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Histologic changes in tissue components of the hyperplastic thyroid gland during its involution in the rat.

Male Fischer rats were fed a low-iodine diet containing thiouracil for 21 days to produce hyperplastic thyroid glands, and then fed a high-iodine diet for various time intervals, from 5 hr to 180 days, in order to study the morphological changes that occur during involution. Thyroids were fixed by perfusion fixation and embedded in Epon. Sections were examined by light microscopy. Initially at 0 days of involution (at the time of the change to the high-iodine diet), follicular lumens were very narrow and capillary lumens were very wide. The capsule was thick and infiltrated with mononuclear leukocytes. No obvious changes occurred for 1 day after the change in diet, but shortly thereafter capillary lumens began to narrow. By 4 days, most capillary lumens were close to normal size; capillaries formed a more or less normal bed except that many were embedded in a relatively thick or wide interfollicular matrix. This matrix was largely gone by 21 days. Between 1 and 21 days, follicular lumens dilated progressively as colloid accumulated. The density of staining of the accumulated colloid varied from follicle to follicle, and this variation was also observed in older controls. Inflammatory cells gradually disappeared from the capsule and most were gone by 15 days. Starting at approximately 15 days and continuing to 180 days, one or more disintegrating cells were found in some lumen profiles. Colloid goiters were not observed in these rats even after several months of involution. Some lumens were rather large, however, and small fractions of the follicles, both small and large, were bounded by flat cells and resembled "cold" follicles morphologically.

Animals↗

Comparison of a special class of epithelial cells in hyperplastic thyroids undergoing involution and in thyroids in hypophysectomized rats.

In male Fischer rats, a class of follicles with flat epithelium is present as a minor component of thyroid glands in which most of the follicles have cuboidal epithelium. These follicles occur in thyroids that have been made hyperplastic by feeding the rats thiouracil for 21 days and then allowing involution for 21 days or more. They also occur in older control rats. The follicles resemble in morphology, at the light-microscope level, the so-called "cold" follicles that occur in aged mice. We have examined the ultrastructure of the flat cells in these follicles and compared it with that of the flat cells occurring in the thyroid follicles of hypophysectomized rats. The cells in involution have abundant rough endoplasmic reticulum (RER) and few lysosomes and, in these respects, differ markedly from cells in hypophysectomized rats. The follicles with flat cells are surrounded by a normal incidence of blood capillaries, so that the cells do not appear to be deprived of access to an adequate supply of materials necessary to satisfy their metabolic requirements. Their abundant RER suggests that they have thyroid-stimulating hormone (TSH) receptors, so that the flat cell may be the result of some process occurring at a step distal to receptor coupling with TSH. Their occurrence in young rats after the induction of hyperplasia may be a consequence of cell multiplication producing a clone of neighboring abnormal cells that have an abnormally small cell height.

Animals↗

Observations on the fine structure of propylthiouracil-induced "brown degeneration" in the zona reticularis of mouse adrenal cortex.

Propylthiouracil (6-propyl-2-thiouracil), an anti-thyroid agent, was fed to mice in a concentration equal to 0.1% of their diet for periods of 10 and 15 weeks. The cells of the inner zone of the adrenal cortex were examined with the electron microscope. In animals receiving propylthiouracil for ten weeks mitochondria were altered and the smooth endoplasmic reticulum (SER) showed a marked focal proliferation. In contrast to control animals rough endoplasmic reticulum was abundant and was frequently associated with the hyperplastic SER. After 15 weeks these alterations were no longer present but had been replaced by a spectrum of "brown degeneration." The less affected cells were characterized by increased numbers of liposomes and lysosomes and the more affected cells by liposomal and mitochondrial degeneration. These observations emphasize that "brown degeneration" is a true degenerative process and not a spontaneous proliferation of ceroid pigment. It is suggested that the changes described may be directly related to an alteration in cholesterol metabolism.

