GSH Transferase Activity Research Articles

Physiology, Biochemistry, and Transcriptomics Jointly Reveal the Phytotoxicity Mechanism of Acetochlor on Pisum sativum L.

Acetochlor, as a commonly used pre-emergent herbicide, can be toxic to crops and affect production if used improperly. However, the toxic mechanism of acetochlor on plants is not fully understood. The present study used a combination of transcriptomic analysis and physiological measurements to investigate the effects of short-term (15-day) exposure to different concentrations of acetochlor (1, 10, 20 mg/kg) on the morphology, physiology, and transcriptional levels of pea seedlings, aiming to elucidate the toxic response and resistance mechanisms in pea seedlings under herbicide stress. The results showed that the toxicity of acetochlor to pea seedlings was dose-dependent, manifested as dwarfing and stem base browning with increasing concentrations, especially at 10 mg/kg and above. Analysis of the antioxidant system showed that from the 1 mg/kg treatment, malondialdehyde, superoxide dismutase, peroxidase, and glutathione peroxidase in peas increased with increasing concentrations of acetochlor, indicating oxidative damage. Analysis of the glutathione (GSH) metabolism system showed that under 10 mg/kg treatment, the GSH content of pea plants significantly increased, and GSH transferase activity and gene expression were significantly induced, indicating a detoxification response in plants. Transcriptomic analysis showed that after acetochlor treatment, differentially expressed genes in peas were significantly enriched in the phenylpropane metabolic pathway, and the levels of key metabolites (flavonoids and lignin) were increased. In addition, we found that acetochlor-induced dwarfing of pea seedlings may be related to gibberellin signal transduction. Environ Toxicol Chem 2024;43:2005-2019. © 2024 SETAC.

Dietary and Lifestyle Factors Modulate the Activity of the Endogenous Antioxidant System in Patients with Age-Related Macular Degeneration: Correlations with Disease Severity.

Age-related macular degeneration (AMD) is a common cause of blindness in the elderly population, but the pathogenesis of this disease remains largely unknown. Since oxidative stress is suggested to play a major role in AMD, we aimed to assess the activity levels of components of the antioxidant system in patients with AMD. We also investigated whether lifestyle and dietary factors modulate the activity of these endogenous antioxidants and clinical parameters of disease severity. We recruited 330 patients with AMD (39 with early, 100 with intermediate and 191 with late form of AMD) and 121 controls in this study. At enrolment, patients’ dietary habits and physical activity were assessed, and each study participant underwent a thorough ophthalmologic examination. The activity of several components of the antioxidant system were measured in red blood cells and platelets using both kinetic and spectrophotometric methods. Patients with AMD consumed much lower levels of fatty fish and eggs than the control group (p = 0.008 and p = 0.04, respectively). In the nAMD group, visual acuity (VA) correlated positively with green vegetable consumption (Rs = +0.24, p = 0.004) and omega-3-rich oil intake (Rs = +0.17, p = 0.03). In the AMD group, the total physical activity MET score correlated positively with VA (Rs = +0.17, p = 0.003) and correlated negatively with the severity of AMD (Rs = −0.14, p = 0.01). A multivariate analysis of patients and controls adjusted for age, sex, and smoking status (pack-years) revealed that AMD was an independent variable associated with a lower RBC catalase (β = −0.37, p < 0.001) and higher PLT catalase (β = +0.25, p < 0.001), RBC GPx (β = +0.26, p < 0.001), PLT GPx (β = +0.16, p = 0.001), RBC R-GSSG (β = +0.13, p = 0.009), PLT R-GSSG (β = +0.12, p = 0.02) and RBC GSH transferase (β = +0.23, p < 0.001) activity. The activities of components of the antioxidant system were associated with disease severity and depended on dietary habits. The observed substantial increase in the activity of many critical endogenous antioxidants in patients with AMD further indicates that the required equilibrium in the antioxidant system is disturbed throughout the course of the disease. Our findings explicitly show that a diet rich in green vegetables, fish and omega-3-rich oils, supplemented by physical exercise, is beneficial for patients with AMD, as it might delay disease progression and help retain better visual function.

