Biodegradation and metabolic pathway of quinalphos by Cunninghamella elegans ATCC36112.

Quinalphos is a long-term, wide-spectrum organophosphate insecticide with residual problems in the natural environment. Cunninghamella elegans (C. elegans) is a member of Mucoromycotina. Since the degradation products of its exogenous compounds are similar to those of mammals, it is often used to simulate the metabolism pathways of mammals. In this study, the detailed metabolic pathways of quinalphos were investigated with C. elegans. Quinalphos was degraded by 92% in 7 days, while ten metabolites were produced. The metabolites were analyzed and identified by GC-MS. To determine the responsible enzymes in quinalphos metabolism, piperonyl butoxide (PB) and methimazole included in the culture flasks, and the kinetic responses of quinalphos and its metabolites by C. elegans were measured. Results indirectly demonstrated that cytochrome P450 monooxygenases were involved in the metabolism of quinalphos, but that methimazole inhibited the metabolism less efficiently. Comprehensive metabolic pathways can be deduced from the detailed analysis of metabolite profiles in control and inhibitor assays.

Methimazole and sodium perchlorate exert anti-thyroidal effects in the T3-induced Xenopus laevis metamorphosis assay: A rapid assay for screening thyroid disrupting chemicals.

Thyroid disrupting chemicals (TDCs) have received much attention due to their potential adverse effects on animal and human health, which calls for rapid screen assays to identify them. The triiodothyronine (T3)-induced Xenopus metamorphosis assay (TiXMA) we developed previously has been successfully applied to the detection of the TDCs disrupting thyroid hormone (TH) signaling. Here, we attempted to expand the application of the TiXMA to the screening of the TDCs interfering with the hypothalamic-pituitary-thyroid (HPT) axis. Two well-known TH synthesis inhibitors methimazole (MMI) and sodium perchlorate (SP) were employed to test the sensitivity of the TiXMA to the TDCs interfering with the HPT axis. As expected, we observed that the two chemicals concentration-dependently antagonized T3-induced morphological changes and body weight reduction of X. laevis tadpoles following 96 h-exposure, in parallel with blocked thyroid development and down-regulated tshß expression in the brain. All the data show that both MMI and SP exert inhibitory effects on T3-induced metamorphosis, indicating that the TiXMA is capable of screening the TDCs interfering with the HPT axis. In comparison with Amphibian Metamorphosis Assay (AMA), a 21-day assay for screening the TDCs interfering with the HPT axis, the TiXMA has a remarkable advantage of shorter exposure duration (96 h).

Territory aggression and energy budget in food-restricted striped hamsters.

Food resource availability is one of the most important factors affecting interindividual competition in a variety of animal species. However, the energy budget and territory aggression strategy of small mammals during periods of food restriction remain uncertain. In this study, metabolic rate, body temperature, territory aggression behavior, and fat deposit were measured in male striped hamster (Cricetulus barabensis) restricted by 20% of ad libitum food intake with or without supplementary methimazole. Serum thyroid hormone (tri-iodothyronine, T3 and thyroxine, T4), and cytochrome c oxidase (COX) activity in liver, brown adipose tissue, and skeletal muscle, were also measured. Attack latency, total attack times and duration, and the interval duration between attacks of resident hamsters were not significantly changed during food restriction, which was not significantly affected by supplementary methimazole. Metabolic rate and body temperature was significantly increased in food-restricted hamsters following introduction of an intruder, which was not completely blocked by supplementary methimazole. Serum T3 and T4 levels and BAT COX activity were not significantly changed following aggression, and were significantly decreased by supplementary methimazole. These findings suggest that striped hamsters increase energy expenditure for territory aggression during food restriction, and consequently lead to excessive energy depletion. Territory aggression behavior may decrease the capacity to cope with food shortage, which may be independent of thyroid hormone.

Role of CYP2A6 in Methimazole Bioactivation and Hepatotoxicity.

Methimazole (MMI) is a widely used antithyroid drug, but it can cause hepatotoxicity by unknown mechanisms. Previous studies showed that the hepatic metabolism of MMI produces N-methylthiourea, leading to liver damage. However, the specific enzyme responsible for the production of the toxic metabolite N-methylthiourea is still unclear. In this study, we screened cytochromes P450 (CYPs) in N-methylthiourea production from MMI. CYP2A6 was identified as the key enzyme in catalyzing MMI metabolism to produce N-methylthiourea. When mice were pretreated with a CYP2A6 inhibitor, formation of N-methylthiourea from MMI was remarkably reduced. Consistently, the CYP2A6 inhibitor prevented MMI-induced hepatotoxicity. These results demonstrated that CYP2A6 is essential in MMI bioactivation and hepatotoxicity.

