Inhibition Of Glutathione Peroxidase Activity Research Articles

The combination of ascorbate and menadione causes cancer cell death by oxidative stress and replicative stress

The combination of ascorbate and menadione (VC:VK3 = 100:1) is an investigational treatment for cancer under clinical trials. Dehydroascorbic acid (DHA), the oxidized form of ascorbate, can be taken up by cells via glucose transporters, over-expressed in many cancer cells. It has been known that the combination of VC/VK3 kills cancer cells by inducing hydrogen peroxide (H2O2) via a redox cycling reaction. However, the mechanism has not been fully understood yet. Intracellularly, DHA is reduced to ascorbate by NADPH via GSH and glutaredoxin as well as by thioredoxin (Trx) and the selenoenzyme thioredoxin reductase (TrxR). These two systems are also critical as electron donors for ribonucleotide reductase (RNR), which produces deoxyribonucleotides de novo for DNA replication and DNA repair and is highly expressed in tumor cells. We found that RNR was highly sensitive to VC/VK3 in vitro with similar effects as observed with H2O2. In cancer cells, VC/VK3 inhibited RNR mainly by targeting its R2 subunit. More importantly, both the Trx and GSH systems were oxidized by the combination, which resulted in the loss of GSH, increased protein glutathionylation, and highly oxidized Trx1. The mechanism of cell death induced by VC/VK3 was also elucidated. We found that VC/VK3 inhibited glutathione peroxidase activity and led to an elevated level of lipid peroxidation, which triggered apoptosis-inducing factor (AIF) mediated cell death pathway. Therefore, the combination not only induced replicative stress by inhibiting RNR, but also oxidative stress by targeting anti-oxidant systems and triggered AIF-mediated cancer cell death.

Antioxidant compound (E)-2-benzylidene-4-phenyl-1,3-diselenole protects rats against thioacetamide-induced acute hepatotoxicity.

The aim of this study was to investigate whether (E)-2-benzylidene-4-phenyl-1,3-diselenole (BPD) protects against hepatotoxicity induced by thioacetamide (TAA). On the first day of treatment, male adult Wistar rats received BPD (10 or 50 mg·kg-1). On the second day, the rats received a single intraperitoneal injection of TAA (400 mg·kg-1). Twenty-four hours after TAA administration, biochemical determinations and liver histological analysis were carried out. BPD (50 mg·kg-1) reduced plasma aspartate and alanine aminotransferase, alkaline phosphatase, and lactate dehydrogenase activities increased by TAA exposure. Treatment with BPD was effective against increased lipid peroxidation levels and attenuated a decrease in hepatic reduced glutathione and ascorbic acid levels as well as an inhibition of glutathione peroxidase activity caused by TAA exposure. The higher dose of BPD protected against the inhibition of hepatic δ-aminolevulinic dehydratase activity induced by TAA. Finally, histopathological examination of the liver showed that BPD markedly ameliorated TAA-induced hepatic injury. In conclusion, BPD protected against hepatotoxicity and oxidative stress caused by TAA exposure in rats.

Generation of Redox Imbalance Mediates the Cytotoxic Effect of Malabaricone-A in a Multidrug Resistant Cell Line.

Multidrug resistance (MDR) refers to cross-resistance to a range of structurally and functionally unrelated compounds, and is accompanied by an elevated expression of ATP driven cell-membrane transporters. The cytotoxicity of Malabaricone-A (MAL-A), a diarylnonanoid derived from Myristica malabarica was demonstrated in leukemic cell lines, but its effectiveness in drug-resistant cancer cell lines has not been evaluated. Accordingly, this study tested its cytotoxic potential in a T-lymphoblastic leukemic cell line, CCRF CEM and its MDR counterpart, CEM/ADR5000. The effectiveness of MAL-A was 1.8 fold higher in CEM/ADR5000 than CCRF CEM cell line, the IC50 being value 5.40 ± 1.41 vs. 9.72 ± 1.08 µg/ml, respectively, suggesting that MAL-A demonstrated 'collateral sensitivity'. This cytotoxicity of MAL-A was attributed to an enhanced generation of oxidative stress, as the IC50 value increased following the addition of an anti-oxidant, N-acetyl cysteine (NAC). Furthermore, MAL-A depleted glutathione and inhibited glutathione peroxidase activity, which too contributed towards generation of a redox imbalance. This culminated in an apoptosis mediated cell death as evident by mitochondrial membrane depolarization, enhanced caspase-3 activity, increased externalization of phosphatidylserine and an increase in the sub G0/G1 population. Collectively, compounds with pro-oxidant activity have promising therapeutic potential in drug resistant phenotypes, worthy of future pharmacological consideration.