Activation versus inhibition of microsomal glutathione S-transferase activity by acrolein. Dependence on the concentration and time of acrolein exposure

The toxicity of acrolein, an α,β-unsaturated aldehyde, is due to its soft electrophilic nature and primarily involves the adduction of protein thiols. The thiol glutathione (GSH) forms the first line of defense against acrolein. The present study confirms that acrolein added to isolated rat liver microsomes can increase microsomal GSH transferase (MGST) activity 2–3 fold, which can be seen as a direct adaptive increase in the protection against acrolein. At a relatively high exposure level, acrolein appeared to inhibit MGST. The activation is due to adduction of thiol groups, and the inactivation probably involves adduction of amino groups in the enzyme by acrolein. The preference of acrolein to react with thiol groups over amino groups can explain why the enzyme is activated at a low exposure level and inhibited at a high exposure level of acrolein. These opposite forms of direct adaptation on the level of enzyme activity further narrow the thin line between survival and promotion of cell death, governed by the level of exposure.

Characterization of glutathione transferases involved in the pathogenicity of Alternaria brassicicola.

BackgroundGlutathione transferases (GSTs) represent an extended family of multifunctional proteins involved in detoxification processes and tolerance to oxidative stress. We thus anticipated that some GSTs could play an essential role in the protection of fungal necrotrophs against plant-derived toxic metabolites and reactive oxygen species that accumulate at the host-pathogen interface during infection.ResultsMining the genome of the necrotrophic Brassica pathogen Alternaria brassicicola for glutathione transferase revealed 23 sequences, 17 of which could be clustered into the main classes previously defined for fungal GSTs and six were ‘orphans’. Five isothiocyanate-inducible GSTs from five different classes were more thoroughly investigated. Analysis of their catalytic properties revealed that two GSTs, belonging to the GSTFuA and GTT1 classes, exhibited GSH transferase activity with isothiocyanates (ITC) and peroxidase activity with cumene hydroperoxide, respectively. Mutant deficient for these two GSTs were however neither more susceptible to ITC nor less aggressive than the wild-type parental strain. By contrast mutants deficient for two other GSTs, belonging to the Ure2pB and GSTO classes, were distinguished by their hyper-susceptibility to ITC and low aggressiveness against Brassica oleracea. In particular AbGSTO1 could participate in cell tolerance to ITC due to its glutathione-dependent thioltransferase activity. The fifth ITC-inducible GST belonged to the MAPEG class and although it was not possible to produce the soluble active form of this protein in a bacterial expression system, the corresponding deficient mutant failed to develop normal symptoms on host plant tissues.ConclusionsAmong the five ITC-inducible GSTs analyzed in this study, three were found essential for full aggressiveness of A. brassicicola on host plant. This, to our knowledge is the first evidence that GSTs might be essential virulence factors for fungal necrotrophs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0462-0) contains supplementary material, which is available to authorized users.

English

&nbsp; The potent of n-butanol extract of celery (Apium graveolens) seed in ameliorating the lipid peroxidation and antioxidant status was investigated in streptozotocin-induced diabetic rats. Thirty two mature male rats were assigned to four groups, non-diabetic control and three diabetic groups. Diabetes was induced by single injection with streptozotocin (60 mg/kg b.w.,&nbsp;i.p.). Rats &ge; 200 mg/dl of blood glucose were used as diabetic. Diabetic groups (D, B, and I) were drenched with drinking water, n-butanol extract (60 mg/kg, b.w.), or injected with insulin (4 IU/animal), respectively for 21 days. On day 22, body weight gain was registered and male rats were sacrificed. Blood and liver subcellular fluid was obtained to assess blood glucose level and subcellular activity of Alanine aminotranferease (ALT), Aspartate aminotransferase (AST), catalase, Super oxide dismutase (SOD), Glutathione (GSH)-transferase and -reductase, and assessment of Malondialdehyde (MDA) and glutathione concentrations. Diabetic rats (D) showed marked increased blood glucose, decreased weight gain, increased activity of ALT, SOD, CAT, GSH-transferase, decreased GSH-reductase and normal AST. N-butanol extract of celery seed (B) or insulin (I) therapy moderated blood glucose within normal range, enhanced body weight gain and normalized the activities of all antioxidant enzymes. In conclusion, n-butanol extract of celery seed have potent role in ameliorating stressful complications accompanied by diabetes mellitus. &nbsp; Key words:&nbsp;Celery,&nbsp;Apium graveolens, diabetes mellitus, antioxidants.