Antithyroid Drugs Inactivate TSH Binding to the TSH Receptor by their Reducing Action.

Backgroud and Objective: Antithyroid drugs (ATDs) [methylmercaptoimidazole (MMI) and propylthiouracil (PTU) ] are used to treat hyperthyroidism in Graves' disease. The effect of ATDs and reducing agents (mercaptoethanol, dithiothreitol and cysteine) on bovine (b) TSH binding to human (h) and porcine (p) TSH receptor (R) was examined. METHODS AND RESULTS: (1) ATDs was pre-incubated with hTSHR coated tube for 1- 4 h, washed free of ATDs, and then 125I-bTSH binding to hTSHR after 1 h incubation was examined. MMI (10-40 mM) decreased 125I-bTSH binding in a dose-dependent manner and binding decreased proportionally as preincubation time increased from 1 to 4 h. PTU (10mM) slightly decreased binding, When reducing agents were pre-incubated with hTSHR for 2 h, 125I-bTSH binding similarly decreased. (2) Porcine thyroid membrane was pre-incubated with both agents for 2 h. Then, the washed or unwashed membrane was incubated with 125I-bTSH for 1 h. 125I-bTSH binding in both methods decreased. (3) When the effect of ATDs or reducing agents on the biological activity of 125I-bTSH and thyroid stimulating antibody (TSAb) was examined after gel-filtration of 125I-bTSH- and TSAb- treated with both reagents for 1 h, no inactivation was observed. (4) ATDs showed similar reducing action as reducing agents because iodine (I+) was reduced to I- by ATDs. CONCLUSION: ATDs inactivate the TSH-binding site of TSHR by reduction, although ATDs do not inactivate bTSH and TSAb activity. This suggests that TSAb would not stimulate the thyroid due to the inactivation of the TSHR when ATDs are administered to patients with Graves' disease.

Assessment of liver slices for research on metabolic drug-drug interactions in cattle.

1. Precision-cut liver slices (PCLS) from food-producing animals have not been extensively used to study xenobiotic metabolism, and thus information on this field of research is sparse. 2. The aims of the present work were to further validate the technique of production and culture of bovine PCLS and to characterize the metabolic interaction between the anthelmintic albendazole (ABZ) and the flavin-monooxygenase (FMO) inhibitor methimazole (MTZ). 3. Nine steers were used as donors. PCLS were produced and incubated under two methods: a dynamic organ culture (DOC) incubator and a well-plate (WP) system. 4. Tissue viability, assessed through both structural and functional markers, was preserved throughout 12 h of incubation. ABZ was metabolized to its (+) and (-) albendazole sulfoxide stereoisomers (ABZSO) in bovine PCLS. The interaction between ABZ and MTZ resulted in a reduction (p < 0.001) in the rates of appearance of (+) ABZSO. Conversely, in presence of MTZ, the rates of appearance of (-) ABZSO increased under both systems (p < 0.05). 5. Both culture systems were suitable for assessing the interaction between ABZ and MTZ. 6. Overall, the results presented herein show that PCLS are a useful and reliable tool for short-term studies on metabolic drug-drug interactions in the bovine species.

A comparison of potency differences among thyroid peroxidase (TPO) inhibitors to induce developmental toxicity and other thyroid gland-linked toxicities in humans and rats.

