Methylthiouracil Research Articles

Determination of naturally occurring thyreostats in bovine milk by high performance liquid chromatography combined with fluorescence detection

This presented study describes a method based on high performance liquid chromatography combined with fluorescence detection (HPLC-FLD) using N-(2-iodoacetyl)-1-pyrenemethylamine (NIPA) as a novel fluorescence labeling reagent for the determination of thyreostats in bovine milk. Five thyreostats, belonging to the group of imidazole and thiouracil, were investigated in this work: tapazole (TAP), thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU) and phenylthiouracial (PhTU). Thyreostats were specifically purified by a silver ion solid phase extraction (Ag-SPE) cartridge and then labeled using NIPA. The labeled derivatives showed excellent fluorescence property with maximum excitation and emission wavelengths of 330 nm and 375 nm, respectively. The labeled derivatives were separated on a reversed-phase Eclipse SB-C18 column within 12 min. Excellent linearity (R2 > 0.995) of all thyreostats was achieved with the limits of detection (LODs) and the limits of quantitation (LOQs) in the low micrograms per liter range of 0.21–0.30 μg/L and 0.70–1.00 μg/L, respectively. Satisfactory recoveries in the range of 93.5–98.0% were obtained for all thyreostats. The developed method has been successfully applied to analyze thyreostats in bovine milk with good applicability. Thirty bovine milk samples have been investigated, and varying levels of thiouracil were detected in thirteen of these samples. The highest level in the raw milk reached a value of 4.5 μg/L. To our best knowledge, this study is the first to report the presence of naturally occurring thiouracil in milk by HPLC-FLD analysis.

Residue analysis of thyreostats in baby foods via matrix solid phase dispersion and liquid chromatography – dual-polarity electrospray – tandem mass spectrometry

ABSTRACTThis paper describes a rapid method for confirming residues of thyreostats in meat-based baby foods by using liquid chromatography – dual polarity electrospray – tandem mass spectrometry (LC-ES(±)-MS/MS). Six thioureylenes, belonging to the group of thiouracil and imidazole, were selected for this work: thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU), phenylthiouracil (PhTU), mercaptobenzimidazole (MBI) and tapazole (TAP).The amphoteric nature of these compounds allows their electrospray detection in both positive and negative ionisation. Nevertheless, MS detection is not favoured by their low molecular weights, while their chromatographic retention is also thwarted by their high polarity. A pentafluorophenyl (PFP) core-shell phase column was selected to avoid peak asymmetry or peak splitting, and a dual-polarity ionisation method was optimised to obtain a sensitivity as high as possible. The method was validated according to the Commission Decision 657/2002/EC. A simple and fast procedure based on matrix solid phase dispersion (MSPD) was optimised to extract analytes from baby foods with recoveries exceeding 82%. Limit of decision (CCα) and detection capability (CCβ) were lower than the permissible maximum concentration (10 ng g−1). The validated method was then applied to assess the potential occurrence of the six selected thyreostats in nine commercial products. All the samples were found free of contamination.

Suppressive effects of methylthiouracil on polyphosphate-mediated vascular inflammatory responses.

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.

Anti-inflammatory effects of methylthiouracil in vitro and in vivo

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.

Methylthiouracil, a new treatment option for sepsis

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.

Ameliorative effect of methylthiouracil on TGFBIp-induced septic responses

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.

Analysis of antithyroid drugs in surface water by using liquid chromatography–tandem mass spectrometry

This paper describes development and validation of a new method for the simultaneous determination of six antithyroid drugs (ATDs) in surface waters by using liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS). Target compounds include two ATD classes: thiouracil derivatives (thiouracil (TU), methyl-thiouracil (MTU), propyl-thiouracil (PTU), phenyl-thiouracil (PhTU)) and imidazole derivatives (tapazole (TAP), and mercaptobenzimidazole (MBI)). Sensitivity and selectivity of the LC-multiple reaction monitoring (MRM) analysis allowed applying a simple pre-concentration procedure and “shooting” the concentrated sample into the LC–MS/MS system without any other treatment. Recoveries were higher than 75% for all analytes. Intra-day precision and inter-day precision, calculated as relative standard deviation (RSD), were below 19 and 22%, respectively. Limits of detection (LODs) ranged from 0.05 to 0.25μg/L; limits of quantitation (LOQs) varied between 0.15 and 0.75μg/L. The validated method was successfully applied to the analysis of ATD residues in surface water samples collected from the Tiber River basin and three lakes of Lazio (central Italy). The analytes were quantified based on matrix-matched calibration curves with mercaptobenzimidazole-d4 (MBI-d4) as the internal standard (IS). The most widespread compound was TAP, one of the most common ATDs used in human medicine, but also TU and MBI were often detected in the analysed samples.

