Effects of propylthiouracil on D-Galactosamine hepatotoxicity in the rat evidence for a non-thyroidal effect

The cytoprotective effects of propylthiouracil (PTU) were studied in rats treated with the hepatotoxin D-galactosamine (D-GNH₂). Five days of PTU pretreatment prior to D-GNH₂ caused hypothyroidism and a significant reduction in liver injury as assessed by serum transaminase levels. When PTU was administered as a single dose with d-gnh₂, significant decreases in transaminase also occurred at times when thyroid function was unchanged. Furthermore, aminopyrine oxidation showed significant impairment after D-GNH₂ and was normalized by one dose of PTU. Further studies were carried out in thyroidectomized rats. PTU caused significant reductions in transaminase levels when given for 5 days pretreatment or as a single dose. Animals receiving pretreatment with PTU plus thyroxine (T₄) also had significant decreases in serum transaminase. The antithyroid drug methimazole also had a hepatoprotective effect, while two other potent antithyroid compounds (2-thiouracil and 2-thiobarbituric acid) did not. These data suggest that PTU can protect against liver injury induced by D-GNH₂, that the effect is independent of thyroid function, and that this effect is not common to all thiolcontaining antithyroid drugs.     

Effects of developmental hypothyroidism induced by maternal administration of methimazole or propylthiouracil on the immune system of rats

Methimazole (MMI) and propylthiouracil (PTU) are popularly used antithyroid drugs (ATDs) for the treatment of Graves' hyperthyroidism. The aim of the present study was to determine the effects of ATDs on the developing immune system of the rats. Maternal Sprague-Dawley rats were given drinking water containing 200 ppm of MMI, 12 ppm of PTU (high-dose PTU), or 3 ppm of PTU (low-dose PTU) between gestational day (GD) 10 and postnatal week (PNW) 3. Exposure to the ATDs was ceased upon weaning at PNW3, and the male offspring were sampled at PNWs 3 or 11. The serum thyroid-related hormone levels and the hematological components in the offspring were then determined. The expressions of surface markers in the spleen, thymus and peripheral blood were determined using flowcytometry. The weights of the body, spleen and thymus and the splenic and thymic cell numbers were decreased in the MMI-treated and the high-dose PTU-treated animals at PNWs 3 and 11. The serum levels of thyroid-related hormones were depressed in the MMI and high-dose PTU groups. FACS analysis revealed that the ATDs caused proportional changes in the lymphoid cell subpopulations. The proportion of B cells among the total lymphocytes was significantly decreased at PNW3, whereas that of T cells, especially of inactive T cells, was dramatically increased. Moreover, the proportion of CD4(+)CD25(+) regulatory T cells was significantly increased in the spleen and peripheral blood at PNW3. Most of the above-described changes had recovered to normal levels at PNW11. These results suggest that ATDs might have temporal immunomodulatory effects on the developing immune system.     

Mechanism of inactivation of thyroid peroxidase by thioureylene drugs

We have investigated the mechanism by which the thioureylene drugs, 1-methyl-2-mercap-toimidazole (MMI) and 6-n-propylthiouracil (PTU), inactivate thyroid peroxidase (TPO). Our results indicate that inactivation of TPO by MMI and PTU involves a reaction between the drugs and the oxidized heme group produced by interaction between TPO and H₂O₂. This conclusion is supported by the following observations. First, addition of a low concentration of H₂O₂ to a solution of TPO shifted λ_max of the Soret band from 411 to 420 nm, reflecting the formation of an oxidized form of TPO (TPO_ox). Addition of MMI or PTU to TPO_ox, produced a Soret spectrum that was significantly different from the spectrum of native TPO or TPO_ox, whereas addition of MMI or PTU to native TPO produced no significant change in the heme spectrum. Second, studies with radiolabeled MMI and PTU combined with simultaneous assays of enzyme activity (guaiacol assay) showed that firm binding of the drugs to TPO and inactivation of the enzyme occurred on addition of the drugs to TPO_ox. However, neither binding nor inactivation occurred on addition of the drugs to native TPO. Third, the presence of a low concentration of iodide prevented the shift in the Soret spectrum, the binding of labeled drug, and the loss of enzyme activity associated with the addition of thioureylene drugs to TPO + H₂O₂. Under these conditions we assume that the enzyme was present as TPO · I_ox, a form in which the heme is present in the same reduced state as in native TPO. This would explain the protective action of iodide on the inactivation of TPO_ox, by MMI and PTU.     