Adrenal Cortex↗

Chemotherapeutic agents, XVIII: Synthesis of pi-deficient pyrimidines and fused pyrimidines as leishmanicidal agents.

Synthesis of 6-aryl-5-cyano-2-thiouracils 1a-d from the condensation-cyclization of an aromatic aldehyde, thiourea and ethyl cyanoacetate has been described. Alkylation of 1a-d under different reaction conditions with mono- and dihalo-alkanes yielded 2, 3, and 6. Interaction of 1 with POCl3 provided halopyrimidines 8a,b. Nucleophilic substitution on 8 and 3 with aromatic amines gave 9a-d and 7a-d respectively. 6-Chloro-5-nitro-3-methyluracil (11) obtained by nitration of 10 underwent nucleophilic substitution with amines providing 12. Some of the compounds screened as leishmanicides did not exhibit any significant activity.

Animals↗

Non-nucleoside HIV reverse transcriptase inhibitors, Part 6[1]: synthesis and anti-HIV activity of novel 2-[(arylcarbonylmethyl)thio]-6-arylthio DABO analogues.

2-(Arylcarbonylmethyl)thio-6alpha-naphthylmethyl derivatives of dihydro-alkoxy-benzyl-oxopyrimidines (DABO) were newly found to exhibit activity against both HIV-1 and HIV-2. To further explore their structure-activity relationship, the modified S-DABO analogues (5a-g and 6e-f) with a 1-naphthylthio or phenylthio group at the C-6 position were synthesized. S-Alkylation of 5-ethyl-2-thiouracil with substituted 2-bromo-acetophenones provided crude 2-[(arylcarbonylmethyl)thio]-5-ethyl-(3H)-uracil 2a-e, which was directly subjected to toluenesulfonylation with TsCl to afford disulfonate 4a-e. Substitution of 4a-e with arylthiol afforded the desired S-DABO analogues 5a-g and 6e-f. The compounds were evaluated for their in vitro anti-HIV activity in MT-4 cells. The IC(50) values for anti-HIV-1 activity fall into the range 0.37-29.50 microM, and the IC(50) values for anti-HIV-2 activity fall into the range 23.11-181.07 microM. The results indicated that these compounds are moderately active against HIV-1 and HIV-2.

Anti-HIV Agents↗

Chemotherapeutic agents, XXI: Synthesis of Pi-deficient pyrimidines as leishmanicides.

Synthesis of 5-cyano-6-(3-pyridyl)-2-thiouracil (1) has been described from 3-pyridinecarboxaldehyde, thiourea, and ethyl cyanoacetate. Alkylation of 1 with mono- and dihaloalkanes under different conditions, provided 2a,b, 3a,b, and 4a-i. Halogenation of 4g with POCl3 yielded 5 which underwent nucleophilic substitution with amines to give 6a-g. Fusion of 4a with aromatic and heterocyclic amines at 160 degrees C gave the substitution products 7a-c. Some of the compounds were screened for antileishmanial activity but only one of them (6d) demonstrated very significant activity.

Animals↗

Regulation of neuron numbers in Xenopus laevis: effects of hormonal manipulation altering size at metamorphosis.

Xenopus laevis tadpoles reared in a 0.01% solution of 6-n-propyl-2-thiouracil (PTU) are blocked in their development at larval stage 54 but continue to increase in size. When released from the effects of PTU they metamorphose into frogs of sizes significantly larger than those of their untreated siblings. Using this size difference to examine the hypothesis that neuron numbers are matched to the size of their postsynaptic targets during neuronal cell death, we measured the following on stage 66 frogs metamorphosing from PTU-treated and untreated tadpoles: lumbar lateral motor column (L-LMC) motoneuron number and mean nuclear cross-sectional area; thoracic and lumbar dorsal root ganglion (DRG) cell number and mean nuclear cross-sectional area; and muscle fiber number in two representative thigh muscles. A few measurements of neuron number and cell size were also made on untreated and PTU-treated stage 54 tadpoles. The most striking correlations observed were not between peripheral size and neuron numbers but between peripheral size and neuron size. Motoneuron numbers were not increased in the PTU-treated animals, perhaps because the increase in peripheral size involved an increase in muscle fiber diameter rather than an increase in muscle fiber number. Thoracic DRG cell number, but not the sum of thoracic and lumbar DRG cell numbers, was increased. In general, our findings do not support the hypothesis that neuron numbers are matched to peripheral size by a process regulating the amount of cell death that occurs during metamorphic stages in Xenopus laevis.