Analysis of the effects of iron and vitamin C co-supplementation on oxidative damage, antioxidant response and inflammation in THP-1 macrophages

Objectives The aims of the study were to test the susceptibility of THP-1 macrophages to develop oxidative stress and to deploy antioxidant defense mechanisms that insure the balance between the pro- and antioxidant molecules. Design and methods Differentiated THP-1 were incubated in the presence or absence of iron-ascorbate (Fe/As) (100/1000 μM) and the antioxidants Trolox, BHT, α-Tocopherol and NAC. Results Fe/As promoted the production of lipid peroxidation as reflected by the formation of malondialdehyde and H 2O 2 along with reduced PUFA levels and elevated glutathione disulfide/total glutathione ratio, a reliable index of cellular redox status. THP-1 macrophages developed an increase in cytoplasmic SOD activity due in part to high cytoplasmic SOD1. On the other hand, a decline was noted in mRNA and protein of extra-cellular SOD3, as well as the activity of GSH-peroxidase, GSH-transferase and ATOX-1 expression. Conclusions Macrophages activated under conditions of oxidative stress do not adequately deploy a powerful endogenous antioxidant response, a situation that can lead to an enhanced inflammatory response.

Pleiotropic effects of hexavalent chromium (CrVI) in Mytilus galloprovincialis digestive gland

Hexavalent Chromium Cr(VI) is an important contaminant considered as a model oxidative toxicant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. On the other hand, in mammals the reduced form Cr(III) is considered an essential microelement, involved in regulation of lipid and carbohydrate metabolism; moreover, recent evidence suggests that Cr may have endocrine effects. In this work, the effects of Cr(VI) were investigated in the digestive gland of the marine bivalve Mytilus galloprovincialis. Mussels were exposed to 0.1–1–10–100 μg Cr(VI) L −1 animal −1 for 96 h. At 100 μg L −1, a large increase in total Cr tissue content was observed; in these conditions, the lysosomal membranes were completely destabilized, whereas other lysosomal biomarkers (neutral lipids-NL and lipofuscin-LF), as well as different enzyme activities and gene expression were unaffected, this indicating severe stress conditions in the tissue. On the other hand, at lower concentrations, changes in other histochemical, biochemical and molecular endpoints were observed. In particular, at both 1 and 10 μg L −1, lysosomal destabilization was associated with significant NL and LF accumulation; however, no changes in catalase and GSH transferase (GST) activities were observed. At the same concentrations, GSSG reductase (GSR) activity was significantly increased, this probably reflecting the recycling of GSSG produced in the GSH-mediated intracellular reduction of Cr(VI). Increased activities of the key glycolytic enzymes PFK (phosphofructokinase) and PK (pyruvate kinase) were also observed, indicating that Cr(VI) could affect carbohydrate metabolism. Cr(VI) induced downregulation or no effects on the expression of metallothioneins MT10 and MT20, except for an increase in MT20 transcription in males. Moreover, significant up-regulation of the Mytilus estrogen receptor MeER2 and serotonin receptor (5-HTR) were observed in both sexes. The results demonstrate that exposure to Cr(VI) in the low ppb range did not result in strong toxicity or oxidative stress conditions in mussel digestive gland. On the other hand, our data support the hypothesis that low concentrations of the metal can exert pleiotropic effects on mussel physiology, from modulation of lipid and carbohydrate metabolism, to effects on the expression of estrogen-responsive genes.

Structure and Function of YghU, a Nu-Class Glutathione Transferase Related to YfcG from Escherichia coli

The crystal structure (1.50 Å resolution) and biochemical properties of the GSH transferase homologue, YghU, from Escherichia coli reveal that the protein is unusual in that it binds two molecules of GSH in each active site. The crystallographic observation is consistent with biphasic equilibrium binding data that indicate one tight (K(d1) = 0.07 ± 0.03 mM) and one weak (K(d2) = 1.3 ± 0.2 mM) binding site for GSH. YghU exhibits little or no GSH transferase activity with most typical electrophilic substrates but does possess a modest catalytic activity toward several organic hydroperoxides. Most notably, the enzyme also exhibits disulfide-bond reductase activity toward 2-hydroxyethyl disulfide [k(cat) = 74 ± 6 s(-1), and k(cat)/K(M)(GSH) = (6.6 ± 1.3) × 10(4) M(-1) s(-1)] that is comparable to that previously determined for YfcG. A superposition of the structures of the YghU·2GSH and YfcG·GSSG complexes reveals a remarkable structural similarity of the active sites and the 2GSH and GSSG molecules in each. We conclude that the two structures represent reduced and oxidized forms of GSH-dependent disulfide-bond oxidoreductases that are distantly related to glutaredoxin 2. The structures and properties of YghU and YfcG indicate that they are members of the same, but previously unidentified, subfamily of GSH transferase homologues, which we suggest be called the nu-class GSH transferases.