Effects of methylthiouracil on the proliferation and apoptosis of rat bone marrow stromal cells.

The aim of the present study was to investigate the effects of methylthiouracil (MTU) on the proliferation and apoptosis of rat bone marrow stromal cells (BMSCs). Rat BMSCs were isolated, cultured in vitro and treated with various concentrations of MTU. Cell growth curves were determined using the Cell Counting Kit-8 method and the effect of MTU on BMSCs in a logarithmic growth phase was observed. BMSC apoptosis following MTU treatment was detected by flow cytometry. The experimental results demonstrated that the proliferation-inhibition effect was gradually enhanced with increasing MTU concentrations and the extension of treatment time. Statistically significant differences were observed between the treatment and the control groups (P<0.05). In addition, the BMSC apoptosis rate gradually increased with increasing drug concentrations and treatment time extension; statistically significant differences were observed between the treatment and the control groups (P<0.05). Therefore, the results of the present study demonstrated that MTU inhibited the proliferation of BMSCs and promoted apoptosis, indicating the cytotoxic effects of MTU on BMSCs.

THE IDENTIFICATION OF THYROTROPIN-SECRETING CELLS IN THE PITUITARY GLAND OF THE HERMAPHRODITE SYNCHRONOUS TELEOST SERRANUS SCRIBA (L.)

SUMMARY Adult specimens of the hermaphrodite synchronous teleost, Serranus scriba were treated with methyl-thiouracil in order to identify the TSH secreting cells in the adenohypophysis. The thyroid of treated animals becames goitrous with hypertrophy of the follicles, which became lined with columnar epithelium. In the adenohypophysis the PAS and TP cells of the rostro-ventral cords accumulated their secretion. The basophils of peripheral cords of the gland and those distributed along the ramification of the neurohypophysis in the adenohypophysis were not affected by this treatment. Morphological changes of the thyroid gland during reproduction were also studied when an increase in the number of thyroid follicles was observed. During this period they were lined with a columnar epithelium. These changes are in correlation with the number of basophil cells of the adenohypophysis.

Determination of propylthiouracil and methylthiouracil in human serum using high-performance liquid chromatography with chemiluminescence detection

Based on the sensitizing effect of formaldehyde on the chemiluminescence (CL) reaction of propylthiouracil (PTU) and methylthiouracil (MTU) with acidic potassium permanganate and the combination technique of high-performance liquid chromatography (HPLC), a sensitive, selective and simple post-column CL detection method for determining PTU and MTU is described. The optimal conditions for the CL detection and HPLC separation were carried out. The linear ranges were 0.1–20 μg mL −1 for MTU and 0.1–10 μg mL −1 for PTU, the detection limits were 0.03 μg mL −1 for PTU, 0.03 μg mL −1 for MTU and the quantification limits were 0.1 μg mL −1 for PTU, 0.1 μg mL −1 for MTU. The method has been satisfactorily applied for the determination of MTU and PTU in human serum samples.

An Analytical Method to Screen for Six Thyreostatic Drug Residues in the Thyroid Gland andMuscle Tissues of Food Producing Animals by Liquid Chromatography with Ultraviolet Absorption Detection and Liquid Chromatography/Mass Spectrometry

A method was developed and validated to screen for residues of the thyreostatic drugs, tapazole (TAP), mercaptobenzimidazole (MBI), thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PrTU), and phenylthiouracil (PhTU) in bovine, equine, ovine, and porcine thyroid and muscle tissues at concentrations > or = 5 ng/g using 2-methoxy-mercaptobenzimidazole (MeMBI) and dimethylthiouracil (DMTU) as internal standards. In this method, the drugs were solvent extracted from thyroid and muscle tissue and cleaned up on an amino-propyl solid-phase extraction (SPE) cartridge. The unretained fraction containing TAP and MBI and the internal standard, MeMBI, was collected as Fraction 1. The retained fraction containing TU, MTU, PrTU, PhTU, and the internal standard, DMTU, was eluted with 3% acetic acid-isopropanol as Fraction 2. Fraction 1 was further cleaned up on an alumina B SPE cartridge and analyzed by gradient elution on a C18 high-performance liquid chromatography (HPLC) column with ultraviolet detection at wavelengths of 255 and 300 nm. Fraction 2 was taken to dryness, derivatized with 4-chloro-7-nitrobenzo-2-furazan at pH 8, and analyzed by gradient elution on a C18 LC column with mass spectrometry (MS) detection. Any "presumptive positive" test results were submitted for further analysis by LC/MS/MS. The validated method was applied to the analysis of over 300 thyroid tissue samples.

