cells.

Thyrotropin releasing-hormone (TRH) increased the activity of cytrochrome C oxidase in a concentration of 0.01 and 1.0 microgram/ml in the adenohypophysis of rats fed methylthiouracil for 6 weeks. This effect of TRH on the activity of the enzyme was blocked with T4 added to the incubation medium in a concentration of 20 microgram/ml. Actinomycin D (20 MICrogram/ml) prevented the block of the enzyme with thyroxin. In a concentration of 0.01 microgram/ml TRH, and in a concentration of 2.0 microgram/ml T4 failed to change the activity of cytochrome oxidase in the adenohypophysis of normal and partially thyroidectomized rats.

Chronic investigations (up to 3 years) conducted in dogs have evidenced that periodic disturbances of vascular permeability produced by using dicoumarin with a long-term blocking of the thyroid function greatly aggravate pathological manifestations induced by the action of 6-methylthiouracil. They result in the development in the animals of endogenous hypercholesterinemia, elevated content in the blood of beta-lipoproteins and in a periodic rise of the arterial pressure. These changes were particularly spectacular at the final stage of the experiment in animals with marked manifestations of atherosclerosis and were accompanied by reduced stand-by potentialities of the adrenal gland, disruption of metabolic processes in the myocardium along with the slowed down resorption of NaI131 from the heart muscle. The sensitivity of the heart to adrenalin was down too.

Histologic-cytological and morphometrical changes were investigated in the adrenal cortex of male Wistar-rats following pinealectomy and application of melatonin in eu-, hypo-, and hyperthyroid situations. A rat experiment (at an average of 45 d) to find a possible functional connection between the pineal gland and the adrenal cortex was carried out. In the literature, there are only a few of informations about the role of the pineal in regulating ACTH secretion. The results are very contrarily. We found that pinealectomy is connected with a progressive transformation and melatonin with a little regressive transformation in the adrenal cortex. But, it is not evident, that the glomerular zone is activated after both pinealectomy and application of melatonin. In our opinion, the glomerular zone and the secretion of aldosterone increased after as well pinealectomy as melatonin. Application of melatonin diminishes the function of the pineal gland (see group 4-pinealectomy plus melatonin-where was found a progressive transformation). Under these experimental conditions, one can speak of a "pharmacological pinealectomy" after application of melatonin alone. However, the effect of melatonin on the fascicular zone and the glomerular zone is different. The effects of pinealectomy or application of melatonin in combination with methylthiouracil or thyroxin are relatively unimportant.

The effects of Crinofizin (Cfz), cortisone, triiodothyronine (T3) and methylthiouracil (MTU), alone or in association with one another, on collagen metabolism of impuberal and adult rabbits were tested. These effects were assessed by the hydroxyprolinuria (Hyp) assay corroborated with observations regarding mortality, body weight, diuresis and excretion of nitrogenous substances. Cfz given alone stimulated weight gain and did not alter characteristically the Hyp. Cortisone and T3 given in various doses and ways without Cfz induced general phenomena of the antianabolic or catabolic type, affecting characteristically the Hyp, which was low after administration of cortisone and increased after T3; association of Cfz to both cortisone and T3 had general protecting and corrective effects and brought Hyp towards or at normal values. Chronic administration of MTU with or without Cfz did not have significant effects. The corrective value of Cfz by anabolic and anti-catabolic actions on collagen metabolism disturbed by cortisone or T3 in excess is discussed.

The mutagenic effect of carbimazol and methylthiouracil on mice fetal blood in the 15th and 16th day of intrauterine life, as compared to the effect on maternal bone marrow and control animals, was studied using the micronucleus test. The incidence of micronucleated cells was significantly higher in the fetal blood than in the maternal bone marrow. The finding demonstrates that the method of transplacentar mutagenesis is a reliable test for estimating the mutagenic risk of such drugs as carbimazol and methylthiouracil in current therapeutical use.