Human placental glutathione S-transferase activity and polycyclic aromatic hydrocarbon DNA adducts as biomarkers for environmental oxidative stress in placentas from pregnant women living in radioactivity- and chemically-polluted regions

This study was designed to analyze the effect of environmental oxidative stress on human placental monooxygenases, glutathione S-transferase (GST) activity and polycyclic aromatic hydrocarbon (PAH)–DNA adducts in human term placentas from radioactivity-contaminated and chemically-polluted areas of the Ukraine and Belarus, and to compare these biomarkers to the newborn's general health status. Placental PAH–DNA adduct formation, GST activity, 7-ethoxycoumarin O-deethylase (ECOD) activity, and thiobarbituric reactive substances (TBARS), an index of lipid peroxidation, were measured in groups of women exposed to different levels of radioactivity and PAH pollution. The in vitro metabolism data, obtained from 143 human placental samples at term, were compared to indices of maternal and newborn health. The highest ECOD activity was recorded in placentas obtained from chemically-polluted areas and a radioactivity-contaminated area; the ECOD activity was 7-fold and 2-fold higher compared to the region considered to be “clean”. Newborns with the most compromised health status displayed the greatest down-regulation of GST activity (144–162 mU mg protein −1 vs. 258–395 mU mg protein −1), enhanced ECOD activity and the highest level of PAH–DNA adduct formation. The highest level of TBARS was observed in women exposed to the highest levels of radiation. The efficiency of placental detoxification negatively correlated with maternal age and the health status of the newborn. Environmental oxidative stress was related to an increase in anemia, threatened abortions, toxemia, fetal hypoxia, spontaneous abortions and fetal hypotrophy. Our data suggest that chemically- or radioactivity-induced oxidative stress enhance cytochrome P450-mediated enzymatic activities potentially resulting in increased formation of reactive metabolites. The activity of GSH-transferase is not enhanced. This imbalance in detoxification capacity can be measured as increased production of PAH–DNA adducts, decreased lipid peroxidation and compromised fetal health.

Analysis of the Structure and Function of YfcG from Escherichia coli Reveals an Efficient and Unique Disulfide Bond Reductase

YfcG is one of eight glutathione (GSH) transferase homologues encoded in the Escherichia coli genome. The protein exhibits low or no GSH transferase activity toward a panel of electrophilic substrates. In contrast, it has a very robust disulfide-bond reductase activity toward 2-hydroxyethyldisulfide on par with mammalian and bacterial glutaredoxins. The structure of YfcG at 2.3 A-resolution from crystals grown in the presence of GSH reveals a molecule of glutathione disulfide in the active site. The crystallographic results and the lack of functional cysteine residues in the active site of YfcG suggests that the reductase activity is unique in that no sulfhydryl groups in the YfcG protein are covalently involved in the redox chemistry.

Bisphenol-A alters gene expression and functional parameters in molluscan hepatopancreas

Bisphenol-A (BPA) is a well-known xenoestrogen in mammalian systems that can affect reproduction also in aquatic organisms. In this work the possible effects of BPA were investigated in the hepatopancreas of the bivalve mollusc Mytilus galloprovincialis: mussels were injected with different amounts of BPA (3–60 ng/g dw tissue) and tissues sampled at 24 h post-injection. Expression of different Mytilus genes was evaluated by RT-Q-PCR: BPA exposure increased the expression of MeER2 and induced downregulation of antioxidant genes, catalase and metallothioneins. Moreover, BPA induced changes in activity of catalase, GSH transferase (GST) and GSSG reductase (GSR), and in total glutathione content. A decrease in lysosomal membrane stability and increased neutral lipid accumulation were also observed. The results were compared with those obtained with similar concentrations of 17β-estradiol. These data demonstrate that BPA can alter gene expression, activities of enzymes involved in redox balance, and lysosomal function in molluscan hepatopancreas, a tissue involved in the control of metabolism and gamete maturation. Overall, these data indicate that BPA, at environmentally relevant concentrations, can have both estrogen-like and distinct effects in invertebrates like in vertebrates.