Voltammetric Determination of Methylthiouracil in Animal Feed Using Carbon Fiber Microelectrodes

Cylindrical carbon fiber microelectrodes (CFMEs) (8 μm in diameter, 8 mm in length) are used to develop a voltammetric method for the determination of the thyreostatic drug methylthiouracil (MTU) in animal feed samples. Methanol, containing 0.05 mol L−1 tetrabutylammonium perchlorate as supporting electrolyte, was selected as working medium because of the high MTU solubility in this solvent, which is also used for the extraction of MTU from real samples. Reproducible electroanalytical responses were obtained with no need of applying cleaning treatments to the CFME. The net-current square-wave (SW) oxidation signal of MTU was selected to be exploited analytically. Under the optimized SW conditions, a linear calibration plot for MTU was obtained in the 1.0×10−6–1.0×10−4 mol L−1 range, with a detection limit of 3.8×10−7 mol L−1. The SW voltammetric behavior of other thyreostatic drugs was checked. A simple extraction procedure with methanol, followed by a clean up step, was used for the determination of MTU in a commercial dry fodder sample spiked with this drug at the 500 μg g−1 level. The detection limit in this type of sample was found to be 8.6×10−6 mol L−1.

Histomorphometry and quantification of nucleolar organizer regions in bovine thyroid containing methylthiouracil residues.

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.

Altura do epitélio folicular da tireóide na triagem para detecção de resíduos de antitireoidianos em bovinos

A altura do epitélio folicular da tireóide, mensurada em secções histológicas coradas pela hematoxilina e eosina, foi testada como característica de triagem para a pesquisa química de resíduos de antitireoidianos em 53 bovinos. Cinco glândulas, contendo resíduos detectados por cromatografia de camada delgada de alta resolução (HPTLC), eram provenientes de cinco novilhos experimentalmente tratados com suspensão oral de metiltiouracil (5g/animal/dia), durante 20 dias, interrompido cinco dias antes do abate. As demais glândulas, sem resíduos detectados por HPTLC, provinham de seis controle experimentais que receberam apenas placebo e de 42 animais abatidos em matadouros industriais de Minas Gerais (n=25) e Goiás (n=17). Todas as glândulas positivas apresentaram hiperplasia intensa e a altura do epitélio folicular foi significativamente maior, embora as 42 glândulas negativas também se mostrassem moderadamente hiperplásicas. Com base nas respostas típicas individuais de 47 tireóides com bócio parenquimatoso e de seis histologicamente normais, ficou estabelecido o limite de 16mim para a altura do epitélio, acima do qual toda glândula deverá ser considerada suspeita e testada quimicamente. Foi determinada a correlação entre a altura do epitélio folicular e outras características indiretas de detecção de resíduos, concluindo-se que a altura do epitélio presta-se para a triagem de programas oficiais de controle de resíduos de drogas antitireoidianas na carne.

Determination of thyreostat residues from bovine matrices using high-performance liquid chromatography

High-performance liquid chromatography (HPLC) methodologies were evaluated for the detection and quantification of thyreostatic drug residues in cattle serum and thyroid tissue. The paper details a protocol, using a simple ethyl acetate extraction for the determination of thiouracil, tapazole, methyl thiouracil, propyl thiouracil and phenyl thiouracil in thyroid tissue. Using two sequential HPLC injections, and quantitative analysis, in two steps, all five thyreostats were detectable at concentrations greater than 2.45–4.52 ng/g. Modifications to a published method for detection of thyreostatic residues in serum involving the addition of mercaptoethanol and a freezing step are described. The modifications improved sensitivity and allowed detection of the five thyreostats at levels greater than 16.98–35.25 ng/ml. Young bulls were treated with thyreostats to demonstrate the validity of the methodologies described. Administered thyreostats were not absorbed equally by the test animals and the compounds were not all detected in the serum samples removed at 7 days following drug withdrawal. These experiments indicate the necessity to be able to detect thyreostat residues in a variety of matrices.