Anti-Cancer Effect of Thiacremonone through Down Regulation of Peroxiredoxin 6

Thiacremonone (2, 4-dihydroxy-2, 5-dimethyl-thiophene-3-one) is an antioxidant substance as a novel sulfur compound generated from High-Temperature-High-Pressure-treated garlic. Peroxiredoxin 6 (PRDX6) is a member of peroxidases, and has glutathione peroxidase and calcium-independent phospholipase A2 (iPLA2) activities. Several studies have demonstrated that PRDX6 stimulates lung cancer cell growth via an increase of glutathione peroxidase activity. A docking model study and pull down assay showed that thiacremonone completely fits on the active site (cys-47) of glutathione peroxidase of PRDX6 and interacts with PRDX6. Thus, we investigated whether thiacremonone inhibits cell growth by blocking glutathione peroxidase of PRDX6 in the human lung cancer cells, A549 and NCI-H460. Thiacremonone (0–50 μg/ml) inhibited lung cancer cell growth in a concentration dependent manner through induction of apoptotic cell death accompanied by induction of cleaved caspase-3, -8, -9, Bax, p21 and p53, but decrease of xIAP, cIAP and Bcl2 expression. Thiacremonone further inhibited glutathione peroxidase activity in lung cancer cells. However, the cell growth inhibitory effect of thiacremonone was not observed in the lung cancer cells transfected with mutant PRDX6 (C47S) and in the presence of dithiothreitol and glutathione. In an allograft in vivo model, thiacremonone (30 mg/kg) also inhibited tumor growth accompanied with the reduction of PRDX6 expression and glutathione peroxidase activity, but increased expression of cleaved caspase-3, -8, -9, Bax, p21 and p53. These data indicate that thiacremonone inhibits tumor growth via inhibition of glutathione peroxidase activity of PRDX6 through interaction. These data suggest that thiacremonone may have potentially beneficial effects in lung cancer.

Involvement of mGlu5 receptor in 3-nitropropionic acid-induced oxidative stress in rat striatum

Objectives:The excitotoxin 3-nitropropionic acid (3-NP) induces a suitable experimental model of Huntington’s disease (HD). This compound induces neurodegeneration via glutamatergic activation and oxidative stress, suggesting that the metabotropic glutamate receptor blockage and free radical scavenging are potential therapeutic targets in HD. In this study, we evaluated the role of 3-[(2-methyl-1,3-thiazol-4-yl) ethynyl]-pyridine (MTEP), a selective mGlu5 receptor antagonist, in a 3-NP model of HD.Methods:We administered 3-NP (20 mg/kg, intraperitoneal) to rats for 4 days. MTEP at doses of 2·5 and 5 mg/kg was administered 30 min before 3-NP. Behavioral tests and biochemical experiments were performed to assess the effects of 3-NP and the ability of MTEP to ameliorate these changes.Results:3-NP administration induced body weight loss, decreased locomotor activity, and inhibition of succinate dehydrogenase and Na+–K+ adenosine triphosphate (ATP)ase activities in rat striatum. We also observed increases in reactive species (RS) levels and glutathione reductase activity, decreased non-protein thiol levels, and an inhibition of glutathione peroxidase activity in the striatum of rats treated with 3-NP. Notably, all of these effects were attenuated by MTEP treatment.Discussion:Our results demonstrate the neuroprotective effect of MTEP and reinforce the involvement of mGluR5 in 3-NP-induced oxidative stress in rat striatum.