Animals↗

Effects of thyroid status and choice of USP preparation on the pharmacokinetics of propylthiouracil in the beagle dog.

In vitro dissolution profile testing of 12 generic 6-propyl-2-thiouracil (PTU) formulations indicated significant differences in their solubilities in water and in 0.1N hydrochloric acid. Two products, representing the extremes of solubility, were subsequently tested for bioavailability. Bioavailability data from euthyroid, hyperthyroid, and hypothyroid beagle dogs indicated that the rate of PTU absorption varied appreciably between the two generic products studied. These results were consistent with the in vitro dissolution data. Comparisons of the pharmacokinetic parameters obtained for the euthyroid beagle dog to those reported for euthyroid man12 indicated that the beagle dog is an adequate model for PTU disposition in man. These combined results suggest that all generic formulations of PTU are not the same and that the most soluble product may be desirable in situations where rapid absorption is important, such as thyroid storm.

Animals↗

Effect of hypothyroidism induced by propylthiouracil on ovarian function and structure in offspring from treated mothers (Rats).

The aim of the present study was to investigate the effects of hypothyroidism induced during the pre- and postnatal periods of life on ovarian function and structure in offspring (pups) 120 days of age. Three groups were used. In the prenatal group, treatment was given from conception to parturition. In the postnatal group, treatment was given from parturition to 25 days postpartum. Hypothyroidism was induced by administration of 0.1% 6-n-propyl-2-thiouracil (PTU) in the drinking water of mothers. Body weights of the offspring were measured weekly. In each group, ten offspring were sacrificed at 120 days of age. Postnatal PTU treated pups showed delay in eye opening, teething, fur development, and weaning (35-37 days) compared to control animals (28-30 days). Body weight of offspring in the postnatal PTU treatment group was significantly decreased (P < 0.001), while the prenatal PTU treatment group showed a significant increase (P < 0.0001) compared to control animals. There was a significant (P < 0.05) reduction in paired ovarian weight of offspring in the postnatal PTU treatment group compared to control animals. Diameter of the ovaries was not affected by any treatment. Regarding the morphometery, only offspring in the prenatal PTU treatment group showed a significant (P < 0.001) increase in the diameter of graafian follicles. No significant difference was observed in morphometery of the granulosa layer, primary, and developing follicles of control and all treated groups. Number of primary, developing, and graafian follicles of all the treated groups was similar to that of the control group. The corpora lutea of the postnatal PTU treated group contained a population of large numbers of luteal cells compared to the control group. The prenatal PTU treated group did not exhibit a profound effect on ovarian morphology, histology, and morphometery. No difference was found in the serum estradiol concentration of control and PTU treated groups. J. Exp. Zool. 293:407-413, 2002.

Animals↗

Effect of hypothyroidism induced by propylthiouracil and thiourea on male and female reproductive systems of neonatal mice.