With the object of investigating the so called 'feedback' mechanism, whereby hormones from the various endocrine glands influence the hypothalamus in its regulation of the hypophysial tropic hormones, the activity of the nucleus preopticus (NPO) has been studied by means of karyometry under experimental conditions to understand the endocrine nature of the hypothalamic nuclei. (1) Oestrogen and testosterone propionate caused an increase in nuclear volume of the NPO cells, which is related to the synthesis of secretion. An increase of the nuclear volume is attributed to an increase of secretion. (2) Methylthiouracil (MTU) and thiourea bring about shrinkage in nuclear dimensions (p less than 0.001). Depletion of NSM is noticed in the hypothalamo-hypophysial neurosecretory system after MTU administration. (3) Two to five days' administration brings about depletion of NSM in the cells of the NPO. Eight to 15 days' metopiron treatment enhanced the activity of NPO cells. (4) Hypophysectomy caused shrinkage of the NPO cell nuclei, depletion of AF-stainable material in the cells of the NPO. ACTH treatment reversed these changes and brought about hypertrophy of the neurosecretory cells. (5) Neomercazole treatment and radiothyroidectomy bring about shrinkage in the NPO cell nuclei. Thyroidectomized toads when injected with thyroxine showed an enlargement of their nuclei.

The effects of an isocaloric control diet of high-protein and low-protein diets (in which protein amounted to 16, 62 and 2% of total calorie content, respectively) with and without simultaneous administration of Thyreotom or methylthiouracil on total body composition have been studied in growing male Wistar rats. In spite of higher average Kcal intake (per day and 100 g of body weight), the rats on the high-protein diet were weighing less than the control animals at the end of the experiment. As compared to the control animals, the rats on the high-protein diet as well as those on the low-protein diet showed a greater relative weight of the thyroid gland. The protein percentage of the body was not significantly increased by an increase in protein supply; likewise, it was not significantly reduced by a decrease in protein supply. The body fat content was by 2/3 lower in the rats on the lowprotein diet than in the control animals. This difference was statistically significant. Thyroid hormones (1 mug liothyronine + 4 mug levothyroxine/100 g body weight, administered orally for 3 weeks) produced a slight increase of body protein in the rats on the high-protein diet; they increased it significantly in the rats on the low-protein diet. Body fat was correspondingly reduced. Methylthiouracil reduces the body fat content in the control animals and in the rats on the high-protein diet; it increased the body fat content in the rats on the low-protein diet. Our results confirm the partial findings of human pathology and demonstrate clearly that nutritional diseases, especially protein deficiency, worsen considerably in case of concomitant disorders of thyroid hormone metabolism.

Histologic-cytological and morphometrical changes were investigated in the adrenal cortex of male Wistar-rats in experimental hypo- and hyperthyroid situation. Under hypothyroid metabolism's situation (methylthiouracil-application or thyroidectomy) a regressive transformation was found: It is a decrease of secretion of the fascicular zone. The glomerular zone was activated. After long-time application of high dosis of thyroxin, the histological findings show some criteria of an increasing function and also of a beginning exhaustion, too. It is evident, that the duration of the hyperthyroid situation is important. Thyroxin is an unspecific stressor. The long-time application of high thyroxin dosis induced a regressive transformation and atrophic changes in the adrenal cortex. The nuclei of the fascicular and the glomerular zone were diminuated significantly.

Age peculiarities of interaction of the hypothalamo-hypophysial system and the thyroid gland with consideration to direct and feed-back control were studied. Significant alterations appear in old age in the hypothalamo-hypophysial system and the thyroid gland at the level of direct and feed-back control; there is an increase of sensitivity of the thyroid gland tissue to TSH, and of the hypothalamus-hypophysis--to the T4 effect. This rise of sensitivity is largely determined by the activation of the T4 deiodination processes in the adenohypophysis. The reactive capacity of the thyroid gland and of the hypothalamo-hypophysial complex to the corresponding influence is decreased.

It was found that the H2O2 generating system containing D-aspartate oxidase isolated from the thyroid gland and D-aspartate, takes part in oxidation of iodides. The molecular I2 formed under experimental conditions is subsequently incorporated into thyroid proteins. Thiosemicarbazide, thiourea, methylthiouracyl, sulphathiazole, thiocyanate and azides were found to have an inhibiting effect on iodide oxidation. Methimazole inhibits both the oxidation of iodide and incorporation of 131I into protein. The iodide incorporation was inhibited by catalase. The findings of these investigations suggest an indirect participation of D-aspartate oxidase in the synthesis of the thyroid hormone by supplying the essential substrate for the iodide oxidation, H2O2.