Hossein Babaei

Ameliorative Effects of Taurine Against Methimazole-Induced Cytotoxicity in Isolated Rat Hepatocytes

Methimazole is used as an antithyroid drug to control the symptoms of hyperthyroidism and maintain patients in a euthyroid state. Administration of this drug is associated with agranulocytosis and hepatotoxicity, which are the two most significant adverse effects. The present investigation was conducted to study the protective role of taurine against cytotoxicity induced by methimazole and its proposed reactive intermediary metabolite, N-methylthiourea, in an in vitro model of isolated rat hepatocytes. At different points in time, markers such as cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential, and hepatocyte glutathione content were evaluated. Treating hepatocytes with methimazole resulted in cytotoxicity characterized by the reduction in cell viability, an increase in ROS formation and lipid peroxidation, mitochondrial membrane potential collapse, and a reduction in cellular glutathione content. Furthermore, a significant amount of oxidized glutathione (GSSG) was formed when rat hepatocytes were treated with methimazole. N-methylthiourea toxicity was accompanied by a reduction in cellular GSH content, but no significant changes in lipid peroxidation, ROS formation, GSSG production, or changes in mitochondrial membrane potential were detected. Administration of taurine (200 μM) effectively reduced the toxic effects of methimazole or its metabolite in isolated rat hepatocytes.

Cytoprotective Effects of Taurine against Toxicity Induced by Isoniazid and Hydrazine in Isolated Rat Hepatocytes

Isoniazid is one of the most commonly used drugs to treat tuberculosis. Its administration is associated with a high incidence of hepatotoxicity. The aim of this study was to establish the protective effects of taurine against cytotoxicity induced by isoniazid and its suspected toxic metabolite hydrazine in isolated rat hepatocytes by measuring reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial depolarisation, reduced glutathione (GSH), and oxidised glutathione (GSSG). Isoniazid caused no significant ROS formation in normal hepatocytes, but in glutathione-depleted cells it was considerable. Hydrazine caused ROS formation and lipid peroxidation in both intact and glutathione-depleted cells. Both isoniazid and hydrazine caused mitochondrial membrane depolarisation. Hydrazine lowered cellular GSH reserve and increased GSSG. Taurine (200 μmol L-1) and N-acetylcysteine (200 μmol L-1) effectively countered the toxic effects of isoniazid and/or hydrazine by decreasing ROS formation, lipid peroxidation, and mitochondrial damage. Taurine prevented depletion of GSH and lowered GSSG levels in hydrazine-treated cells. This study suggests that the protective effects of taurine against isoniazid and its intermediary metabolite hydrazine cytotoxicity in rat hepatocytes could be attributed to antioxidative action. Sažetak Izoniazid je jedan od najčešćih lijekova za tuberkulozu, ali se njegova primjena povezuje s veoma učestalom hepatotoksičnosti. Cilj je ovog istraživanja bio ocijeniti djelotvornost taurina u zaštiti izoliranih hepatocita štakora od citotoksičnosti izazvane izoniazidom i njegovim toksičnim metabolitom hidrazinom. U tu smo svrhu utvrdili razine reaktivnih kisikovih spojeva (ROS), lipidnu peroksidaciju, depolarizaciju mitohondrija, reducirani glutation (GSH) te oksidirani glutation (GSSG). Izoniazid nije doveo do značajnoga nastanka ROS-a u normalnih hepatocita, ali je zato bio značajan u stanica osiromašenih glutationom. I izoniazid i hidrazine doveli su do depolarizacije membrane mitohondrija. Hidrazin je smanjio staničnu rezervu GSH i povećao razinu GSSG. Taurin (200 μmol L-1) i N-acetilcistein (200 μmol L-1) uspješno su zaštitili od toksičnoga djelovanja izoniazida i/ili hidrazina, smanjivši nastanak ROS-a, lipidnu peroksidaciju i oštećenje mitohondrija. Taurin je spriječio potpuni gubitak GSH-a te snizio razine GSSG-a u stanica tretiranih hidrazinom. Rezultati našeg istraživanje upućuju na to da se zaštitno djelovanje taurina od stanične toksičnosti izoniazida i hidrazina može pripisati njegovu andioksidacijskome djelovanju.