Antioxidative effect of sesame coat on LDL oxidation and oxidative stress in macrophages

The effects of ethanolic extract of sesame ( Sesamum indicum L.) coat (EESC) on oxidation of low-density lipoprotein (LDL) and production of nitric oxide in macrophages were investigated. The results show that EESC in the range of 0.01–0.8 mg/ml markedly inhibited copper-induced LDL oxidation and H 2O 2-induced cell damage. In addition, EESC at 0.1 mg/ml not only enhanced glutathione (GSH) levels, but also increased activity of GSH peroxidase, GSH reductase, GSH transferase and catalase by 3.32-, 3.39-, 1.60- and 2.26-fold compared to the control, respectively. Moreover, EESC showed dose-dependent inhibitory effects on nitric oxide production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages and in sodium nitroprusside (SNP), a NO donor. The generation of prostaglandin E2 (PGE2) in stimulated macrophages was also reduced by EESC. The induction of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) proteins in activated macrophages was inhibited by EESC. In addition, the levels of iNOS and COX-2 mRNA in activated macrophages were decreased by EESC when determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Furthermore, LPS-induced degradation of IκB-α was down regulated by EESC, indicating that EESC prevented LPS-induced NF-κB activation. These results implied that EESC could exhibit a protective action on biomolecules and generation of inflammatory mediators in vitro.

Protective effects of burdock ( Arctium lappa Linne) on oxidation of low-density lipoprotein and oxidative stress in RAW 264.7 macrophages

The protective effects of burdock ( Arctium lappa Linne) on oxidation of low-density lipoprotein (LDL) and nitric oxide production were investigated. The results showed that methanolic extracts of burdock (MEB) and their major components, chlorogenic acid (CHA) and caffeic acid (CA), showed marked antioxidant activity against oxidative damage of liposome ( p < 0.05), deoxyribose ( p < 0.05) and protein ( p < 0.05). In addition, at a concentration of 500 μg/ml, the inhibitory effect of MEB on LDL oxidation was 66.9% compared to the control ( p < 0.05). MEB, at 200 μg/ml, not only enhanced GSH levels, but also increased activity of GSH reductase, GSH peroxidase, GSH transferase and catalase, which were 3.82-, 24.9-, 4.35- and 3.02-fold compared to the control ( p < 0.05), respectively. MEB directly scavenged nitric oxide in a concentration-dependent fashion ( p < 0.05). Moreover, MEB showed a reducing effect on nitric oxide production of lipopolysaccharide (LPS)-induced RAW 264.7 cells. The expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in activated RAW 264.7 cells were inhibited by MEB. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that the expression of iNOS and COX-2 mRNA in activated macrophages were suppressed by a high concentration (500 μg/ml) of MEB. Furthermore, a downregulated degradation of IκB-α by MEB was found, indicating that MEB reduced iNOS enzyme expression as a result of preventing NF-κB activation. These results suggest that MEB displays an inhibitory action on biomolecules and has a bioactive action for attenuating excessive NO generation at inflammatory site as well as in cardiovascular disease.

Cytoprotection by Melatonin, Precursors and Metabolites in an In Vitro Model of Neurotoxicity Induced by Okadaic Acid

Recently, it has been emphasized that oxidative stress could play a synergistic role with tau phosphorylation, cell shape changes, cytoskeletal disruption and cell death in Alzheimers disease. Okadaic acid induces oxidative damage, cell shape alterations, tau hyperphosphorylation and cytoskeletal disorganization similar to Alzheimers disease. In this sense, different evidence suggests that indolamines may be useful for protection against oxidative stress and cellular disorganization. In this study, we looked at the effects of melatonin, 5-hydroxytryptophan (5OHTrp), 5-hydroxytryptamine (5HT), N-acetyl-5- hydroxytryptamine (NA5OHT) or 6-hydroxymelatonin (6OHMEL) (10-5 M) on the oxidative changes and cell shape alterations produced by okadaic acid (50 nM) in N1E-115 neuroblastoma cells. The effects of okadaic acid were evaluated as changes in the quantity of lipid peroxidation products, reduced glutathione (GSH) content, activity of anti-oxidative enzymes and major length axes (as an index of cell shape changes). Okadaic acid gives rise to lipid peroxidation products (P < 0.001), GSH consumption (P < 0.001), major length axis (P < 0.001) and a reduction in the activity of GSH-transferase, GSH-reductase and catalase between 43.33 - 55.17%. These changes were completely prevented by melatonin, whereas 5OHTrp, 5HT, NA5OHT and 6OHMEL partially blocked the alterations induced by okadaic acid. In conclusion, our data indicates: (i) The importance of oxidative stress in both this experimental model and in the development and course of neurodegenerative diseases, especially Alzheimers. (ii) The protective effects of indolamines (iii) and that melatonin is much more efficient than its precursors and metabolites in reducing the extent of oxidative stress and cell shape changes induced by okadaic acid. Keywords: alzheimers disease, cell shape, okadaic acid, oxidative stress, ne neuroblastoma cells