The effect of hypothyroidism induced by 6-propyl-2-thiouracil (PTU) or thiourea (TU) on the development of the reproductive system in male and female neonatal ICR mice was investigated. PTU or TU was injected subcutaneously into experimental animals from postnatal day 1 (PD1) onward. The histological changes of the reproductive organs, formation of ovarian follicles, and spermatogenesis were examined on PD 14, 21, and 28, and the fertility of the hypothyroid mice in adulthood was followed. It was found that PTU or TU treatment did not produce an effect on the histology of the neonatal uterus and oviduct. In contrast, the drugs induced a decrease in the number of primordial follicles, multilaminar follicles, and Graafian follicles in the ovary. The number of follicles with degenerated follicular cells was increased. In the testis both PTU and TU treatments brought about a decrease in the number of seminiferous tubules with developing spermatids although the mean diameter of seminiferous tubules and the histology of the testis, epididymis, seminal vesicle, and coagulating gland was unaffected. The mating between hypothyroid females and euthyroid males and that between hypothyroid males and euthyroid females were normal with regard to the pregnancy rate, litter size, and sex ratio of offspring. The somatic growth of the resulting offspring was normal. It is concluded that the retarding effect on ovarian and testicular development in mice during neonatal period was not serious enough to adversely affect reproduction in the hypothyroid animals.

Animals↗

Bioavailability of propylthiouracil in humans.

Single lots of five commercially available 50-mg propyl-thiouracil formulations were evaluated in vitro and in vivo. Each product met the USP XIX specifications for drug content, content uniformity, and disintegration time. However, major differences were noted among products in their rate and extent of dissolution. Statistically significant differences (p less than 0.05) were observed in vivo among the drug formulations at all but one of the sampling times, as determined from crossover blood level studies in 12 healthy male volunteers. The differences among the areas under the plasma level-time curves for the various products were not statistically significant. No statistically significant correlations were found between the in vitro and in vivo parameters studied.

Biological Availability↗

Mitigation of thyroxine-induced hyperglycaemia by two plant extracts.

Extracts of Trigonella foenum-graecum (TFG) seed and Allium sativum (AS) bulb were evaluated for their efficacy to ameliorate l-thyroxine (l-T4) induced hyperglycaemia in rats. Simultaneously, the serum cholesterol concentration, a supporting parameter for thyroid function, was also estimated. Thyroxine treatment in rats (300 microg/kg b. wt./day) increased the levels of both the thyroid hormones, namely thyroxine (T4) and tri-iodothyronine (T3) with a concomitant elevation in serum glucose concentration and a reduction in serum cholesterol level. Administration of TFG (220 mg/kg/day) and AS (500 mg/kg/day) extracts in hyperthyroid animals decreased the serum glucose concentration as well as the serum thyroid hormones. For comparison, propyl thiouracil (PTU), an antithyroid compound, was used as the standard at a daily dose of 10 mg/kg. The reductions in serum glucose and thyroid hormone concentrations in the plant extract treated groups were comparable to that in PTU treated animals. Our findings indicate that TFG seed and AS bulb extracts may prove to be effective in the treatment of thyroxine-induced hyperglycaemia.

Alanine Transaminase↗

Guggulu (Commiphora mukul) potentially ameliorates hypothyroidism in female mice.

The efficacy of guggulu, the gum resin of Commiphora mukul in regulating hypothyroidism was evaluated in female mice. In addition to estimating serum levels of thyroxine and triiodothyronine, hepatic 5' monodeiodinase, hepatic glucose-6-phospatase and lipid-peroxidation (LPO), the activities of the anti-oxidative enzymes, superoxide dismutase (SOD) and catalase (CAT), were investigated. While 6-n-propyl-2-thiouracil (PTU, 10.00 mg/kg/d for 30 days) induced hypothyroidism in mice, as evidenced by a decrease in thyroid hormone concentration and in hepatic 5'D-I activity, simultaneous administration of guggulu (200 mg/kg/d for 30 days) reversed this effect, indicating its potential to stimulate thyroid function. Although in PTU treated animals a marginal increase in hepatic LPO was observed, when simultaneously treated with guggulu, it was decreased. A parallel increase in the activity of endogenous antioxidants, SOD and CAT, in the latter group indicated the safe and antiperoxidative nature of the drug. These findings suggest the possible use of guggulu in the amelioration of hypothyroidism.