Organoselenium Bis Selenide Attenuates 3-Nitropropionic Acid-Induced Neurotoxicity in Rats

This study was designed to evaluate the effects of bis selenide on Huntington disease (HD)-like signs induced by 3-nitropropionic acid (3-NP) in rats. To this aim, rats were treated for 4days with bis selenide (5 or 20mg/kg/day, per oral) 30min before 3-NP (20mg/kg/day, intraperitoneally). The body weight gain, locomotor activity, motor coordination, and biochemical parameters in striatal preparations were assessed 24h after the last injection of 3-NP. The highest dose of bis selenide was effective in protecting against body weight loss and motor coordination deficit induced by 3-NP. The impairment of locomotor activity caused by 3-NP was abolished by bis selenide at both doses. Bis selenide (5 and 20mg/kg) partially restored succinate dehydrogenase activity inhibited after 3-NP exposure. The dose of 20mg/kg of bis selenide recovered partially δ-aminolevulinic acid dehydratase activity, and totally Na(+), K(+)-ATPase activity, two sulfhydryl enzymes sensitive to oxidizing agents, which had their activities inhibited by 3-NP. Also, 3-NP led to an increase in protein carbonyl levels and glutathione reductase activity and inhibited catalase activity-alterations that were reversed by bis selenide administration at both doses. The highest dose of bis selenide was effective against the increase of RS levels, the depletion of reduced glutathione content, and the inhibition of glutathione peroxidase activity induced by 3-NP. Bis selenide was not effective against inhibition of SOD activity caused by 3-NP. These findings demonstrate that bis selenide elicited protective effects against HD-like signs induced by 3-NP in rats.

Antioxidant mechanisms of iso-6-cassine in suppressing seizures induced by pilocarpine

The aim of this study was to evaluate the in vitro antioxidant effects of 12-[(2R,5R,6R)-5-hydroxy-6-methylpiperidin-2-yl]dodecan-2-one (iso-6-cassine; ISO) and the anticonvulsant effects of ISO on pilocarpine-induced seizures in rats. Wistar rats were treated with 0.9% saline (i.p., control group), pilocarpine (400 mg/kg, i.p., pilocarpine group), and the association of ISO (1.0 mg/kg, i.p.) plus pilocarpine (400 mg/kg, i.p.), 30 min after administration of ISO (ISO plus pilocarpine group). After the treatments all groups were observed for 1h. The antioxidant effect of ISO on the pilocarpine model was assessed by determining the activity of glutathione peroxidase (GPx), glutathione-S-transferase (GST) and catalase (CAT) as well as the levels of reactive species (RS) and lipid peroxidation (LP). In vitro, ISO (5 μM) reduced RS and LP. ISO (1.0 mg/kg) and abolished seizures and death induced by pilocarpine in rats. ISO protected against the increase in the RS and LP levels, GST activity as well as the inhibition of GPx activity caused by pilocarpine. In addition, ISO increased the catalase activity in hippocampus of seized rats. In conclusion, the dta suggest that ISO can present anticonvulsant and antioxidant properties in the pilocarpine model of seizures in rats.

Effects of olive leaf polyphenols against H₂O₂ toxicity in insulin secreting β-cells.

In pancreatic β-cells, although H₂O₂ is a metabolic signal for glucose stimulated insulin secretion, it may induce injury in the presence of increased oxidative stress (OS) as in the case of diabetic chronic hyperglycemia. Olea europea L. (olive) leaves contain polyphenolic compounds that may protect insulin-secreting cells against OS. The major polyphenolic compound in ethanolic olive leaf extract (OLE) is oleuropein (about 20%), thus we compared the effects of OLE with the effects of standard oleuropein on INS-1 cells. The cells were incubated with increasing concentrations of OLE or oleuropein for 24 h followed by exposure to H₂O₂ (0.035 mM) for 45 min. H₂O₂ alone resulted in a significantly decreased viability (MTT assay), depressed glucose-stimulated insulin secretion, increased apoptotic and necrotic cell death (AO/EB staining), inhibited glutathione peroxidase activity (GPx) and stimulated catalase activity that were associated with increased intracellular generation of reactive oxygen species (ROS) (fluorescence DCF). OLE and oleuropein partly improved the viability, attenuated necrotic and apoptotic death, inhibited the ROS generation and improved insulin secretion in H₂O₂-exposed cells. The effects of oleuropein on insulin secretion were more pronounced than those of OLE, while OLE exerted a stronger anti-cytotoxic effect than oleuropein. Unlike OLE, oleuropein had no significant preserving effect on GPx; however, both compounds stimulated the activity of catalase in H₂O₂-exposed cells. These findings indicate different modulatory roles of polyphenolic constituents of olive leaves on redox homeostasis that may have a role in the maintenance of β-cell physiology against OS.