The potencies of resorcinol, 6-propylthiouracil (PTU) and methimazole (MMI) for inducing developmental toxicity and neurotoxicity were compared in pregnant rats, regarded as valid model for human thyroid toxicity. Profound differences on maternal thyroid hormone levels (THs), maternal toxicity as well as developmental and neurotoxicity sequelae occurred. Resorcinol affected none of those end points. PTU and MMI caused significant effects. Therapy with either PTU or MMI during the first trimester of human pregnancy can cause reductions of maternal THs, accompanied by disruptions of prenatal development. Clinical MMI studies show sporadic evidence of teratogenic effects, with equivocal relation to thyroid peroxidase (TPO) inhibition. In recent decades no MMI associated prenatal toxicity has been reported, an outcome possibly related to carefully managed therapy. Orally administered resorcinol was rapidly absorbed, metabolized and excreted and was undetectable in the thyroid. In contrast, PTU or MMI accumulated. Resorcinol's potency to inhibit TPO was profoundly lower than that of PTU or MMI. Quantum chemical calculations may explain low resorcinol reactivity with TPO. Thus, distinctions in the target organ and the TPO inhibitory potency between these chemicals are likely contributing to different reductions of maternal THs levels and affecting the potency to cause developmental toxicity and neurotoxicity.

Drug oxygenation activities mediated by liver microsomal flavin-containing monooxygenases 1 and 3 in humans, monkeys, rats, and minipigs.

Liver microsomal flavin-containing monooxygenases (FMO, EC 1.14.13.8) 1 and 3 were functionally characterized in terms of expression levels and molecular catalytic capacities in human, cynomolgus monkey, rat, and minipig livers. Liver microsomal FMO3 in humans and monkeys and FMO1 and FMO3 in rats and minipigs could be determined immunochemically with commercially available anti-human FMO3 peptide antibodies or rat FMO1 peptide antibodies. With respect to FMO-dependent N-oxygenation of benzydamine and tozasertib and S-oxygenation of methimazole and sulindac sulfide activities, rat and minipig liver microsomes had high maximum velocity values (Vmax) and high catalytic efficiency (Vmax/Km, Michaelis constant) compared with those for human or monkey liver microsomes. Apparent Km values for recombinantly expressed rat FMO3-mediated N- and S-oxygenations were approximately 10-100-fold those of rat FMO1, although these enzymes had similar Vmax values. The mean catalytic efficiencies (Vmax/Km, 1.4 and 0.4 min(-1)µM(-1), respectively) of recombinant human and monkey FMO3 were higher than those of FMO1, whereas Vmax/Km values for rat and minipig FMO3 were low compared with those of FMO1. Minipig liver microsomal FMO1 efficiently catalyzed N- and S-oxygenation reactions; in addition, the minipig liver microsomal FMO1 concentration was higher than the levels in rats, humans, and monkeys. These results suggest that liver microsomal FMO1 could contribute to the relatively high FMO-mediated drug N- and S-oxygenation activities in rat and minipig liver microsomes and that lower expression of FMO1 in human and monkey livers could be a determinant factor for species differences in liver drug N- and S-oxygenation activities between experimental animals and humans.

Development of a thyroperoxidase inhibition assay for high-throughput screening.

High-throughput screening (HTPS) assays to detect inhibitors of thyroperoxidase (TPO), the enzymatic catalyst for thyroid hormone (TH) synthesis, are not currently available. Herein, we describe the development of a HTPS TPO inhibition assay. Rat thyroid microsomes and a fluorescent peroxidase substrate, Amplex UltraRed (AUR), were employed in an end-point assay for comparison to the existing kinetic guaiacol (GUA) oxidation assay. Following optimization of assay metrics, including Z', dynamic range, and activity, using methimazole (MMI), the assay was tested with a 21-chemical training set. The potency of MMI-induced TPO inhibition was greater with AUR compared to GUA. The dynamic range and Z' score with MMI were as follows: 127-fold and 0.62 for the GUA assay, 18-fold and 0.86 for the 96-well AUR assay, and 11.5-fold and 0.93 for the 384-well AUR assay. The 384-well AUR assay drastically reduced animal use, requiring one-tenth of the rat thyroid microsomal protein needed for the GUA 96-well format assay. Fourteen chemicals inhibited TPO, with a relative potency ranking of MMI > ethylene thiourea > 6-propylthiouracil > 2,2',4,4'-tetrahydroxy-benzophenone > 2-mercaptobenzothiazole > 3-amino-1,2,4-triazole > genistein > 4-propoxyphenol > sulfamethazine > daidzein > 4-nonylphenol > triclosan > iopanoic acid > resorcinol. These data demonstrate the capacity of this assay to detect diverse TPO inhibitors. Seven chemicals acted as negatives: 2-hydroxy-4-methoxybenzophenone, dibutylphthalate, diethylhexylphthalate, diethylphthalate, 3,5-dimethylpyrazole-1-methanol, methyl 2-methyl-benzoate, and sodium perchlorate. This assay could be used to screen large numbers of chemicals as an integral component of a tiered TH-disruptor screening approach.