EFFECTS OF ENZYME INDUCTION AND/OR GLUTATHIONE DEPLETION ON METHIMAZOLE-INDUCED HEPATOTOXICITY IN MICE AND THE PROTECTIVE ROLE OF N-ACETYLCYSTEINE

PURPOSE Methimazole is the most convenient drug used in the management of hyperthyroid patients. However, associated with its clinical use is hepatotoxicity as a life threatening adverse effect. The exact mechanism of methimazole-induced hepatotoxicity is still far from clear and no protective agent has been developed for this toxicity. METHODS This study attempts to evaluate the hepatotoxicity induced by methimazole at different experimental conditions in a mice model. Methimazole-induced hepatotoxicity was investigated in different situations such as enzyme induced and/or glutathione depleted animals. RESULTS Methimazole (100 mg/kg, i.p) administration caused hepatotoxicity as revealed by increase in serum alanine aminotransferase (ALT) activity as well as pathological changes of the liver. Furthermore, a significant reduction in hepatic glutathione content and an elevation in lipid peroxidation were observed in methimazole-treated mice. Combined administration of L-buthionine sulfoximine (BSO), as a glutathione depletory agent, caused a dramatic change in methimazole-induced hepatotoxicity characterized by hepatic necrosis and a severe elevation of serum ALT activity. Enzyme induction using phenobarbital and/or β-naphtoflavone beforehand, deteriorated methimazole-induced hepatotoxicity in mice. N-acetyl cysteine (300 mg/kg, i.p) administration effectively alleviated hepatotoxic effects of methimazole in both glutathione-depleted and/or enzyme induced animals. CONCLUSION The severe hepatotoxic effects of methimazole in glutathione-depleted animals, reveals the crucial role of glutathione as a cellular defense mechanism against methimazole-induced hepatotoxicity. Furthermore, the more hepatotoxic properties of methimazole in enzyme-induced mice, indicates the role of reactive intermediates in the hepatotoxicity induced by this drug. The protective effects of N-acetylcysteine could be attributed to its radical/reactive metabolite scavenging, and/or antioxidant properties as well as glutathione replenishment activities.

Mechanisms of methimazole cytotoxicity in isolated rat hepatocytes

Abstract 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.

Development and evaluation of Leishmania infantum rK26 ELISA for serodiagnosis of visceral leishmaniasis in Iran

The purpose of this study was to prepare recombinant K26 antigen from Leishmania infantum and evaluate its performance by enzyme-linked immunosorbent assay (ELISA) test for serodiagnosis of visceral leishmaniasis (VL) in endemic regions of Iran. The results were compared with those obtained by direct agglutination test (DAT) and whole cell ELISA using crude parasite antigen. Of 93 sera from patients with confirmed VL, 90 sera were positive with rK26 ELISA (sensitivity=96.8%), whereas 85 sera were positive with DAT (sensitivity=91.4%) and 89 sera were positive with whole cell ELISA (sensitivity=95.7%). Of 130 subjects who either had other infectious diseases (n=30) or were healthy (n=100), rK26 ELISA were negative in all cases (specificity=100%), whereas DAT were negative in 116 cases (specificity=89.2%) and whole cell ELISA was negative in 114 cases (specificity=87.7%). The results of this study indicate that the rK26 ELISA is more sensitive and specific than conventional methods and could be used for reliable diagnosis of VL caused by Leishmania infantum.