Neurochemical Evidence that Lysine Inhibits Synaptic Na+,K+-ATPase Activity and Provokes Oxidative Damage in Striatum of Young Rats In vivo

Lysine (Lys) accumulation in tissues and biological fluids is the biochemical hallmark of patients affected by familial hyperlysinemia (FH) and other inherited metabolic disorders. In the present study we investigated the effects of acute administration of Lys on relevant parameters of energy metabolism and oxidative stress in striatum of young rats. We verified that Lys in vivo intrastriatal injection did not change the citric acid cycle function and creatine kinase activity, but, in contrast, significantly inhibited synaptic Na(+),K(+)-ATPase activity in striatum prepared 2 and 12 h after injection. Moreover, Lys induced lipid peroxidation and diminished the concentrations of glutathione 2 h after injection. These effects were prevented by the antioxidant scavengers melatonin and the combination of α-tocopherol and ascorbic acid. Lys also inhibited glutathione peroxidase activity 12 h after injection. Therefore it is assumed that inhibition of synaptic Na(+),K(+)-ATPase and oxidative damage caused by brain Lys accumulation may possibly contribute to the neurological manifestations of FH and other neurometabolic conditions with high concentrations of this amino acid.

Vitamin C, resveratrol and lipoic acid actions on isolated rat liver mitochondria: all antioxidants but different

Modulating mitochondrial antioxidant status is a nutritional issue of great interest in the treatment or prevention of several oxidative stress related diseases such as obesity. Thus, the aim of the present study was to analyze the effects of three antioxidants on hepatic mitochondrial function and antioxidant status. Isolated rat liver mitochondria were incubated with vitamin C, resveratrol and lipoic acid. The activity of antioxidant enzymes (manganese superoxide dismutase and glutathione peroxidase), ROS generation and respiratory parameters (RCR, P/O ratio and respiratory states) were measured. Vitamin C influenced mitochondrial function by decreasing of ROS generation (P < 0.0001), by stimulating the activity of manganese superoxide dismutase (197.60 ± 35.99%; P < 0.001) as well as glutathione peroxidase (15.70 ± 5.76%; P < 0.05) and by altering the activity of the electron transport chain, mainly by decreasing the P/O ratio (P < 0.05). Resveratrol induced a significant increase in manganese superoxide dismutase activity (160 ± 11.78%; P < 0.0001) and a decrease in ROS generation (P < 0.05 to P < 0.0001). By contrast, lipoic acid inhibited glutathione peroxidase activity (16.48 ± 3.27%; P < 0.05) and induced the uncoupling of the electron transport chain (P < 0.01). Moreover, this antioxidant induced a strong decrease in the P/O ratio (P < 0.05 to P < 0.0001). In conclusion, our results suggest that the three tested antioxidants produced direct effects on mitochondrial function, although the magnitude and intensity of these actions were significantly different, which may have implications when administrated as antioxidants.

Dysregulated glutathione metabolism links to impaired insulin action in adipocytes

Oxidative stress plays an important role in obesity-related metabolic diseases. Glutathione peroxidase (GPX) is an antioxidant enzyme downregulated in adipose tissue of obese mice. However, the role of GPX in adipocytes remains elusive. The objective of this study was to clarify the pathophysiological changes in GPX activity and glutathione metabolism and their roles in the pathogenesis of insulin resistance in adipocytes. To achieve this goal, we measured cellular GPX activity, glutathione (GSH) contents, GSH/GSSG ratio, and mRNA expression of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme for de novo GSH synthesis, in adipose tissue of control and ob/ob mice and in 3T3-L1 adipocytes treated with insulin, H(2)O(2), free fatty acid (FFA), or TNFalpha. Furthermore, we investigated the effects of GPX inhibition with a specific GPX inhibitor or RNA interference against GPX, H(2)O(2), and reduced GSH on insulin signaling in 3T3-L1 adipocytes. ob/ob Mice showed not only a decrease in cellular activity of GPXs (GPX1, -4, and -7) but also an increase in gamma-GCS expression, resulting in increased GSH contents in adipose tissue. These alterations in glutathione metabolism were also observed during differentiation of 3T3-L1 cells and their exposure to insulin, FFA, or H(2)O(2). Inhibition of GPX activity, addition of GSH, and H(2)O(2) resulted in impaired insulin signaling in 3T3-L1 adipocytes. These results suggest that decreased GPX activity and increased gamma-GCS expression lead to overaccumulation of GSH, which might be involved in the pathogenesis of insulin resistance in obesity.