Animals↗

Radioactivity-based and spectrophotometric assays for isoorotate decarboxylase: identification of the thymidine salvage pathway in lower eukaryotes.

A few organisms, notably some fungi, have the ability to metabolize thymidine to uracil, thus conserving the pyrimidine ring for subsequent metabolic use. Neurospora crassa possesses this pathway, termed the thymidine salvage pathway, and can utilize thymidine as a total pyrimidine source. The enzyme isoorotate decarboxylase (IDCase) completes this pathway via the enzymatic removal of the carboxylate from isoorotate to yield uracil. We describe in this communication two assays for IDCase and their application to determine activity levels, kinetic constants, and inhibitory properties. One uses [carboxy-14C]isoorotate from which the enzymatically generated 14CO2 is collected and quantitated. The second assay utilizes the spectral difference between 2-thioisoorotate and its decarboxylated product, 2-thiouracil. The spectral difference is greatest at 334 nm, out of the range of absorbance of total protein and thus usable for a spectrophotometric assay. The assays are sufficiently sensitive and accurate to be used in the measurement of Km values for both substrates. IDCase activity is found to be significantly higher in N. crassa strains lacking uc-1, a putative regulatory gene, suggesting a degree of metabolic control over this pathway. 5-Nitrouracil is found to inhibit IDCase with an estimated Ki value that is too low for accurate determination.

Ammonium Sulfate↗

Influence of PROP-sensitivity on taste perceptions and hedonics in French women. A study performed without retronasal olfaction.

Detection threshold for the taste of PROP (6-n-propyl thiouracil) in aqueous solution was determined in 173 French Caucasian women deprived of retronasal olfaction by blowing an air stream into the nostrils. As expected, the detection thresholds were bimodally distributed, although as many as 73% of the subjects had thresholds above the antimode concentration and therefore qualified as non-tasters. Detection threshold, difference threshold, intensity perceptions and preference ratings were then determined for NaCl, sucrose, caffeine, Na saccharin and naringin in 20 tasters and 20 non-tasters. No differences were found between tasters and non-tasters for detection or difference thresholds of the various tastants. Intensity and preference ratings for solutions of NaCl, saccharin or caffeine were not influenced by taster status. Although ratings of sweetness intensity for sucrose solutions did not differ between tasters and non-tasters, concentrated sucrose solutions were more disliked by non-tasters than by tasters. Non-tasters rated naringin solutions as more bitter than tasters, but naringin preference ratings were independent of taster status. Some of these findings were unexpected and further studies are required to find out whether they stem from the odour-preventing procedure or are of biological or cultural origin.

Adult↗

Myocardial adaptation to long-term action of substances associated with decreased intensity of cardiac function.

The effect of prolonged treatment of rats with 6-n-propyl-2-thiouracil (PTU), verapamil, or propranolol on cardiac pump function and the properties of myofibrils and mitochondria was studied. After 6-8 weeks of treatment, the heart rate and maximal cardiac output of the isolated heart of rats treated with verapamil or propranolol were higher than those in the control group. The PTU treatment was followed by lower heart rate and maximal work. Calcium sensitivity (pCa50 value) of skinned ventricular fibers was higher in all experimental groups compared to the control by 0.07-0.15 units. Myofibrillar Mg2+, Ca(2+)-ATPase activity measured in isolated Triton-skinned cardiomyocytes was considerably lower after PTU treatment than that in respective controls (0.128 +/- 0.013 vs 0.178 +/- 0.010 mumol Pi/min/mg protein). In contrast, long-term treatment with verapamil or propranolol was accompanied by increased activity to 0.223 +/- 0.018 and 0.254 +/- 0.015 mumol Pi/min/mg protein, respectively. Neither the basal mitochondrial respiration rate of saponin-skinned cardiac fibers nor its enhancement after addition of low ADP concentration or creatine was significantly altered in any experimental group. Also no difference between control and experimental groups was observed in the total activity of creatine kinase or relative percentage of its isoenzymes extracted from cardiac tissue. Thus the changes in cardiac pump function after prolonged treatment with agents decreasing cardiac function may be attributed to concomitant alterations of myofibrils while mitochondria remain relatively intact.