The pharmacokinetics of methimazole in a novel lipophilic formulation administered transdermally to healthy cats.

AIM: To determine the pharmacokinetics of a novel lipophilic formulation of transdermal methimazole compared to oral carbimazole. METHODS: Healthy cats received 5 mg carbimazole orally every 12 hours for 13 treatments (n=6), then received transdermal methimazole (n=5) at a dose of 5 mg, then 10 mg, once daily on the pinna for 7 days, with 21 days between treatments. Concentrations of methimazole in serum over 24 hours and at 148 hours were determined by high performance liquid chromatography. RESULTS: Concentrations of methimazole in serum for the first 24 hours were not reliably detected in all cats treated with 5 mg methimazole transdermally, while for those receiving 5 mg carbimazole orally and 10 mg methimazole transdermally all cats had detectable concentrations of methimazole in serum. The maximum concentration and area under the curve were lower in cats receiving 10 mg methimazole transdermally (108 (SD 25) ng/mL and 2544 (SD 216) mg-hour/mL, respectively) than those receiving 5 mg oral carbimazole (355 (SD 113) ng/mL and 31,866 (SD 439) ng-hour/mL, respectively) (p<0.05). The time at maximal concentration and elimination half-life were longer for 10 mg transdermal methimazole (5.2 (SD 1.1) hours and 13 (SD 3) hours, respectively) compared to 5 mg oral carbimazole (2.1 (SD 1.6) hours and 5.1 (SD 1.2) hours, respectively). At 148 hours, mean concentrations of methimazole in serum were higher in cats receiving 10 mg methimazole transdermally (506 (SD 165) ng/mL) than for 5 mg oral carbimazole (255 (SD 28) ng/mL) or 5 mg transdermally (204 (SD 76) ng/mL). The mean relative bioavailability of 10 mg transdermal methimazole compared to oral carbimazole was 48 (min 43, max 55)%. CONCLUSION: Transdermal methimazole at a dose of 10 mg administered to the pinnae of healthy cats once daily in a novel lipophilic formulation has half the relative bioavailablity compared to 5 mg oral carbimazole. CLINICAL RELEVANCE: Transdermal methimazole can be absorbed from the skin of healthy cats.

Maternal transfer of methimazole and effects on thyroid hormone availability in embryonic tissues.

Methimazole (MMI) is an anti-thyroid drug used in the treatment of chronic hyperthyroidism. There is, however, some debate about its use during pregnancy as MMI is known to cross the mammalian placenta and reach the developing foetus. A similar problem occurs in birds, where MMI is deposited in the egg and taken up by the developing embryo. To investigate whether maternally derived MMI can have detrimental effects on embryonic development, we treated laying hens with MMI (0.03% in drinking water) and measured total and reduced MMI contents in the tissues of hens and embryos at different stages of development. In hens, MMI was selectively increased in the thyroid gland, while its levels in the liver and especially brain remained relatively low. Long-term MMI treatment induced a pronounced goitre with a decrease in thyroxine (T4) content but an increase in thyroidal 3,5,3'-triiodothyronine (T3) content. This resulted in normal T3 levels in tissues except in the brain. In chicken embryos, MMI levels were similar in the liver and brain. They gradually decreased during development but always remained above those in the corresponding maternal tissues. Contrary to the situation in hens, T4 availability was only moderately affected in embryos. Peripheral T3 levels were reduced in 14-day-old embryos but normal in 18-day-old embryos, while brain T3 content was decreased at all embryonic stages tested. We conclude that all embryonic tissues are exposed to relatively high doses of MMI and its oxidised metabolites. The effect of maternal MMI treatment on embryonic thyroid hormone availability is most pronounced for brain T3 content, which is reduced throughout the embryonic development period.

Exploiting endobiotic metabolic pathways to target xenobiotic antioxidants to mitochondria.