Enzymatic S-methylation of 6-n-propyl-2-thiouracil and other antithyroid drugs.

Mouse kidney thiol transmethylase and S-adenosylmethionine were incubated with the radioactive antithyroid drugs, 2-thiouracil (TU), 6-propyl-2-thiouracil (PTU), methimazole (MMI). 6-methyl-2-thiouracil (6-methyl TU) or thiourea. Radioactive metabolites were produced with TU, PTU and 6-methyl TU and, in each case. were identified as the corresponding S-methyl derivatives. No measurable metabolism of MMI or thiourea was observed. Kinetic studies with the partially purified enzyme demonstrated K_m values for TU, PTU and 6-methyl TU of 1 × 10^?3 M, 2.5 × 10^?3 M and 1.54 × 10^?3 M respectively. Extensive investigation with PTU demonstrated that methylation was to the sulfur rather than the nitrogen of the thiopyrimidine and that the pH optimum for PTU was 8.0. Methylation of PTU was proportional to enzyme concentration, with little spontaneous methylation occurring, and was not reversible. TU and 6-methyl TU inhibited PTU metabolism and were apparently competitive substrates. Thiouracil nucleoside and thiouracil nucleotide were not substrates for the enzyme. Studies with porcine thyroid peroxidase demonstrated that S-methylation of PTU. TU and MMI abolished the antiperoxidase activity observed with the parent compound. The results obtained demonstrate that S-methylation is a general pathway of metabolism for thiopyrimidine antithyroid drugs, but not for thiourea or MMI, which markedly decreases the antiperoxidase activity of the parent compound.     

Glucuronide conjugation of 6-n-propyl-2-thiouracil and other antithyroid drugs by guinea pig liver microsomes in vitro.

Guinea pig liver microsome UDP glucuronyl transferase and UDPGA were incubated with the radioactive antithyroid drugs 6-n-propyl-2-thiouracil (PTU). 1-methy1-2-mercaptoimidazole (methimazole, MMI) and 2-thiouracil (TU). Radioactive metabolites were produced with PTU and thiouracil and, in each case, were identified as the corresponding β-glucuronide conjugate. No measurable glucuronidation of MMI was observed. Kinetic studies with the microsomal preparation demonstrated a K_m rmvalue of 7.2 × 10^?4 M for PTU and 6.7 × 10^?3 M for thiouracil. Glucuronide conjugation of PTU was linear for 1 hr. declining thereafter while conjugation of phenolphthalein was linear for 2 hr. Conjugation of phenolphthalein by microsomes stored in 0.154 M KCl at ?20° for 14 days was 41 per cent higher than in fresh microsomes, whereas conjugation of PTU was 67.4 per cent lower. PTU glucuronidation did not occur in the absence of UDPGA and was essentially linear with respect to enzyme concentrations. Under the same conditions, spontaneous N-glucuronidation of PTU by glucuronate was not measurable. The pH optimum for PTU glucuronidation was 8.0 and similar to the broad optimums of 7.3 to 7.9 for UDP glucuronyl transferases from a variety of sources rather than to non-enzymatic N-glucuronidation, which has a reported pH optimum of 3–4. The conjugating enzyme for PTU was located primarily in the guinea pig liver microsomes with this fraction exhibiting 75 per cent of the total activity of whole homogenates. PTU conjugation was inhibited by MMI but not by thiouracil, thiourea or 6-methy1-2-thiouracil. The results obtained demonstrate that β-glucuronide conjugation of the antithyroid drugs PTU and thiouracil, but not MMI. is readily catalyzed by a guinea pig liver microsomal UDP glucuronyl transferase in vitro.     

Apigenin loaded nanoparticle delayed development of hepatocellular carcinoma in rats

Hepatocellular carcinoma (HCC) is one of the major causes of cancer related death globally. Apigenin, a dietary flavonoid, possesses anti-tumor activity against HCC cells in-vitro. Development, physicochemical characterization of apigenin loaded nanoparticles (ApNp), biodistribution pattern and pharmacokinetic parameters of apigenin upon intravenous administration of ApNp, and effect of ApNp treatment in rats with HCC were investigated. Apigenin loaded nanoparticles had a sustained drug release pattern and successfully reached the hepatic cancer cells in-vitro as well as in liver of carcinogenic animals. ApNp predominantly delayed the progress of HCC in chemical induced hepatocarcinogenesis in rats. Quantification of apigenin by liquid chromatography–mass spectroscopy (LC-MS/MS) showed that apigenin availability significantly increased in blood and liver upon ApNp treatment. Apigenin loaded nanoparticle delivery substantially controlled the severity of hepatocellular carcinoma and could be a future hope for lingering the survival in hepatic cancer patients.     

