High-throughput screening suggests glutathione synthetase as an anti-tumor target of polydatin using human proteome chip.

Polydatin (PD) is a bio-active ingredient with known anti-tumor effects. However, its specific protein targets yet have not been systematically screened, and the molecular anti-tumor mechanism is still unclear. Here, proteomic-chip was efficiently used to screen potential targets of PD. First, we investigated through animal experiment and proteomics studies, and found that polydatin play an important role in tumor cells. Then, the red-green fluorescent of polydatin was compared comprehensively to screen its targets on chip, followed by bioinformatics analysis. Glutathione synthetase (GSS) was selected as candidate research target. After a series of molecular biological experiments GSS was confirmed a target protein for PD in vitro. Moreover, we also found that PD can significantly inhibit the activity of GSS in vitro and live cells. Our findings reveal that PD could be a selective small-molecule GSS enzyme activity inhibitor and GSS could be a potential therapeutic target in cancer.

Exogenous glutathione improves intracellular glutathione synthesis via the γ-glutamyl cycle in bovine zygotes and cleavage embryos.

Excess reactive oxygen species (ROS) generated in embryos during in vitro culture damage cellular macromolecules and embryo development. Glutathione (GSH) scavenges ROS and optimizes the culture system. However, how exogenous GSH influences intracellular GSH and improves the embryo developmental rate is poorly understood. In this study, GSH or GSX (a stable GSH isotope) was added to the culture media of bovine in vitro fertilization embryos for 7 days. The cleavage rate, blastocyst rate, and total cell number of blastocysts were calculated. Similarly to GSH, GSX increased the in vitro development rate and embryo quality. We measured intracellular ROS, GSX, and GSH for 0-32-hr postinsemination (hpi) in embryos (including zygotes at G1, S, and G2 phases and cleaved embryos) cultured in medium containing GSX. Intracellular ROS significantly decreased with increasing intracellular GSH in S-stage zygotes (18 hpi) and cleaved embryos (32 hpi). γ-Glutamyltranspeptidase ( GGT) and glutathione synthetase ( GSS) messenger RNA expression increased in zygotes (18 hpi) and cleaved embryos treated with GSH, consistent with the tendency of overall GSH content. GGT activity increased significantly in 18 hpi zygotes. GGT and GCL enzyme inhibition with acivicin and buthionine sulfoximine, respectively, decreased cleavage rate, blastocyst rate, total cell number, and GSH and GSX content. All results indicated that exogenous GSH affects intracellular GSH levels through the γ-glutamyl cycle and improves early embryo development, enhancing our understanding of the redox regulation effects and transport of GSH during embryo culture in vitro.

Establishment of a drug-induced rhabdomyolysis mouse model by co-administration of ciprofloxacin and atorvastatin.

Rhabdomyolysis is one of the serious side effects of ciprofloxacin (CPFX), a widely used antibacterial drug; and occasionally, acute kidney injury (AKI) occurs. Often, rhabdomyolysis has occurred in patients taking CPFX co-administered with statins. The purpose of this study is to establish a mouse model of drug-induced rhabdomyolysis by co-administration of CPFX and atorvastatin (ATV) and to clarify the mechanisms of its pathogenesis. C57BL/6J mice treated with L-buthionine-(S,R)-sulfoximine (BSO), a glutathione synthesis inhibitor, were orally administered with CPFX and ATV for 4 days. Plasma levels of creatinine phosphokinase (CPK) and aspartate aminotransferase (AST) were significantly increased in the CPFX and ATV-co-administered group. Histopathological examination of skeletal muscle observed degeneration in gastrocnemius muscle and an increased number of the satellite cells. Expressions of skeletal muscle-specific microRNA and mRNA in plasma and skeletal muscle, respectively, were significantly increased. The area under the curve (AUC) of plasma CPFX was significantly increased in the CPFX and ATV-co-administered group. Furthermore, cytoplasmic vacuolization and a positively myoglobin-stained region in kidney tissue and high content of myoglobin in urine were observed. These results indicated that AKI was induced by myoglobin that leaked from skeletal muscle. The established mouse model in the present study would be useful for predicting potential rhabdomyolysis risks in preclinical drug development.

Buthionine sulfoximine is a multitarget inhibitor of trypanothione synthesis in Trypanosoma cruzi.