Characterization of UPF peptides, members of the glutathione analogues library, on the basis of their effects on oxidative stress-related enzymes

Previously the authors have designed and synthesized a library of antioxidative glutathione analogues called UPF peptides which are superior to glutathione in hydroxyl radical elimination. This paper is a follow-up study which investigated the effects of the most promising members of the library (UPF1 and UPF17) on oxidative stress-related enzymes. At concentrations used in vivo experiments neither UPF peptide influenced the activity of glutathione peroxidase (GPx) when purified enzyme or erythrocyte lysate was used. At higher concentrations they inhibited GPx activity. UPF peptides had no effect on glutathione reductase (GR) activity. Also they, as well as glutathione itself, slightly increased MnSOD activity in human brain mitochondria and inhibited oxidative burst caused by neutrophil NAD(P)H oxidase. RT-PCR measurements showed that UPF1 and UPF17 have no effect on GPx and MnSOD expression level in human blood mononuclear cells. The results of this study confirm that investigated UPF peptides do not interfere with the enzymatic mechanisms of antioxidative defence and can be used as themselves or as a lead for the protector molecule design against excessive oxidative stress.

Tanshinone IIA reduces macrophage death induced by hydrogen peroxide by upregulating glutathione peroxidase

Aims Tanshinone IIA is an important ingredient in the herb danshen ( Salvia miltiorrhiza), which has been used to treat cardiovascular diseases such as atherosclerosis and angina for hundreds of years in China. There are numerous reports that TIIA has anti-oxidant properties but the chemical structure indicates that TIIA is fully oxidized. Here, we test the hypothesis that TIIA alters the expression and/or activity of specific anti-oxidation enzymes to protect cells from oxidant damage. Main methods We utilized J774 macrophages to model cellular responses to TIIA when challenged with H 2O 2. Expression and activity levels of several anti-oxidation enzymes were investigated and the only system modulated by TIIA was glutathione peroxidase (GPx). Key findings GPx-1 mRNA levels were significantly increased by TIIA but not the vitamin E analogue, Trolox. GPx activities were also significantly increased by TIIA. Mercaptosuccinic acid inhibited GPx activity and the protective effect of TIIA was attenuated. Thus, TIIA protects cultured macrophages from H 2O 2-induced cell death and protection is mediated in large part by TIIA induction of GPx gene expression and activity. Significance Because of the importance of GPx in health and because TIIA is able to modulate GPx activity to some extent in cell culture, we suggest that TIIA is a worthwhile candidate for further study in animal models of atherosclerosis and eventually in human prospective trials.

CR-6 protects glutathione peroxidase activity in experimental diabetes

Antioxidants can be useful as a supportive therapy in diabetes, and we try to elucidate some of the mechanisms by which these compounds are able to protect from diabetic complications. For this purpose we have assayed, in vitro and in vivo, the ability of CR-6 (3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2 H)-benzopyran), an antioxidant able to scavenge nitrogen reactive species, to protect glutathione peroxidase (GPx) activity. Glucose, in vitro, inhibited GPx activity in a concentration-dependent manner; CR-6 was able to protect GPx activity from glucose-induced inactivation. Alloxan-induced experimental diabetes in mice promoted oxidative stress in the retina and hippocampus, after 3 weeks of hyperglycemia. CR-6 administration prevented not only the alterations of oxidative stress markers (tissue GSH and malondialdehyde (MDA) concentration and GPx activity) but also the impairment of retinal function (as assessed by the modifications in electroretinogram b-wave amplitude). The findings herein confirm the role of nitrogen reactive species in diabetes; therefore, antioxidants effectively quenching these species, such as CR-6, should be considered for the adjuvant treatment of diabetes.

Antioxidant responses to benzo[a]pyrene, tributyltin and their mixture in the spleen of Sebasticus marmoratus