Effects of exogenous triiodothyronine (T 3) and a goitrogen, methylthiouracil (MTU), on thyroid gland function in sheep

The rationale that conjoint use of feedback inhibition could alter the effectiveness of a goitrogen, methylthiouracil (MTU), for achieving pharmacological suppression of thyroid gland function was tested in sheep. Experiment 1 determined the appropriate daily dose of T 3 for achieving feedback suppression of thyroid secretion. Experiment 2 tested the hypothesis that prior treatment with T 3 at such dosage would speed up the thyroid suppressive effect of subsequent administration of MTU, and Experiment 3 determined whether T 3 exerted feedback inhibition on the anterior pituitary gland. In Experiment 1, ewes ( n=6) treated with doses of 0, 1, 5 and 25 μg T 3 per kg live weight as a daily s.c. injection for 2 weeks had a dose-related increase in heart rate and decrease in plasma T 4 concentration. Ewes in Experiment 2 received combinations of daily s.c. injections of T 3 at 5 μg per kg and daily oral dosing with 35 mg per kg MTU ( n=6). Suppression of plasma T 4 concentration by T 3 did not improve the effect of subsequent treatment with MTU. In Experiment 3, plasma TSH responses to TRH stimulation (200 μg, i.v.) were reduced by treatment with T 3, confirming its feedback inhibition of the anterior pituitary gland. It is concluded that, in spite of the inhibitory actions of T 3 on plasma T 4 concentration in sheep, its use prior to treatment with MTU did not affect the resultant suppression of thyroid hormone secretion.

Elimination profile of methylthiouracil in cows after oral administration.

In addition to methods for the determination of residues, there is an important need for knowledge of the fate and excretion of growth promoting substances in fattening animals. In court, often simple questions are asked, such as, over what period of time can the drug be detected?, is it possible to find residues after 2 months, etc. These questions can only be answered by conducting animal experiments. Data on excretion and distribution of thiouracils in cows are rather scarce. At the beginning of the 1980s, animal experiments with methylthiouracil (MTU) were carried out in the Laboratory of Chemical Analysis. These experiments showed that treatment of cows with MTU results in the rapid appearance of the drug in plasma, urine and milk, whereas MTU selectively accumulates in the thyroid gland. The results of these experiments were only published in media with limited access (thesis, abstracts) and also there has been a considerable improvement in data handling with computer programs in the last 15 years. This investigation reinterprets the 'old' analytical data from animal experiments using a pharmacokinetic software package.

The mouse micronucleus test of five possible human carcinogens (ethylene thiourea (ETU), methylthiouracil (MTU), metronidazole (MTZ), 2-nitropropane (2NP) and sodium phenobarbital (PB))

The mouse micronucleus test of five possible human carcinogens (ethylene thiourea (ETU), methylthiouracil (MTU), metronidazole (MTZ), 2-nitropropane (2NP) and sodium phenobarbital (PB))

Cell proliferation and differentiation of cancerous and non-cancerous thyroid tissues of rats.

The cell proliferative activity and capability of cytodifferentiation of cancerous and non-cancerous thyroid tissues were studied using 3H-thymidine (3H-TdR) autoradiography and immunohistochemistry for thyroglobulin. Cancerous lesions were induced by diisopropanolnitrosamine (DIPN) followed by methylthiouracil (MTU). Regenerative thyroid tissues were obtained through wounding. All the rats treated successively with DIPN and MTU were found to have carcinomas that invaded the surrounding tissue or blood vessels. By cumulative labeling with 3H-TdR, the maximal cell cycle times (Tc) of invasive and non-invasive parts of carcinomas were estimated to be 120hr and 180hr, respectively, whereas the Tc of regenerative and hyperplastic thyroid tissues were estimated as 113hr and 519hr, respectively. However, the duration of S phase (Ts) was greater in cancers (15-35hr) than in non-cancerous tissues (3-4hr), and the cancerous tissues showed lower expression of thyroglobulin than the non-cancerous thyroid tissues. These may reflect some injury in the DNA of cancer cells.

Induction of hypothyroidism in goats by parenteral administration of antithyroid drugs

Hypothyroidism was induced in four female goats by daily subcutaneous injection of one of two antithyroid agents for 20 days. Two animals were injected with methyl thiouracil (MT) at the rate of either 10 mg/kg b.wt. per day or 6 mg/kg b.wt. per day whereas the other two goats were injected with thiourea at doses of either 125 mg/kg b.wt. per day or 100 mg/kg b.wt. per day. By day 19 of the treatment, the percentage reductions in pretreatment plasma levels of total triiodothyronine (tT 3) and total thyroxine (tT 4) in the various treatments were as follows: (1) high level thiourea, tT 3> 95; tT 4> 90; (2) low level thiourea, tT 3 86; tT 4 74; (3) high level MT, tT 3 80; tT 4 58; (4) low level MT, tT 3 (16th day 47); tT 4 59. Thiourea appears to be more effective than MT in reducing tT 3 and tT 4 levels. Our attempts to administer antithyroid drugs parenterally to induce hypothyroidism within a short period in goats may be of importance for carrying out experimental studies on the pathophysiology of hypothyroidism.