Buthionine sulfoximine (BSO) induces decreased glutathione (GSH) and trypanothione [T(SH)2 ] pools in trypanosomatids, presumably because only gamma-glutamylcysteine synthetase (γECS) is blocked. However, some BSO effects cannot be explained by exclusive γECS inhibition; therefore, its effect on the T(SH)2 metabolism pathway in Trypanosoma cruzi was re-examined. Parasites exposed to BSO did not synthesize T(SH)2 even when supplemented with cysteine or GSH, suggesting trypanothione synthetase (TryS) inhibition by BSO. Indeed, recombinant γECS and TryS, but not GSH synthetase, were inhibited by BSO and kinetics and docking analyses on a TcTryS 3D model suggested BSO binding at the GSH site. Furthermore, parasites overexpressing γECS and TryS showed ~ 50% decreased activities after BSO treatment. These results indicated that BSO is also an inhibitor of TryS.

In silico interaction of methyl isocyanate with immune protein responsible for Mycobacterium tuberculosis infection using molecular docking.

This article reports in silico analysis of methyl isocyanate (MIC) on different key immune proteins against Mycobacterium tuberculosis. The analysis shows that MIC is released in the Bhopal gas tragedy in 1984, which is highly toxic and extremely hazardous to human health. In this study, we have selected immune proteins to perform molecular docking with the help of Autodock 4.0. Results show that the CD40 ligand and alpha5beta1 integrin have higher inhibition compared to plasminogen activator urokinase, human glutathione synthetase, mitogen-activated protein kinase (P38 MAPK 14), surfactant protein-B, -D (SP-D), and pulmonary SP-D. MIC interacted with His-125, Try-146 residue of CD40 ligand and Ala-149, and Arg-152 residue of alpha5beta1 integrin and affects the proteins functioning by binding on their active sites. These inhibitory conformations were energetically and statistically favored and supported the evidence from wet laboratory experiments reported in the literature. We can conclude that MIC directly or indirectly affects these proteins, which shows that survivals of the disaster suffer from the diseases like tuberculosis infection and lung cancer.

Screening and Identification of Inhibitors Against Glutathione Synthetase, A Potential Drug Target of Plasmodium falciparum.

Malaria is the world's most fatal disease - causing up to 2.7 million deaths annually all over the world. The ability of organisms to develop resistance against existing antimalarial drugs exacerbates the problem. There is a clear cut need for more effective, affordable and accessible drugs that act by novel modes of action. Glutathione synthetase (GS) from Plasmodium falciparum represents an important potential drug target due to its defensive role; hence ceasing the respective metabolic step will destroy the parasite. A three dimensional model of Plasmodium GS was constructed by de novo modelling method and potential GS inhibitors were identified from a library of glutathione (GSH) analogues retrieved from Ligand-info database and filtered using Lipinski and ADME rules. Two common feature pharmacophore models were generated from the individual inhibitor clusters to provide insight into the key pharmacophore features that are crucial for the GS inhibition. Molecular docking of selective compounds into the predicted GS binding site revealed that the compound CMBMB was the best GS inhibitor when compared to the standard reference Chloroquine (CQ). This was taken as indicating that CMBMB was the best effective and safest drug against P. falciparum.

Cell toxicity of 2-hydroxyethyl methacrylate (HEMA): the role of oxidative stress.

2-Hydroxyethyl methacrylate (HEMA) is a methacrylate monomer used in polymer-based dental-restorative materials. In this study, the viability of human lung epithelial cells, BEAS-2B, was investigated after exposure to this monomer. Exposure to HEMA reduced the viability of the BEAS-2B cells as a result of increased apoptosis, interruption of the cell cycle, and decreased cell proliferation. Depletion of cellular glutathione and increased levels of reactive oxygen species (ROS) were seen after exposure of BEAS-2B cells to HEMA. The glutathione synthase inhibitor, L-buthioninesulfoximine (BSO), was used to study whether the reduced viability was caused by glutathione depletion and increased levels of ROS. Similarly to incubation with HEMA, incubation with BSO resulted in glutathione depletion and increased ROS levels, without increasing cell death or inhibiting cell growth. The results indicate that HEMA-induced cell damage is not caused exclusively by these mechanisms. Mechanisms other than glutathione depletion and ROS formation seem to be of importance for the toxic effect of HEMA on lung epithelial cells.

Oxidant-mediated modification of the cellular thiols is sufficient for arginase activation in cultured cells.