It has been reported that there is an interaction between Benzo[a]pyrene (BaP), a widespread carcinogenic polycyclic aromatic hydrocarbon, and tributyltin (TBT), an organometal used as an antifouling biocide. This study was therefore designed to examine the potential in vivo influence of BaP, TBT and their mixture on splenic antioxidant defense systems of Sebastiscus marmoratus. The fish were exposed to water containing environmentally relevant concentrations of BaP, TBT and their mixture. Spleens were collected for biochemical analysis after exposure for 7, 25, 50 d and after recovery for 7, 20 d. Cotreatment with BaP and TBT for 7 d potentiated the induction of glutathione peroxidase (GPx) activity by BaP or TBT alone. The cotreatment for 25 and 50 d resulted in inhibition of GPx activity, which was similar to the effect of TBT. Splenic glutathione S-transferase (GST) activities were significantly elevated in S. marmoratus exposed to BaP starting from 7 d and remained high up to 25 d. However, no further activity change was found with prolonged exposure. Cotreatment of BaP and TBT primarily inhibited the GST activity, which was similar to the effect of TBT. Cotreatment with BaP and TBT for 25 or 50 d potentiated the depletion of GSH (glutathione) by BaP or TBT alone. MDA (malondialdehyde) contents in spleen of S. marmoratus were not significantly altered compared with the control during the test period. Spleen, as an immune organ, is sensitive to exposure of BaP or TBT. It should have an effective mechanism to counteract oxidative damage. Antioxidative defense systems in spleen of S. marmoratus should be considered as potential biomarkers. Short-term exposure of BaP or TBT could result in induction of antioxidant defense system. A significant decrease of these indices, such as GSH, GST, GPx might indicate more severe contamination.

Expression and regulation of antioxidant enzymes in the developing limb support a function of ROS in interdigital cell death

Vertebrate limb development is a well-studied model of apoptosis; however, little is known about the intracellular molecules involved in activating the cell death machinery. We have shown that high levels of reactive oxygen species (ROS) are present in the interdigital ‘necrotic’ tissue of mouse autopod, and that antioxidants can reduce cell death. Here, we determined the expression pattern of several antioxidant enzymes in order to establish their role in defining the areas with high ROS levels. We found that the genes encoding the superoxide dismutases and catalase are expressed in autopod, but they are downregulated in the interdigital regions at the time ROS levels increased and cell death was first detected. The possible role of superoxide and/or peroxide in activating cell death is supported by the protective effect of a superoxide dismutase/catalase mimetic. Interestingly, we found that peroxidase activity and glutathione peroxidase-4 gene ( Gpx4) expression were restricted to the non-apoptotic tissue (e.g., digits) of the developing autopod. Induction of cell death with retinoic acid caused an increase in ROS and decrease in peroxidase activity. Even more inhibition of glutathione peroxidase activity leads to cell death in the digits, suggesting that a decrease in antioxidant activity, likely due to Gpx4, caused an increase in ROS levels, thus triggering apoptosis.

The Bipyridyl Herbicide Paraquat Produces Oxidative Stress-Mediated Toxicity in Human Neuroblastoma SH-SY5Y Cells: Relevance to the Dopaminergic Pathogenesis

Paraquat (PQ) is a cationic nonselective bipyridyl herbicide widely used to control weeds and grasses in agriculture. Epidemiologic studies indicate that exposure to pesticides can be a risk factor in the incidence of Parkinson's disease (PD). A strong correlation has been reported between exposure to paraquat and PD incidence in Canada, Taiwan, and the United States. This correlation is supported by animal studies showing that paraquat produces toxicity in dopaminergic neurons of the rat and mouse brain. However, it is unclear how paraquat triggers toxicity in dopaminergic neurons. Based on the prooxidant properties of paraquat, it was hypothesized that paraquat may induce oxidative stress-mediated toxicity in dopaminergic neurons. To explore this possibility, dopaminergic SH-SY5Y cells were treated with paraquat, and several biomarkers of oxidativestress were measured. First, a specific dopamine transporter inhibitor GBR12909 significantly protected SY5Y cells against the toxicity of paraquat, indicating that paraquat exerts its toxicity by a mechanism involving the dopamine transporter (DAT). Second, paraquat increased intracellular levels of reactive oxygen species (ROS), but decreased the levels of glutathione. Third, paraquat inhibited glutathione peroxidase activity, but did not affect glutathione reductase activity. On the other hand, paraquat increased GST activity by 24 h, after which GST activity returned to the control value at 48 h. Fourth, paraquat dissipated mitochondrial transmembrane potential (MTP). Fifth, paraquat produced increases of malondialdehyde (MDA) and protein carbonyls, as well as DNA fragmentation, indicating oxidative damage to major cellular components. Sixth, paraquat increased the protein level of heme oxygenase-1 (HO-1). Taken together, these findings verify our hypothesis that paraquat produces oxidative stress-mediated toxicity in SH-SY5Y cells. Thus, current findings suggest that paraquat may induce the pathogenesis of dopaminergic neurons through oxidative stress.