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber–Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu 2+ ions either alone or in the presence of ascorbate (Cu 2+/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu 2+ alone displayed Cu 2+-binding, which was significantly increased in the presence of Zn 2+, Hg 2+, Cd 2+, Ag +1 and As 3+. Treatment of microsomes with Cu 2+ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu 2+ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu 2+ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu 2+ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.

Progressive iron accumulation induces a biphasic change in the glutathione content of neuroblastoma cells

Glutathione (GSH) constitutes the single most important antioxidant in neurons, whereas iron causes oxidative stress that leads to cell damage and death. Although GSH and iron produce opposite effects on redox cell status, no mechanistic relationships between iron and GSH metabolism are known. In this work, we evaluated in SH-SY5Y neuroblastoma cells the effects of iron accumulation on intracellular GSH metabolism. After 2 d exposure to increasing concentrations of iron, cells underwent concentration-dependent iron accumulation and a biphasic change in intracellular GSH levels. Increasing iron from 1 to 5 μM resulted in a marked increase in intracellular oxidative stress and increased GSH levels. Increased GSH levels were due to increased synthesis. Further increases in iron concentration led to significant reduction in both reduced (GSH) and total (GSH + (2 × GSSG)) glutathione. Cell exposure to high iron concentrations (20–80 μM) was associated with a marked decrease in the GSH/GSSG molar ratio and the GSH half-cell reduction potential. Moreover, increasing iron from 40 to 80 μM resulted in loss of cell viability. Iron loading did not change GSH reductase activity but induced significant increases in GSH peroxidase and GSH transferase activities. The changes in GSH homeostasis reported here recapitulate several of those observed in Parkinson’s disease substantia nigra. These results support a model by which progressive iron accumulation leads to a progressive decrease in GSH content and cell reduction potential, which finally results in impaired cell integrity.

Reactions of nifurtimox with critical sulfhydryl-containing biomolecules: their potential toxicological relevance.

Nifurtimox (Nfx) is a drug used in the treatment of Chagas' disease, an endemic parasitic disease from Latin American countries. It produces undesirable side-effects in patients, frequently forcing the treatment to be stopped. Its toxic mechanism is not fully understood. In this work we describe purely chemical reactions of Nfx with relevant cellular sulfhydryl (SH) compounds. The compounds tested were glutathione (GSH), cysteine (RSH), lipoic acid (LA) and coenzyme A (CoA). All reacted with Nfx to give nitrite (NO(-) (2)). The relative reaction rates were CoA>LA>GSH>RSH. In studies with GSH and RSH the formation of nitrite was accompanied by decreases in Nfx concentration and increases in the formation of a reaction product revealed by HPLC. We failed to show the presence of liver cytosolic GST (GSH transferase activity)-mediated formation of NO2- from Nfx. These NO(-) (2)-releasing processes occurred under in vivo conditions in Nfx-treated Sprague-Dawley male rats (240-260 g body weight) at a dose of 100 mg Nfx kg(-1) p.o. In urine samples NO(-) (2) excretion was accompanied by unchanged drug and two unidentified more polar metabolites detectable by HPLC. The Nfx reactions with critical SH from molecules such as GSH, RSH, LA and CoA, and potentially others containing SH residues (e.g. enzymes or structural proteins), might have toxicological relevance not only for the Nfx side-effects but also for the chemotherapeutic effects on Trypanosoma cruzi. In addition, Nfx reactions with GSH might be crucial in Nfx detoxification.

Parallel evolutionary pathways for glutathione transferases: structure and mechanism of the mitochondrial class kappa enzyme rGSTK1-1.