Pregnancy outcome, thyroid dysfunction and fetal goitre after in utero exposure to propylthiouracil: a controlled cohort study

AIMS

Propylthiouracil (PTU) is presently considered to be the treatment of choice for hyperthyroidism in pregnancy. It is known to cross the human placenta, and therefore may affect the fetus. The major aims of this study were to evaluate the rate of major anomalies and to report the rate of fetal goitre, accompanied by hypothyroidism, in fetuses/ newborns of mothers after in utero exposure to PTU.

METHODS

Prospective observational controlled cohort study of PTU-exposed pregnancies of women counselled by the Israeli Teratology Information Service between the years 1994 and 2004 compared with women exposed to nonteratogens.

RESULTS

We followed up 115 PTU-exposed pregnancies and 1141 controls. The rate of major anomalies was comparable between the groups [PTU 1/80 (1.3%), control 34/1066 (3.2%), P= 0.507]. Hypothyroidism was found in 9.5% of fetuses/neonates (56.8% of whom with goitre). Hyperthyroidism, possibly resulting from maternal disease, was found in 10.3%. Goitres prenatally diagnosed by ultrasound were successfully treated in utero by maternal dose adjustment. In most cases neonatal thyroid functions normalized during the first month of life without any treatment. Median neonatal birth weight was lower [PTU 3145 g (2655–3537) vs. control 3300 g (2968–3600), P= 0.018].

CONCLUSIONS

PTU does not seem to be a major human teratogen. However, it could cause fetal/neonatal hypothyroidism with or without goitre. Fetal thyroid size monitoring and neonatal thyroid function tests are important for appropriate prevention and treatment.     

A new fatty alcohol from Terminalia arjuna leaves with antileishmanial activity

A new fatty alcohol (1) together with ten known compounds was isolated from the leaves of Terminalia arjuna Roxb. Their chemical structures were established on the basis of physical, chemical, and spectroscopic methods to be identified as (E)-3,5,7,16 tetramethylheptadeca-2-en-1-ol (1), apigenin, luteolin, vitexin, isovitexin, luteolin-3′-glucuronide, gallic acid, methyl gallate, ellagic acid, stigmasterol, and β-sitosterol-3-O-glucoside. Apigenin, vitexin, and isovitexin were isolated for the first time from this species, while luteolin-3′-glucuronide was isolated for the first time from the genus Terminalia. Compound 1 and its acetate derivative (2) showed good antileishmanial activity with IC₅₀ values of 9.0 and 2.0 μg/mL, respectively.     

Apigenin ameliorates hepatic lipid accumulation by activating the autophagy-mitochondria pathway

Apigenin, a flavonoid isolated from plants, provides protection against non-alcoholic fatty liver disease. However, the mechanism by which apigenin decreases lipid accumulation in the liver is unclear. In this study, we investigated the molecular mechanism underlying the beneficial effect of apigenin on the hepatic deregulation of lipid metabolism. Oleic acid (OA)-induced lipid accumulation in human hepatoma cells (Huh7 cells) was used as an in vitro model. Western blot analysis was used for evaluating protein expression. Oil red O staining, Nile red staining, and conventional assay kits were used to assess the level of lipids. Immunocytochemistry was performed to observe mitochondrial morphology. Seahorse XF analyzer was used to measure mitochondrial bioenergetics. Treatment with OA induced lipid accumulation in Huh7 cells, which was attenuated by apigenin. Mechanistically, treatment with apigenin increased the expression of autophagy-related proteins including Beclin1, autophagy related gene 5 (ATG5), ATG7, and LC3II, and the formation of autophagolysosomes, leading to an increase in intracellular levels of fatty acids. Inhibition of autophagy by bafilomycin A1 or chloroquine abolished the protection of apigenin in OA-induced lipid accumulation. Apigenin up-regulated the protein expression related to the β-oxidation pathway including acyl-CoA synthetase long chain family member 1, carnitine palmitoyltransferase 1α, acyl-CoA oxidase 1, peroxisome proliferator activated receptor (PPAR) α, and PPARγ coactivator 1-α. Moreover, apigenin increased the mitochondrial network structure and mitochondrial function by increasing the protein expression related to the process of mitochondria fusion and mitochondrial function. Collectively, our findings suggest that apigenin ameliorates hepatic lipid accumulation by activating the autophagy-mitochondrial pathway.     