Adaptation, Physiological↗

Protein tyrosine phosphatase-eta expression is upregulated by the PKA-dependent and is downregulated by the PKC-dependent pathways in thyroid cells.

We have recently reported the isolation of a rat cDNA encoding a receptor-type tyrosine phosphatase, which appears to be a marker of thyroid differentiation. To elucidate the molecular mechanisms underlying r-PTPeta expression in normal thyroid cells both in vitro and in vivo, we investigated the regulation of r-PTPeta expression in cultured thyrocytes (the rat cell line PC Cl 3) and in an animal model of TSH-dependent thyroid goitrogenesis. In vitro studies showed that mRNA expression of r-PTPeta in thyroid cells is induced in a time- and dose-dependent manner by the activation of growth- and differentiation-linked PKA pathways (TSH and forskolin), whereas it is down-regulated by the activation of the proliferative dedifferentiating PKC-dependent transduction pathway (TPA). However, the regulation of r-PTPeta expression by TSH and TPA, respectively, is observed only in normal thyroid cells, but is lost in transformed thyroid cells. In vivo studies with thiouracil-fed rats demonstrated that increased serum levels of TSH up-regulated r-PTPeta mRNA expression in parallel with the stimulation of thyroid growth and function. The reduction of blood TSH levels due to iodide refeeding to goitrous rats determined a marked down-regulation of r-PTPeta expression, in parallel with involution of thyroid hyperplasia. Taken together these results demonstrate that the phosphatase r-PTPeta is regulated by the two main thyroid regulatory pathways and suggest that it may play an important role in the growth and differentiation of thyroid cells.

Animals↗

Thyroid hormone deiodination pathways in brain and liver of rainbow trout, Oncorhynchus mykiss.

Outer-ring (5') deiodination (ORD) and inner-ring (5) deiodination (IRD) of L-thyroxine (T4) and 3,5,3'-triiodo-L-thyronine (T3) were studied in whole-brain microsomes of rainbow trout and compared with liver deiodination. Brain T4ORD activity (apparent Km = 1.2-2.5 nM; V(max) = 0.10-0.14 pmol/hr/mg microsomal protein) was less than T4IRD activity (apparent Km = 4.9; V(max) = 0.32) and T3IRD activity (apparent Km = 5.2-5.4; V(max) = 1.1-2.0); T3ORD activity was negligible. All three brain deiodinase pathways shared the following properties: pH optima between 7.0 and 7.3, activity enhanced by dithiothreitol (10 mM), weak inhibition by 6-n-propyl-2-thiouracil and iodoacetate, but stronger inhibition by aurothioglucose. Based on competitive inhibition, the substrate preference for brain T4ORD was T4 = tetraiodothyroacetic acid (TETRAC) > 3,3',5'-triiodo-L-thyronine (rT3) > 3,5,3'-triiodothyroacetic acid (TRIAC) >> T3 > 3,5-diiodo-L-thyronine (3,5-T2). A comparable substrate preference profile was obtained for liver T4ORD (Km 1 nM). Both T4IRD and T3IRD in brain had similar substrate preference profiles (rT3 > 3,5-T2 > T4 > T3) which differed from that of T4ORD. Negligible T4IRD and T3IRD activities existed in liver. We conclude that for rainbow trout (i) T4ORD systems in brain and liver are similar, and consistent with a common enzyme that does not match exactly either mammalian type I or II deiodinases, (ii) brain T4IRD and T3IRD enzymes share several common properties, and correspond functionally to the mammalian type III deiodinase, and (iii) under normal physiological conditions the predominant deiodinase pathways in brain (T4IRD and T3IRD) are poised toward T4 and T3 degradation, while that in liver (T4ORD) is poised toward T3 generation.

Animals↗