Cytoprotective Effects of Organosulfur Compounds against Methimazole-Induced Toxicity in Isolated Rat Hepatocytes

PURPOSE Methimazole is a drug widely used in hyperthyroidism. However, life threatening hepatotoxicity has been associated with its clinical use. No protective agent has been found to be effective against methimazole induced hepatotoxicity yet. Hence, the capacity of organosulfur compounds to protect rat hepatocytes against cytotoxic effects of methimazole and its proposed toxic metabolite, N-methylthiourea was evaluated. METHODS Hepatocytes were prepared by the method of collagenase enzyme perfusion via portal vein. Cells were treated with different concentrations of methimazole, N methylthiourea, and organosulfur chemicals. Cell death, protein carbonylation, reactive oxygen species formation, lipid peroxidation, and mitochondrial depolarization were assessed as toxicity markers and the role of organosulfurs administration on them was investigated. RESULTS Methimazole caused a decrease in cellular glutathione content, mitochondrial membrane potential (ΔΨm) collapse, and protein carbonylation. In addition, an increase in reactive oxygen species (ROS) formation and lipid peroxidation was observed. Treating hepatocytes with N methylthiourea caused a reduction in hepatocytes glutathione reservoirs and an elevation in carbonylated proteins, but no significant ROS formation, lipid peroxidation, or mitochondrial depolarization was observed. N-acetyl cysteine, allylmercaptan, and diallyldisulfide attenuated cell death and prevented ROS formation and lipid peroxidation caused by methimazole. Furthermore, organosulfur compounds diminished methimazole induced mitochondrial damage and reduced the carbonylated proteins. In addition, these chemicals showed protective effects against cell death and protein carbonylation induced by methimazole metabolite. CONCLUSION Organosulfur chemicals extend their protective effects against methimazole-induced toxicity by attenuating oxidative stress caused by this drug and preventing the adverse effects of methimazole and/or its metabolite (s) on subcellular components such as mitochondria.

Mechanistic Approach for Toxic Effects of Bupropion in Primary Rat Hepatocytes.

Bupropion is a widely prescribed antidepressant/smoke cessation drug. However, hepatotoxicity is one of its side effects reported in some recipients. The mechanisms by which bupropion induces hepatotoxicity is not clear yet. This experiment was intended to assess the cytotoxic mechanisms of bupropion toward primary rat hepatocytes. Additionally, the effect of α-tocopherol succinate (ALPHA-TOS) and N-acetyl cysteine (NAC) and mitochondrial permeability transition (MPT) pore sealing agent cyclosporine A (Cs A) on this toxicity was investigated. Cell death, LDH leakage, reactive oxygen species (ROS) generation, lipid peroxidation (LPO), and mitochondrial depolarization were examined as toxicity indicators. Results revealed that bupropion led to a surge in ROS formation, depletion of intracellular glutathione, elevation of LPO, and mitochondrial collapse. ALPHA-TOS, NAC and Cs A administration diminished the intensity of cellular damage caused by bupropion. This experiment suggests the protective role of ALPHA-TOS, NAC and Cs A against bupropion-mediated cytotoxicity possibly through their reactive radical scavenging properties and their impacts on mitochondria. Furthermore, mitochondria might be contributed to the oxidative stress response and subsequent toxicological results observed by bupropion.

Venlafaxine-Induced Cytotoxicity Towards Isolated Rat Hepatocytes InvolvesOxidative Stress and Mitochondrial/Lysosomal Dysfunction