α-Keto Acids Accumulating in Maple Syrup Urine Disease Stimulate Lipid Peroxidation and Reduce Antioxidant Defences in Cerebral Cortex From Young Rats

Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder caused by deficiency of branched-chain alpha-keto acid dehydrogenase complex activity which leads to tissue accumulation of the branched-chain alpha-keto acids (BCKAs) alpha-ketoisocaproic acid (KIC), alpha-ketoisovaleric acid (KIV) and alpha-keto-beta-methylvaleric acid (KMV) and their respective amino acids. Neuropathologic findings characteristic of the disease are cerebral edema and atrophy, whose pathophysiology is poorly known. In the present study, we investigated the in vitro effect of BCKAs on various parameters of oxidative stress, namely chemiluminescence (CL), thiobarbituric acid-reactive substances (TBA-RS), total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), and the activities of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (SOD) in cerebral cortex of 30-day-old rats. The major effects observed were with KIC, which significantly increased CL and TBA-RS measurements, decreased TRAP and TAR values, and markedly inhibited GPx activity. KMV and KIV increased CL and decreased TRAP and TAR values. In contrast, these compounds did not affect CAT and SOD activities. Taken together, it was shown that: the BCKAs studied stimulated lipid peroxidation and reduced the brain antioxidant defences, suggesting an increased production of free radicals. In case the in vitro effects here detected also occur in vivo in MSUD, it can be presumed that oxidative stress might contribute, at least in part, to the brain damage found in the affected patients.

Aluminium increases xanthine oxidase activity and disturbs antioxidant status in the rat

The mechanisms responsible for the neurotoxic effects of Al remain poorly understood. In order to determine whether Al promotes oxidative stress in vivo, we measured the enzymatic activity of xanthine oxidase (XO), superoxide dismutase (SOD), glutathione peroxidase (GPX), glutathione-S-transferase (GST) and glutathione reductase (GR) in four groups of rats after eight days of intraperitoneal administration of variable concentrations of Al (0, 5, 10, and 15 mg/kg body weight, respectively). XO activity was measured in both plasma and liver samples, and the activities of the remaining enzymes were further determined in the brain and red blood cells (RBC). The most significant changes were observed in XO and GPX activities, that were enhanced and depressed, respectively. In both instances, the enzyme activities were correlated with Al concentrations, either positively (XO) or negatively (GPX). Enhancement of XO and inhibition of GPX activity may lead to the accumulation of intermediate toxic compounds such as hydrogen peroxide and hydroxyl radicals, since SOD activity is increased as well. The latter finding must be taken with some caution because previous studies have shown contradictory results in this field. Our data suggest that Al toxicity could be mediated by its action on both pro- and anti-oxidant enzymes. The biological significance of these findings remains to be established.

α-Melanocyte-stimulating Hormone Reduces Impact of Proinflammatory Cytokine and Peroxide-generated Oxidative Stress on Keratinocyte and Melanoma Cell Lines

We have previously shown that alpha-melanocyte-stimulating hormone (alpha-MSH) can oppose tumor necrosis factor alpha activation of NF-kappaB (1-2 h) and intercellular adhesion molecule 1 up-regulation (mRNA by 3 h and protein by 24 h) in melanocytes and melanoma cells. The present study reports on the ability of four MSH peptides to control intracellular peroxide levels and glutathione peroxidase (GPx) activity in pigmentary and nonpigmentary cells. In human HBL melanoma and HaCaT keratinocytes tumor necrosis factor alpha and H(2)O(2) both activated GPx in a time- and concentration-dependent manner (by 30-45 min). alpha-MSH peptides were found to inhibit the stimulated GPx activity and had biphasic dose-response curves. MSH 1-13 and MSH [Nle(4)-d-Phe(7)] achieved maximum inhibition at 10(-10) and 10(-12) m, respectively. Higher concentrations (10-100 fold) of MSH 4-10 and MSH 11-13 were required to produce equivalent levels of inhibition. alpha-MSH was also capable of reducing peroxide accumulation within 15 min, and again this inhibition was biphasic. The data support a role of alpha-MSH in acute protection of cells to oxidative/cytokine action that precedes NF-kappaB and GPx activation. The rapidity and potency of the response to alpha-MSH in pigmentary and nonpigmentary cells suggest this to be a central role of this peptide in cutaneous cells.