Oxidative stress plays a role in a range of human disease entities. Hence, strategies to target antioxidants to mitochondria are an active area of investigation. Triphenylphosphonium cation-based antioxidants and SS-peptides have been described and show significant uptake by mitochondria and effectiveness in animal models of conditions linked to oxidative stress. We tested the hypothesis that the mitochondrial ß-oxidation pathway could be exploited to activate the antioxidant phenolic and methimazole prodrugs. Most compounds studied underwent mitochondrial biotransformation to release their antioxidant moieties, and some were cytoprotective in a hypoxia-reoxygenation model in rat cardiomyocytes. These results demonstrate the feasibility of exploiting mitochondrial bioactivation reactions for targeted drug delivery.

Mechanisms of methimazole cytotoxicity in isolated rat hepatocytes.

Methimazole is an antithyroid drug widely used in the treatment of hyperthyroidism. Administration of this drug, often in a chronic manner, is associated with several adverse drug reactions in humans, including life-threatening hepatotoxicity. This study attempted to investigate the cytotoxic mechanism(s) of methimazole toward isolated rat hepatocytes. In addition, the role of proposed methimazole intermediary metabolites, such as N-methylthiourea and glyoxal, in the toxicity induced by this drug was evaluated. Isolated hepatocytes were prepared by the collagenase enzyme perfusion method. Cells were treated with methimazole, N-methylthiourea, and other chemicals and markers, such as cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) formation, lipid peroxidation (LPO), and cellular glutathione (GSH) content, were measured. Methimazole-induced cytotoxicity was accompanied by collapse in MMP, increase in ROS formation, and LPO. Further, methimazole caused reduction in GSH reservoirs, and the cytotoxic effect of the drug was much more severe in GSH-depleted cells. N-methylthiourea caused toxicity in lower concentrations than methimazole and reduced hepatocytes glutathione content. The specific flavin-containing monooxygenase inhibitor, N,N-dimethylaniline, attenuated toxicity induced by N-methylthiourea. Administration of glyoxal trapping agents, such as metformin, hydralazine, or N-acetyl cysteine, effectively prevented methimazole toxicity in intact or GSH-depleted rat hepatocytes. This study indicates that methimazole reactive metabolites are responsible for the cytotoxicity induced by this drug, but the role of glyoxal as a metabolite, which causes ROS formation, LPO, and mitochondrial injury, is predominant because the glyoxal-trapping agents diminished these adverse effects.

Serum from methimazole-treated patients induces activation of aryl hydrocarbon receptor, a transcription factor that binds to dioxin-response elements.

BACKGROUND: The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by xenobiotic substances such as dioxin. After activation, it binds to dioxin response elements of DNA, thereby inducing transcription of a variety of xenobiotic metabolizing enzymes. To investigate whether AhR-activating substances accumulate in patients with endocrine disorders, we tested serum samples for AhR-stimulating activity. METHODS: Serum AhR-stimulating activity was evaluated by exposing the HepG2 cells transiently transfected with an AhR-responsive reporter plasmid to serum samples. On the basis of preliminary findings that implicated methimazole (MMI), wild-type and AhR-null mice were treated with MMI, and their plasma AhR-stimulating activities and thyroxine levels were quantified. RESULTS: In 28 randomly chosen patients, 7 out of 10 Graves' disease patients exhibited increased serum AhR-stimulating activity. The increased activity did not correlate with thyroid hormone status. However, we hypothesized that it might be caused by MMI. Subsequent analyses revealed that in 25 of 26 MMI-treated Graves' patients, serum samples collected after the MMI treatment had significantly higher AhR-stimulating activity compared to samples obtained when the same patients were not on MMI. By contrast, serum AhR-stimulating activity was unchanged in samples from the seven patients on propylthiouracil (PTU) compared to serum taken before the PTU treatment. In vitro experiments demonstrated that an MMI metabolite 3-methyl-2-thiohydantoin, but not MMI, activated AhR. MMI increased plasma AhR-stimulating activities and reduced plasma thyroxine concentrations, in both wild-type and AhR-deficient mice. CONCLUSIONS: Graves' patients taking MMI have increased serum AhR-stimulating activity, which is unrelated to thyroid hormone status, but correlates with MMI treatment. The AhR activation is likely caused by 3-methyl-2-thiohydantoin. Further studies are required to determine the potency of 3-methyl-2-thiohydantoin as an AhR activator and the significance of the differences between MMI and PTU observed in this study.

Changes in thyroid peroxidase activity in response to various chemicals.