Increased arginase activity in the vasculature has been implicated in the regulation of nitric oxide (NO) homeostasis, leading to the development of vascular disease and the promotion of tumor cell growth. Recently, we showed that cysteine, in the presence of iron, promotes arginase activity by driving the Fenton reaction. In the present report, we showed that induction of oxidative stress in erythroleukemic cells with the thiol-specific oxidant, diamide, led to an increase in arginase activity by 42% (P = 0.02; vs. control). By using specific antibodies, it was demonstrated that this increase correlated with an increase in arginase-1 levels in the cells and with corresponding decreases in glutathione and protein thiol levels. Treatment of cells with aurothiomalate (ATM), a protein thiol-complexing agent, diminished the activity of arginase and arginase-1 levels by 19.5 and 35.2%, respectively (vs. control) and significantly decreased both glutathione and protein thiol levels, further implicating the thiol redox system in the cellular activation of arginase. Furthermore, diamide significantly altered the kinetics of arginase, resulting in the doubling of its V(max) (vs. control). Our presented data demonstrate, for the first time that the intracellular arginase activation is may be enhanced in part, via a cellular thiol-mediated mechanism.

Crucial role of (homo)glutathione in nitrogen fixation in Medicago truncatula nodules.

Legumes form a symbiotic interaction with bacteria of the Rhizobiaceae family to produce nitrogen-fixing root nodules under nitrogen-limiting conditions. We examined the importance of glutathione (GSH) and homoglutathione (hGSH) during the nitrogen fixation process. Spatial patterns of the expression of the genes involved in the biosynthesis of both thiols were studied using promoter-GUS fusion analysis. Genetic approaches using the nodule nitrogen-fixing zone-specific nodule cysteine rich (NCR001) promoter were employed to determine the importance of (h)GSH in biological nitrogen fixation (BNF). The (h)GSH synthesis genes showed a tissue-specific expression pattern in the nodule. Down-regulation of the γ-glutamylcysteine synthetase (γECS) gene by RNA interference resulted in significantly lower BNF associated with a significant reduction in the expression of the leghemoglobin and thioredoxin S1 genes. Moreover, this lower (h)GSH content was correlated with a reduction in the nodule size. Conversely, γECS overexpression resulted in an elevated GSH content which was correlated with increased BNF and significantly higher expression of the sucrose synthase-1 and leghemoglobin genes. Taken together, these data show that the plant (h)GSH content of the nodule nitrogen-fixing zone modulates the efficiency of the BNF process, demonstrating their important role in the regulation of this process.

Disruption of redox homeostasis and induction of apoptosis by suppression of glutathione synthetase expression in a mammalian cell line.

The stable HepG2 transfectants anti-sensing expression of the glutathione synthetase (GS) gene exhibited delayed cell growth and increased reactive oxygen species (ROS) level. After the treatment with hydrogen peroxide, the intracellular ROS level was much higher in the stable transfectants than in the vector control cells. However, the GSH levels decreased more significantly in the stable transfectants than in the vector control cells, in the presence of hydrogen peroxide. Hydrogen peroxide-induced apoptosis of the stable transfectants was notably higher than that of the vector control cells. The GS anti-sense RNAs rendered the HepG2 cells more sensitive to growth arrest caused by glucose deprivation. They also sensitized the HepG2 cells to cadmium chloride (Cd) and nitric oxide (NO)-generating sodium nitroprusside (SNP). In brief, the results confirm that GS plays an important role in the defense of the human hepatoma cells against oxidative stress by reducing apoptosis and maintaining redox homeostasis.

Depletion of cytosolic or mitochondrial thioredoxin increases CYP2E1-induced oxidative stress via an ASK-1-JNK1 pathway in HepG2 cells.

Thioredoxin is an important reducing molecule in biological systems. Increasing CYP2E1 activity induces oxidative stress and cell toxicity. However, whether thioredoxin protects cells against CYP2E1-induced oxidative stress and toxicity is unknown. SiRNA were used to knockdown either cytosolic (TRX-1) or mitochondrial thioredoxin (TRX-2) in HepG2 cells expressing CYP2E1 (E47 cells) or without expressing CYP2E1 (C34 cells). Cell viability decreased 40-60% in E47 but not C34 cells with 80-90% knockdown of either TRX-1 or TRX-2. Depletion of either thioredoxin also potentiated the toxicity produced either by a glutathione synthesis inhibitor or by TNFα in E47 cells. Generation of reactive oxygen species and 4-HNE protein adducts increased in E47 but not C34 cells with either thioredoxin knockdown. GSH was decreased and adding GSH completely blocked E47 cell death induced by either thioredoxin knockdown. Lowering TRX-1 or TRX-2 in E47 cells caused an early activation of ASK-1, followed by phosphorylation of JNK1 after 48 h of siRNA treatment. A JNK inhibitor caused a partial recovery of E47 cell viability after thioredoxin knockdown. In conclusion, knockdown of TRX-1 or TRX-2 sensitizes cells to CYP2E1-induced oxidant stress partially via ASK-1 and JNK1 signaling pathways. Both TRX-1 and TRX-2 are important for defense against CYP2E1-induced oxidative stress.