Purpose: Depression is a public disorder worldwide. Despite the widespread use of venlafaxine in the treatment of depression, it has been associated with the incidence of toxicities. Hence, the goal of the current investigation was to evaluate the mechanisms of venlafaxine-induced cell death in the model of the freshly isolated rat hepatocytes. Methods: Collagenase-perfused rat hepatocytes were treated with venlafaxine and other agents. Cell damage, reactive oxygen species (ROS) formation, lipid peroxidation, mitochondrial membrane potential decline, lysosomal damage, glutathione (GSH) level were analyzed. Moreover, rat liver mitochondria were isolated through differential centrifugation to assess respiratory chain functionality. Results: Our results demonstrated that venlafaxine could induce ROS formation followed by lipid peroxidation, cellular GSH content depletion, elevated GSSG level, loss of lysosmal membrane integrity, MMP collapse and finally cell death in a concentration-dependent manner. N-acetyl cysteine, taurine and quercetine significantly decreased the aforementioned venlafaxine-induced cellular events. Also, radical scavenger (butylatedhydroxytoluene and α-tocopherol), CYP2E1 inhibitor (4-methylpyrazole), lysosomotropic agents (methylamine and chloroquine), ATP generators (L-gluthamine and fructose) and mitochondrial pore sealing agents (trifluoperazine and L-carnitine) considerably reduced cytotoxicity, ROS generation and lysosomal leakage following venlafaxine treatment. Mitochondrion dysfunction was concomitant with the blockade of the electron transfer complexes II and IV of the mitochondrial respiratory system. Conclusion: Therefore, our data indicate that venlafaxine induces oxidative stress towards hepatocytes and our findings provide evidence to propose that mitochondria and lysosomes are of the primary targets in venlafaxine-mediated cell damage.

Simultaneous Determination of Tetracyclines Residues in Bovine Milk Samples by Solid Phase Extraction and HPLC-FL Method.

INTRODUCTION Tetracyclines (TCs) are widely used in animal husbandry and their residues in milk may resultin harmful effects on human. The aim of this study was to investigate the presence of TCs residues in various bovine milk samples from local markets of Ardabil, Iran. METHODS One hundred and fourteen pasteurized, sterilized and raw milk samples were collected from markets of Ardabil. Tetracycline, Oxytetracycline and Chlortetracycline (TCs) residues extraction carried out by Solid Phase Extraction method. Determination of TCs residues were performed by high performance liquid chromatography (HPLC) method using Fluorescence detector. RESULTS The mean of total TCs residues in all samples (114 samples) was 97.6 ±16.9ng/g and that of pasteurized, sterilized and raw milk samples were 87.1 ± 17.7, 112.0 ± 57.3 and 154.0 ± 66.3ng/g respectively. Twenty five point four percent of the all samples, and 24.4%, 30% and 28.6% of the pasteurized, sterilized and raw milk samples, respectively had higher TCs residues than the recommended maximum levels (100ng/g). CONCLUSION This study indicates the presence of tetracycline residues more than allowed amount. Regulatory authorities should ensure proper withdrawal period before milking the animals and definite supervisions are necessary on application of these drugs.

Rhizomes of Eremostachys laciniata: Isolation and Structure Elucidation of Chemical Constituents and a Clinical Trial on Inflammatory Diseases

PURPOSE The purpose of this study was the isolation and structure elucidation of chemical compounds from the rhizomes of Eremostachys laciniata (L) Bunge (EL), an Iranian traditional medicinal herb with a thick root and pale purple or white flowers as well as the clinical studies on the therapeutic efficacy and safety of topical application of the EL extract in the management of some inflammatory conditions, e.g., arthritis, rheumatoid arthritis and septic arthritis (Riters syndrome). METHODS The structures of the isolated compounds were elucidated unequivocally on the basis of one and two dimensional NMR, UV and HR-FABMS spectroscopic data analyses. A single-blinded randomized clinical trial was carried out with the extract of the rhizomes of E. laciniata (EL) to determine the efficacy and safety of the traditional uses of EL compared to that of piroxicam in treatment of inflammatory diseases, e.g., osteoarthritis, rheumatoid arthritis and Reiters syndrome. RESULTS Eleven iridoid glycosides, two phenylethanoids and two phytosterols were isolated and identified for the first time from the rhizomes of EL. After 14 days of treatment with the EL and piroxicam ointments, all groups showed significant improvements compared to the control groups. EL (5%) ointment induced better initial therapeutic response than piroxicam (5%) onitment. CONCLUSION This clinical trial established that EL was suitable for topical applications as a safe and effective complementary therapy for inflammatory diseases.