Thyroperoxidase (TPO) is a large heme-containing glycoprotein that catalyzes the transfer of iodine to thyroglobulin during thyroid hormone (TH) synthesis. Previously, we established an in vitro assay for TPO activity based on human recombinant TPO (hrTPO) stably transfected into human follicular thyroid carcinoma (FTC-238) cells. It is important to determine whether environmental chemicals can disrupt TPO activity because it is an important factor in the TH axis. In this study, we used our assay to examine the changes in TPO activity in response to various chemicals, including benzophenones (BPs), polycyclic aromatic hydrocarbons (PAHs), and persistent organic pollutants (POPs). Overall, BPs, PAHs, and POPs slightly altered TPO activity at low doses, as compared with the positive controls methimazole (MMI), genistein, and 2,2',4,4'-tetrahydroxy BP. Benzophenone, benzhydrol, 3-methylchloranthracene, pyrene, benzo(k)fluoranthene, benzo(e)pyrene, perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and heptachlor decreased TPO activity, while 2,4-dihydroxy BP, 2,2'-dihydroxy-4-methoxy BP, and dibenzo(a,h)anthracene increased TPO activity. From these data, we can predict the disruption of TPO activity by various chemicals as a sensitive TH end point. TPO activity should be considered when enacting measures to regulate environmental exposure to thyroid-disrupting chemicals.

Methimazole-induced hypothyroidism causes cellular damage in the spleen, heart, liver, lung and kidney.

It is known that a hypothyroidism-induced hypometabolic state protects against oxidative damage caused by toxins. However, some workers demonstrated that antithyroid drug-induced hypothyroidism can cause cellular damage. Our objective was to determine if methimazole (an antithyroid drug) or hypothyroidism causes cellular damage in the liver, kidney, lung, spleen and heart. Twenty-five male Wistar rats were divided into 5 groups: euthyroid, false thyroidectomy, thyroidectomy-induced hypothyroidism, methimazole-induced hypothyroidism (60 mg/kg), and treatment with methimazole (60 mg/kg) and a T4 injection (20 µg/kg/d sc). At the end of the treatments (4 weeks for the pharmacological groups and 8 weeks for the surgical groups), the animals were anesthetized with sodium pentobarbital and they were transcardially perfused with 10% formaldehyde. The spleen, heart, liver, lung and kidney were removed and were processed for embedding in paraffin wax. Coronal sections were stained with hematoxylin-eosin. At the end of treatment, animals with both the methimazole- and thyroidectomy-induced hypothyroidism had a significant reduction of serum concentration of thyroid hormones. Only methimazole-induced hypothyroidism causes cellular damage in the kidney, lung, liver, heart, kidney and spleen. In addition, animals treated with methimazole and T4 showed cellular damage in the lung, spleen and renal medulla with lesser damage in the liver, renal cortex and heart. The thyroidectomy only altered the lung structure. The alterations were prevented by T4 completely in the heart and partially in the kidney cortex. These results indicate that tissue damage found in hypothyroidism is caused by methimazole.

Phase 1 and phase 2 metabolic activities along the small intestine in adult male sheep.

Metabolic activities of several xenobiotic metabolizing enzymes were evaluated in both hepatic and enteric subcellular fractions obtained from Corriedale × Merino crossbreed rams by using a biochemical approach. Microsomes obtained from the different segments of sheep small intestinal mucosa displayed cytochrome P450 (CYP)-dependent N-demethylations but not O-deethylase activities apparently occurred. CYP-mediated N-demethylations neither decreased nor increased along the small intestinal mucosa. Percentages of activity for erythromycin N-demethylase in the small intestine were between 29% (duodenum) and 45% (ileum) from that measured in the liver, whereas those determined for triacetyl-oleandomycin N-demethylation ranged between 10% (duodenum) and 15% (jejunum) of the same hepatic activity. Conversely, metabolic rates for aminopyrine and chlorfeniramine N-demethylations in the gut mucosa ranged between 3% and 7% compared to their respective hepatic enzyme activities. Sheep enteric mucosa also displayed metabolic reactions typically mediated by flavin-containing monooxygenases (FMOs), carbonyl reductases (CBRs), carboxylesterases (CES), glutathione S-transferases (GSTs) and uridine diphosphoglucuronyltransferases (UGTs). The FMO-mediated sulfoxidation of methimazole was 2.6-fold higher (P < 0.01) in the ileal compared to the duodenal mucosa. Percentages of activity for the microsomal CBR-dependent biotransformation of menadione were between 12% (ileum) and 19% (duodenum-jejunum) of the total activity measured in the liver; metabolic rates measured in duodenum and jejunum were ∼1.7-fold higher (P < 0.05) than that observed in the ileum. The microsomal CES activity (using p-nitrophenyl acetate as substrate) was around twofold higher in duodenum (P < 0.05) and jejunum (P < 0.01) in comparison to the ileum. Cytosolic GST-dependent activities (toward 1-chloro, 2,4-dinitrobenzene) were similar in the mucosa of duodenum, jejunum and ileum. Microsomal UGT activities (toward 1-naphthol) in duodenum and jejunum were three- and fourfold higher, respectively, compared to that measured in the ileum. The small intestinal mucosa may play a critical defensive role due to its involvement in the detoxification of toxic compounds prior to absorption. In addition, gut metabolic reactions may contribute to the presystemic metabolism of orally administered drugs. These results are a further contribution to the understanding of the relevance of the extra-hepatic metabolism of xenobiotics in ruminant species.