Characterization of Plasmodium berghei glutathione synthetase.

Plasmodium berghei contained 0.454±0.031 U/mg of glutathione synthetyase (GS). GS was purified using solid ammonium sulfate and Sephadex G-200 from P. berghei infected mouse erythrocytes. SDS-PAGE showed purified GS as a single band protein of 70 kDa and its Km for γ-glutamylcysteine, glycine and ATP being 0.33 mM, 8.3 mM and 0.43 mM respectively with noncompetitive inhibition by GSH. The malaria parasite enzyme was optimally active at 37°C and pH 8.0-8.5. Heavy metals significantly inhibited parasite GS activity.

Autophagy in cancer associated fibroblasts promotes tumor cell survival: Role of hypoxia, HIF1 induction and NFκB activation in the tumor stromal microenvironment.

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB--another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism", and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.

Patulin-induced genotoxicity and modulation of glutathione in HepG2 cells.

Patulin (PAT), a mycotoxin produced by certain species of Penicillium, Aspergillus and Byssochlamys, is mainly found in ripe apple and apple products. In our present study, a significant increase of the micronuclei frequency induced by PAT was found in human hepatoma HepG2 cells. To elucidate the role of glutathione (GSH) in the effect, the intracellular GSH level was modulated by pre-treatment with buthionine-(S, R)-sulfoximine (BSO), a specific GSH synthesis inhibitor, and by pre-treatment with N-acetylcysteine (NAC), a GSH precursor. It was found that depletion of GSH in HepG2 cells with BSO dramatically increased the PAT-induced micronuclei frequencies and that when the intracellular GSH content was elevated by NAC, the chromosome damage induced by PAT was significantly prevented in our test concentrations (0.19-0.75 microM). These results indicate that GSH play an important role in cellular defense against PAT-induced genotoxicity.

Protective mechanisms of N-acetyl-cysteine against pyrrolizidine alkaloid clivorine-induced hepatotoxicity.

Pyrrolizidine alkaloid (PA) clivorine, isolated from traditional Chinese medicinal plant Ligularia hodgsonii Hook, has been shown to induce apoptosis in hepatocytes via mitochondrial-mediated apoptotic pathway in our previous research. The present study was designed to observe the protection of N-acetyl-cysteine (NAC) on clivorine-induced hepatocytes apoptosis. Our results showed that 5 mM NAC significantly reversed clivorine-induced cytotoxicity via MTT and Trypan Blue staining assay. DNA apoptotic fragmentation analysis and Western-blot results showed that NAC decreased clivorine-induced apoptotic DNA ladder and caspase-3 activation. Further results showed that NAC inhibited clivorine-induced Bcl-xL decrease, mitochondrial cytochrome c release and caspase-9 activation. Intracellular glutathione (GSH) is an important ubiquitous redox-active reducing sulfhydryl (--SH) tripeptide, and our results showed that clivorine (50 microM) decreased cellular GSH amounts and the ratio of GSH/GSSG in the time-dependent manner, while 5 mM NAC obviously reversed this depletion. Further results showed that GSH synthesis inhibitor BSO augmented clivorine-induced cytotoxicity, while exogenous GSH reversed its cytotoxicity on hepatocytes. Clivorine (50 microM) significantly induced cellular reactive oxygen species (ROS) generation. Further results showed that 50 microM Clivorine decreased glutathione peroxidase (GPx) activity and increased glutathione S transferase (GST) activity, which are both GSH-related antioxidant enzymes. Thioredoxin-1 (Trx) is also a ubiquitous redox-active reducing (--SH) protein, and clivorine (50 microM) decreased cellular expression of Trx in a time-dependent manner, while 5 mM NAC reversed this decrease. Taken together, our results demonstrate that the protection of NAC is major via maintaining cellular reduced environment and thus prevents clivorine-induced mitochondrial-mediated hepatocytes apoptosis.