Thyroid status affects the rat cardiac β-adrenoceptor system transiently and time-dependently

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Propylthiouracil,and methimazole,and carbimazole-related hepatotoxicity

Introduction: Propylthiouracil (PTU) has been used for the treatment of hyperthyroidism since the 1940s, but over the years reports of significant hepatotoxicity have come forth, particularly in children. This led to a black box warning being issued by the US FDA in 2009, followed by a similar warning by the European Medicines Agency and the United Kingdom Medicines and Healthcare Regulatory Agency later that year.

Areas covered: This article provides a concise review of the data on hepatotoxicity associated with the currently available antithyroid drugs: PTU, methimazole (MMI) and carbimazole. The differences in mechanism are examined in detail, as well as clinical presentation, management and monitoring. Use in special populations and trends in use of antithyroid medication are also discussed.

Expert opinion: PTU is known to cause severe hepatic failure, particularly in children. Its use in children should be avoided. In adults, it is beneficial to use in the first trimester of pregnancy and thyroid storm. In the rest of the adult population, it should be used with caution. Carbimazole and MMI are associated with less severe hepatic injury and should be preferred when choosing thionamides as a treatment option.     

Antithyroid effects of coal-derived pollutants.

Endemic goiter in iodide-sufficient areas of the United States and Colombia has been linked to watersheds rich in coal and shale, which several reports suggest are the source of water-borne goitrogens. In this report the potential antithyroid activities of aqueous coal and shale extracts and of compounds identified in aqueous effluents from coal conversion processes were assayed in thyroid peroxidase (TPO) and thyroid slice systems. Aqueous extracts of coal and black shale were potent inhibitors of TPO or 125I organification by thyroid slices. The most abundant water-soluble compounds derived from coal are dihydroxy-phenols, thiocyanate, disulfides, and hydroxypyridines. The dihydroxyphenols resorcinol, 2-methylresorcinol, and 5-methylresorcinol (orcinol) were 26.7, 22.5, and 7.2 times more potent, respectively, than the antithyroid drug 6-propylthiouracil (PTU). Other dihydroxyphenols and thiocyanate were less potent but comparable in activity to PTU. All dihydroxypyridines and 3-hydroxypyridine produced inhibitory effects comparable to PTU. None of the disulfides inhibited TPO. The antiperoxidase effects of combinations of two dihydroxyphenols or one dihydroxyphenol and SCN were additive, whereas the effects of a combination of four dihydroxyphenols at threshold inhibitory concentrations were synergistic, resulting in net effects equivalent to or greater than the sum of the individual effects. Thus, antithyroid effects may be greatly amplified by exposure to multiple coal-derived goitrogens and could be many times that produced by any one of the contributing pollutants. These results demonstrate that potent water-borne goitrogens are derived from coal and shale and that their contamination of water supplies could pose a serious threat of thyroid disorders.     

Anti-inflammatory effects of apigenin on nicotine- and lipopolysaccharide-stimulated human periodontal ligament cells via heme oxygenase-1

Background and objectivesAlthough apigenin exhibits various biological effects, its anti-inflammatory role in the periodontal field remains unknown. We examined the anti-inflammatory effects of apigenin and the underlying mechanism in nicotine- and lipopolysaccharide (LPS)-stimulated human periodontal ligament (hPDL) cells.Materials and methodsWestern blotting was used to examine the effect of apigenin (10–40 µM) on the LPS- and nicotine-induced expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1), as well as the phosphorylation of mitogen-activated protein kinases (MAPKs), in hPDL cells. Pro-inflammatory mediators, including nitric oxide (NO), prostaglandin E₂ (PGE₂), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), IL-6, and IL-12 were monitored using Griess reagents and ELISA.ResultsIncubation of hPDL cells with apigenin decreased LPS- and nicotine-induced HO-1 protein expression and activity. Apigenin significantly inhibited the nicotine- and LPS-induced production of NO, PGE₂, IL-1β, TNF-α, IL-6, and IL-12, and the upregulation of iNOS and COX-2 in hPDL cells. Hemin, a selective HO-1 inducer, reversed the apigenin-mediated suppression of nicotine- and LPS-induced NO, PGE₂ and cytokine production. Treatment with inhibitors of the phosphoinositide 3-kinase, MAPKs, p38, and JNK, as well as a protein kinase C inhibitor, blocked the anti-inflammatory effects of apigenin in nicotine- and LPS-treated cells.ConclusionsApigenin possesses anti-inflammatory activity in hPDL cells and works through a novel mechanism involving the action of HO-1. Thus, apigenin may have potential benefits as a host modulatory agent in the prevention and treatment of periodontal disease associated with smoking and dental plaque.     