Benzydamine as a useful substrate of hepatic flavin-containing monooxygenase activity in veterinary species.

Benzydamine (BZ), a weak base and an indazole derivative with analgesic and antipyretic properties used in human and veterinary medicine, is metabolized in human, rat, cattle and rabbit to a wide range of metabolites. One of the main metabolites, BZ N-oxide (BZ-NO), is produced in the liver and brain by flavin-containing monooxygenases (FMOs), by liver and brain enzymes. To evaluate the suitability of BZ as an FMO probe in veterinary species, BZ metabolism was studied in vitro using liver microsomes from bovine, rabbit and swine. Kinetic parameters, K(m) and V(max), of BZ-NO production, were evaluated to corroborate the pivotal role of FMOs. Inhibition studies were carried out by heat inactivation and by specific FMO chemical inhibitors: trimethylamine and methimazole. The results confirmed the presence of FMO activity in the liver and the role of BZ as a suitable marker of FMO enzyme activities for the veterinary species considered.

Gene expression profiling and cellular distribution of molecules with altered expression in the hippocampal CA1 region after developmental exposure to anti-thyroid agents in rats.

To determine whether developmental hypothyroidism causes permanent disruption of neuronal development, we first performed a global gene expression profiling study targeting hippocampal CA1 neurons in male rats at the end of maternal exposure to anti-thyroid agents on weaning (postnatal day 20). As a result, genes associated with nervous system development, zinc ion binding, apoptosis and cell adhesion were commonly up- or down-regulated. Genes related to calcium ion binding were up-regulated and those for myelination were often down-regulated. We, then, examined immunohistochemical cellular distribution of Ephrin type A receptor 5 (EphA5) and Tachykinin receptor (Tacr)-3, those selected based on the gene expression profiles, in the hippocampal formation at the adult stage (11-week-old) as well as at the end of exposure. At weaning, both EphA5- and Tacr3-immunoreactive cells with strong intensities appeared in the pyramidal cell layer or stratum oriens of the hippocampal CA1 region. Although the magnitude of the change was decreased at the adult stage, Tacr3 in the CA1 region showed a sustained increase in expressing cells until the adult stage after developmental hypothyroidism. On the other hand, EphA5-expressing cells did not show sustained increase at the adult stage. The results suggest that developmental hypothyroidism caused sustained neuronal expression of Tacr3 in the hippocampal CA1 region, probably reflecting a neuroprotective mechanism for mismigration.

Copper(II) complexes of methimazole, an anti Grave's disease drug. Synthesis, characterization and its potential biological behavior as alkaline phosphatase inhibitor.

Methimazole (MeimzH) is an anti-thyroid drug and the first choice for patients with Grave's disease. Two new copper(II) complexes of this drug: [Cu(MeimzH)(2)(NO(3))(2)]*0.5H(2)O and [Cu(MeimzH)(2)(H(2)O)(2)](NO(3))(2)*H(2)O were synthesized and characterized by elemental analysis, dissolution behavior, thermogravimetric analysis and UV-vis, diffuse reflectance, FTIR and EPR spectroscopies. As it is known that copper(II) cation can act as an inhibitor of alkaline phosphatase (ALP), the inhibitory effect of methimazole and its copper(II) complexes on ALP activity has also been investigated.