Metallothionein alleviates glutathione depletion-induced oxidative cardiomyopathy in murine hearts.

OBJECTIVE: Antioxidant therapy has shown some promise in critical care medicine in which glutathione depletion and heart failure are often seen in critically ill patients. This study was designed to examine the impact of glutathione depletion and the free radical scavenger, metallothionein (MT), on cardiac function. DESIGN: Friend virus B and MT transgenic mice were given the glutathione synthase inhibitor buthionine sulfoximine (buthionine sulfoximine [BSO], 30 mmol/L) in drinking water for 2 wks. MEASUREMENTS: Echocardiographic and cardiomyocyte functions were evaluated, including myocardial geometry, fraction shortening, peak shortening, time-to-90% relengthening (TR90), maximal velocity of shortening/relengthening (+/-dL/dt), intracellular Ca2+ rise, sarcoplasmic reticulum Ca2+ release, and intracellular Ca2+ decay rate. Sacro (endo)plasmic reticulum Ca2+-ATPase function was evaluated by 45Ca uptake. Highly reactive oxygen species, caspase-3, and aconitase activity were detected by fluorescent probe and colorimetric assays. MAIN RESULT: BSO elicited lipid peroxidation, protein carbonyl formation, mitochondrial damage, and apoptosis. BSO also reduced wall thickness, enhanced end systolic diameter, depressed fraction shortening, peak shortening, +/-dL/dt, sarcoplasmic reticulum Ca2+ release, 45Ca uptake, and intracellular Ca2+ decay, leading to prolonged TR90. BSO-induced mitochondrial loss and myofilament aberration. MT transgene itself had little effect on myocardial mechanics and ultrastructure. However, it alleviated BSO-induced myocardial functional, morphologic, and carbonyl changes. Western blot analysis showed reduced expression of sacro (endo)plasmic reticulum Ca2+-ATPase2a, Bcl-2 and phosphorylated GSK-3beta, enhanced calreticulin, Bax, p53, myosin heavy chain-beta isozyme switch, and IkappaB phosphorylation in FVB-BSO mice, all of which with the exception of p53 were nullified by MT. CONCLUSION: Our findings suggest a pathologic role of glutathione depletion in cardiac dysfunction and the therapeutic potential of antioxidants.

Neurotoxicity from glutathione depletion is mediated by Cu-dependent p53 activation.

Loss of intracellular neuronal glutathione (GSH) is an important feature of neurodegenerative disorders including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. The consequences of GSH depletion include increased oxidative damage to proteins, lipids, and DNA and subsequent cytotoxic effects. GSH is also an important modulator of cellular copper (Cu) homeostasis and altered Cu metabolism is central to the pathology of several neurodegenerative diseases. The cytotoxic effects of Cu in cells depleted of GSH are not well understood. We have previously reported that depletion of neuronal GSH levels results in cell death from trace levels of extracellular Cu due to elevated Cu(I)-mediated free radical production. In this study we further examined the molecular pathway of trace Cu toxicity in neurons and fibroblasts depleted of GSH. Treatment of primary cortical neurons or 3T3 fibroblasts with the glutathione synthetase inhibitor buthionine sulfoximine resulted in substantial loss of intracellular GSH and increased cytotoxicity. We found that both neurons and fibroblasts revealed increased expression and activation of p53 after depletion of GSH. The increased p53 activity was induced by extracellular trace Cu. Furthermore, we showed that in GSH-depleted cells, Cu induced an increase in oxidative stress resulting in DNA damage and activation of p53-dependent cell death. These findings may have important implications for neurodegenerative disorders that involve GSH depletion and aberrant Cu metabolism.

Buthionine sulfoximine causes endothelium dependent hyper-relaxation and hypoadiponectinemia.

A close relationship between oxidative stress, endothelial dysfunction, and hypoadiponectinemia has been observed. The present study was performed to investigate how glutathione depletion via buthionine sulfoximine (BSO) administration affects endothelial function and adiponectin levels in rats. Acetylcholine (Ach)-induced vasodilation was significantly enhanced in BSO-treated rats, compared with control rats. This was completely abolished by L-NAME, and Ach-induced vasodilation was not observed in the aorta without endothelium. These results suggest that Ach-induced hyper-relaxation of the aorta in BSO-treated rats is completely dependent on the presence of endothelium and mediated by changes in eNOS activity. Catalase significantly inhibited this relaxation to Ach and no effect of catalase on sodium nitroprusside-induced relaxation of the aorta without endothelium was observed in BSO-treated rats. Thus, hyper-relaxation of the aorta in BSO-treated rats is likely caused by H2O2 in addition to NO produced by the endothelium via an eNOS-dependent mechanism. Hypoadiponectinemia and decreased levels of adiponectin mRNA in adipose tissue were observed in BSO-treated rats. Protein expression of eNOS and SODs (SOD-1 and SOD-2) in the aorta was increased and plasma NOx levels were decreased in BSO-treated rats. Our results suggest that oxidative stress induced by BSO causes eNOS uncoupling and hyper-relaxation by producing H2O2, and that BSO-induced oxidative stress causes hypoadiponectinemia, probably by increasing H2O2 production in adipose tissue.