The Inhibitory Effect of Apigenin on the Agonist-Induced Regulation of Vascular Contractility via Calcium Desensitization-Related Pathways

Apigenin, a natural flavonoid found in a variety of vegetables and fruits, has been shown to possess many biological functions. The present study was undertaken to investigate the influence of apigenin on vascular smooth muscle contractility and to determine the mechanism involved. Denuded aortic rings from male rats were used and isometric contractions were recorded and combined with molecular experiments. Apigenin significantly relaxed fluoride-, thromboxane A₂ mimetic- or phorbol ester-induced vascular contraction, which suggests that apigenin could be an anti-hypertensive that reduces agonist-induced vascular contraction regardless of endothelial nitric oxide synthesis. Furthermore, apigenin significantly inhibited fluoride-induced increases in pMYPT1 levels and phorbol ester-induced increases in pERK1/2 levels, which suggests the mechanism involving the inhibition of Rho-kinase and MEK activity and the subsequent phosphorylation of MYPT1 and ERK1/2. This study provides evidence regarding the mechanism underlying the relaxation effect of apigenin on agonist-induced vascular contraction regardless of endothelial function.     

In vitro antiproliferative characteristics of flavonoids and diazepam on SNU-C4 colorectal adenocarcinoma cells

The need for beneficial use of sedatives in oncologic patients is increasing. Therefore, in this study, antiproliferative characteristics of herbal and synthetic sedatives were examined in vitro in SNU-C4 human colorectal adenocarcinoma cells. Apigenin (50% inhibition concentration, IC₅₀ = 1.8 ± 0.5 μM) and diazepam (IC₅₀ = 7.0 ± 0.5 μM) showed concentration-dependent inhibition of SNU-C4 cancer cell survival. Efficacy of cancer cell survival inhibition by apigenin and diazepam was much lower than that of 5-fluorouracil (5-FU), a known chemotherapeutic drug. However, 10⁻⁶ M concentration of apigenin and diazepam potentiated 5-FU-induced cytotoxicity. In SNU-C4 cells, 10⁻⁶ M concentrations of diazepam, flumazenil (Ro15-1788), Ro5-4864, or PK11195, all ligands for central- or peripheral-type benzodiazepine (BZD) receptors, inhibited cell survival like the flavonoid apigenin (4′,5,7-trihydroxyflavone) and fisetin (3,7,3′,4′-tetrahydroxyflavone). Also like the plant flavonoids, treatment with 10⁻⁶ M concentration of diazepam for 3 days hardly affect the peripheral-type BZD receptor (PBR) messenger RNA (mRNA) expression and inhibited glucose utilization of SNU-C4 cells. Treatment with flavonoids or diazepam for 6 days upregulated PBR mRNA expression and cell cytotoxicity of SNU-C4 cells. Furthermore, treatment with 10⁻⁶ M concentration of apigenin, a natural sedative material originating from traditional herbs, positively modulated BZD-induced antiproliferative cytotoxicity in SNU-C4 cells. Overall, the in vitro antiproliferative activity on SNU-C4 cancer cells of herbal sedatives, such as apigenin, plus additive enhancement of synthetic BZD- and 5-FU-induced antiproliferative activities, were shown. In conclusion, this study provides experimental basis for advanced trial in the future.     