The class kappa glutathione (GSH) transferase is an enzyme that resides in the mitochondrial matrix. Its relationship to members of the canonical GSH transferase superfamily has remained an enigma. The three-dimensional structure of the class kappa enzyme from rat (rGSTK1-1) in complex with GSH has been solved by single isomorphous replacement with anomalous scattering at a resolution of 2.5 A. The structure reveals that the enzyme is more closely related to the protein disulfide bond isomerase, dsbA, from Escherichia coli than it is to members of the canonical superfamily. The structures of rGSTK1-1 and the canonical superfamily members indicate that the proteins folds have diverged from a common thioredoxin/glutaredoxin progenitor but did so by different mechanisms. The mitochondrial enzyme, therefore, represents a fourth protein superfamily that supports GSH transferase activity. The thioredoxin domain functions in a manner that is similar to that seen in the canonical enzymes by providing key structural elements for the recognition of GSH. The hydroxyl group of S16 is within hydrogen-bonding distance of the sulfur of bound GSH and is, in part, responsible for the ionization of the thiol in the E*GSH complex (pKa = 6.4 +/- 0.1). Preequilibrium kinetic experiments indicate that the k(on) for GSH is 1 x 10(5) M(-1) s(-1) and k(off) for GS- is approximately 8 s(-1) and relatively slow with respect to turnover with 1-chloro-2, 4-dinitrobenzene (CDNB). As a result, the KM(GSH) (11 mM) is much larger than the apparent Kd(GSH) (90 microM). The active site has a relatively open access channel that is flanked by disordered loops that may explain the relatively high turnover number (280 s(-1) at pH 7.0) toward CDNB. The disordered loops form an extensive contiguous patch on one face of the dimeric enzyme, a fact that suggests that the protein surface may interact with a membrane or other protein partner.

Protective melatonin effect on oxidative stress induced by okadaic acid into rat brain.

We studied the effect of melatonin on the oxidative changes produced by the intracerebroventricular (i.c.v.) injection of okadaic acid (200 ng/kg BW) in the Wistar rat. The effects of okadaic acid were evaluated as changes in the quantity of lipid peroxides, reduced glutathione content (GSH) and activity of antioxidative enzymes. Okadaic acid caused lipid peroxidation (5.35 +/- 0.47 micro mol/g tissue in the i.c.v. vehicle group versus 10.14 +/- 0.88 micro mol/g tissue in the okadaic acid group, P < 0.001), GSH consumption (0.115 +/- 0.0065 micro mol/g tissue in the i.c.v. vehicle group versus 0.024 +/- 0.0021 micro mol/g tissue, P < 0.001), and a reduction in the activity of GSH-peroxidase, GSH-reductase and GSH-transferase between 60-80%. All these changes were prevented by pre-injection of 4.5 mg melatonin per kg BW 2 hr before okadaic acid. These findings indicate: (i) okadaic acid induces a status of oxidative stress in the brain, characterized by a high level of lipid peroxidation, decreases in GSH content and diminished activities of antioxidative enzymes, and (ii) melatonin prevents the deleterious effects induced by okadaic acid. In conclusion, the results show the ability of melatonin to modify the neural response to okadaic acid with the protective mechanism likely involving the antioxidative processes of melatonin.

Effects of Silymarin MZ-80 on Hepatic Oxidative Stress in Rats with Biliary Obstruction

This study was designed to evaluate the effects of three pharmaceutical forms of silymarin (silymarin MZ-80, silybinin-β-cyclodextrin, and silybinin) on the liver oxidative status in vitro and after oral administration to rats with extrahepatic biliary obstruction (EBO) and sham-operated animals. We evaluated thiobarbituric acid-reactive substances (TBARS), glutathione (GSH + GSSG) and their related enzyme activities (GSH peroxidase, GSSG reductase and GSH transferase). All three compounds inhibited the in vitro production of TBARS (IC₅₀ 56–533 µmol/l). These compounds, mainly silymarin MZ-80, also increased GSH peroxidase and GSH transferase activities. In EBO rats we found increases in TBARS production which was inhibited by 50–70% after treatment. Glutathione was reduced by 55% and elevated by silymarin MZ-80. GSH transferase increased in the group given silymarin MZ-80. We conclude that all three derivatives of silymarin show a clear ability to reduce lipid peroxidation in the liver. Silymarin MZ-80 was the only compound that enhanced the glutathione antioxidant system.