Evidence of a new dechlorinated ochratoxin A derivative formed in opossum kidney cell cultures after pretreatment by modulators of glutathione pathways: correlation with DNA-adduct formation.

Ochratoxin A (OTA), a nephrotoxic mycotoxin probably implicated in human Balkan endemic nephropathy and associated urothelial tumors, induces renal carcinomas in rodents and nephrotoxicity in pigs. OTA induces DNA-adduct formation, but the structure of the adducts and their role in nephrotoxicity and carcinogenicity have only partly been elucidated. In vivo, 2-mercaptoethane sulfonate (MESNA) protects rats against OTA-induced nephrotoxicity but not against carcinogenicity, indicating two different mechanisms leading to nephrotoxicity or carcinogenicity. To better understand how DNA-adduct could be generated, opossum kidney cells (OK) have been treated by OTA alone or in presence of several compounds such as MESNA or N-acetylcysteine (another agent that, like MESNA, reduces oxidative stress by increasing of free thiols in kidney), buthionine sulfoximine (BSO) (an inhibitor of glutathione-synthase), and alpha amino-3-chloro-4,5-dihydro-5-isoxazole acetic acid (ACIVICIN) (an inhibitor of gamma glutamyl transpeptidase). Cytotoxicity of OTA on OK cells was evaluated by applying the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. None of the listed agents diminished OTA cytotoxicity significantly; ACIVICIN even increases OTA cytotoxicity. In contrast, analysis of the HPLC profiles of OTA metabolites produced during these incubations indicated that the pattern, the quantity of metabolites, and the nature of the derivatives were modulated by these agents. Ochratoxin B (OTB), open-ring ochratoxin A (OP-OA), 4 hydroxylated OTA, 10 hydroxylated OTA, OTA without phenylalanine, OTB without phenylalanine, and a dechlorinated OTA metabolite could be identified by nano-ESI-IT-MS.

Characterization of the bifunctional gamma-glutamate-cysteine ligase/glutathione synthetase (GshF) of Pasteurella multocida.

Glutamate-cysteine ligase (gamma-ECL) and glutathione synthetase (GS) are the two unrelated ligases that constitute the glutathione biosynthesis pathway in most eukaryotes, purple bacteria, and cyanobacteria. gamma-ECL is a member of the glutamine synthetase family, whereas GS enzymes group together with highly diverse carboxyl-to-amine/thiol ligases, all characterized by the so-called two-domain ATP-grasp fold. This generalized scheme toward the formation of glutathione, however, is incomplete, as functional steady-state levels of intracellular glutathione may also accumulate solely by import, as has been reported for the Pasteurellaceae member Haemophilus influenzae, as well as for certain Gram-positive enterococci and streptococci, or by the action of a bifunctional fusion protein (termed GshF), as has been reported recently for the Gram-positive firmicutes Streptococcus agalactiae and Listeria monocytogenes. Here, we show that yet another member of the Pasteurellaceae family, Pasteurella multocida, acquires glutathione both by import and GshF-driven biosynthesis. Domain architecture analysis shows that this P. multocida GshF bifunctional ligase contains an N-terminal gamma-proteobacterial gamma-ECL-like domain followed by a typical ATP-grasp domain, which most closely resembles that of cyanophycin synthetases, although it has no significant homology with known GS ligases. Recombinant P. multocida GshF overexpresses as an approximately 85-kDa protein, which, on the basis of gel-sizing chromatography, forms dimers in solution. The gamma-ECL activity of GshF is regulated by an allosteric type of glutathione feedback inhibition (K(i) = 13.6 mM). Furthermore, steady-state kinetics, on the basis of which we present a novel variant of half-of-the-sites reactivity, indicate intimate domain-domain interactions, which may explain the bifunctionality of GshF proteins.