Atom Level Electrotopological State Indexes in QSAR: Designing and Testing Antithyroid Agents

Purpose. To design new antithyroid agents with less side effects, the electrotopological-state (E-state) indexes of thiourylene moiety (S_N&S) was utilized as a guideline to develop five acyclic thio-urylene-type compounds with reduced antioxidant property. Methods. These agents were synthesized and screened for antithyroid activity in rats using ¹²⁵I-thiocyanate discharge technique. In addition, chemiluminescence studies on the activated polymorpho-nuclear leukocytes (PMNLs) were also conducted to reveal antioxidant properties of the tested compounds. Results. A linear relationship between the in vitro literature value of the formation constants of thiourylene-type compounds with iodine (K_c) and the S_N&S was observed and utilized in designing those agents. At least one of the compounds (abouthiouzine) was found to have a potential value as an antithyroid agent. The relative efficacy of abouthiouzine [l-n-butyl-3(isonicotinamido)-2-thiourea], after equimolar dose, was 102% and 51.5% of that of propylthiouracil with respect to the rate of ¹²⁵I-discharge and ¹²⁵I-uptake, respectively. In addition, Chemiluminescence studies on PMNLs revealed that abouthiouzine has slight oxidant property. Such properties may provide advantages in avoiding the iatrogenic hypothyroidism and antithyroid-induced immunological reactions. Conclusions. The E-state approach provides guidelines to economize efforts and cost of designing new antithyroid drugs.     

Effect of thyroidectomy on 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced lipid peroxidation

Thyroid hormones have been implicated in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Therefore, the effects of methimazole (MMI) and propylthiouracil (PTU) induced hypothyroidism and surgical thyroidectomy on several toxic manifestations of TCDD were investigated. Female rats were treated with MMI (0.50 mg/kg) for 10 or 28 days, or PTU (5.0 mg/kg) for 10 days. Other animals were surgically thyroidectomized. The animals received TCDD (100 micrograms/kg) orally or the corn oil vehicle 6 days prior to sacrifice. MMI and PTU decreased serum thyroxine (T4) levels by 27-33% while surgical thyroidectomy decreased T4 levels by 66%. TCDD alone decreased T4 levels by 67%, and similar effects occurred in MMI and PTU treated animals. TCDD produced a 9% increase in serum triiodothyronine (T3) concentrations, and neither MMI or PTU treatment for 10 days modified this effect. Neither antithyroid drug prevented TCDD induced weight loss. TCDD administration resulted in over a 300% increase in hepatic malondialdehyde (MDA) content and a 60% decrease in glutathione peroxidase activity. Neither antithyroid compound affected TCDD-induced alterations in these two parameters. TCDD enhanced MDA content by 220% and inhibited glutathione peroxidase activity by 39% in surgically thyroidectomized rats. Thus, only severe hypothyroidism produced by surgical thyroidectomy was able to partially prevent the effects of TCDD on hepatic MDA content and glutathione peroxidase activity.     

Site of glucuronide conjugation to the antithyroid drug 6-n-propyl-2-thiouracil.

Chromatographyically pure, essentially salt-free radioactive 6-n-propyl-2-thiouracil (PTU) glucuronides were isolated from rat bile and urine and synthesized with guinea pig liver microsomes to determine if more than one PTU glucuronide was formed and to determine which group or groups in the PTU molecule was glucuronidated. Analyses on Bio-Gel P-2 and DEAE-Sephadex A-25 columns and on TLC sheets in five solvent systems demonstrated that the three glucuronide preparations were chromatographically identical. Furthermore, the reactivities of the three glucuronides with 1 N HCl, methyl iodide, sodium azide-iodine reagent, 2,6-dichloroquinone-chloroimide and H₂O₂ were also identical strongly indicating that a single PTU glucuronide was formed. The PTU glucuronide was partially hydrolyzed by 1 N HCl to 6-n-propyl-uracil (PU), a reaction typical of S-conjugated PTU; demonstrated greatly reduced reactivity with methyl iodide whereas the S of PTU was readily methylated; exhibited a negative reaction in the azide-iodine test which was a certain indication that the C—SH or CS group was not present; failed to react with 2,6-dichloroquinone-chloroimide which reacts with the C=S of PTU and provides the basis for a colorimetric assay for PTU; and was not oxidized by H₂O₂ to form sulfate as are all PTU derivatives except S conjugates of PTU. Furthermore PU, which possesses identical potential conjugation sites with the exception of the S, was not glucuronidated under conditions in which PTU was readily conjugated. The results obtained strongly indicate that the glucuronide is conjugated to the S of PTU.