Oxidation of structural cysteine residues in thioredoxin 1 by aromatic arsenicals enhances cancer cell cytotoxicity caused by the inhibition of thioredoxin reductase 1

Thioredoxin systems, composed of thioredoxin reductase (TrxR), thioredoxin (Trx) and NADPH, play important roles in maintaining cellular redox homeostasis and redox signaling. Recently the cytosolic Trx1 system has been shown to be a cellular target of arsenic containing compounds. To elucidate the relationship of the structure of arsenic compounds with their ability of inhibiting TrxR1 and Trx1, and cytotoxicity, we have investigated the reaction of Trx1 system with seven arsenic trithiolates: As(Cys)₃, As(GS)₃, As(Penicillamine)₃, As(Mercaptoethanesulfonate)₃, As(Mercaptopurine)₃, As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃. The cytotoxicity of these arsenicals was consistent with their ability to inhibit TrxR1 in vitro and in cells. Unlike other arsenicals, As(Mercaptopurine)₃ which did not show inhibitory effects on TrxR1 had very weak cytotoxicity, indicating that TrxR1 is a reliable drug target for arsenicals. Moreover, the two aromatic compounds As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃ showed stronger cytotoxicity than the others. As(2-mercaptopyridine)₃ which selectively oxidized two structural cysteines (Cys62 and Cys69) in Trx1 showed mild improvement in cytotoxicity. As(2-mercaptopyridine N-oxide)₃ oxidized all the Cys residues in Trx1, exhibiting the strongest cytotoxicity. Oxidation of Trx1 by As(2-mercaptopyridine)₃ and As(2-mercaptopyridine N-oxide)₃ affected electron transfer from NADPH and TrxR1 to peroxiredoxin 1 (Prx1), which could result in the reactive oxygen species elevation and trigger cell death process. These results suggest that oxidation of structural cysteine residues in Trx1 by aromatic group in TrxR1-targeting drugs may sensitize tumor cells to cell death, providing a novel approach to regulate cellular redox signaling and also a basis for rational design of new anticancer agents.     

Shikonin targets cytosolic thioredoxin reductase to induce ROS-mediated apoptosis in human promyelocytic leukemia HL-60 cells

Shikonin, a major active component of the Chinese herbal plant Lithospermum erythrorhizon, has been applied for centuries in traditional Chinese medicine. Although shikonin demonstrates potent anticancer efficacy in numerous types of human cancer cells, the cellular targets of shikonin have not been fully defined. We report here that shikonin may interact with the cytosolic thioredoxin reductase (TrxR1), an important selenocysteine (Sec)-containing antioxidant enzyme with a C-terminal -Gly-Cys-Sec-Gly active site, to induce reactive oxygen species (ROS)-mediated apoptosis in human promyelocytic leukemia HL-60 cells. Shikonin primarily targets the Sec residue in TrxR1 to inhibit its physiological function, but further shifts the enzyme to an NADPH oxidase to generate superoxide anions, which leads to accumulation of ROS and collapse of the intracellular redox balance. Importantly, overexpression of functional TrxR1 attenuates the cytotoxicity of shikonin, whereas knockdown of TrxR1 sensitizes cells to shikonin treatment. Targeting TrxR1 with shikonin thus discloses a previously unrecognized mechanism underlying the biological activity of shikonin and provides an in-depth insight into the action of shikonin in the treatment of cancer.     

Hyperthermia in combination with oxidative stress induces autophagic cell death in HT-29 colon cancer cells

The purpose of this study was to evaluate the mechanism of ROS-induced hyperthermic cell death in a colon cancer cell line. HT-29 colon cancer cells were exposed to heat (43 degrees C) in the presence of tert-butyl hydroperoxide (t-BOOH). t-BOOH combined with hyperthermia significantly decreased cell viability as compared with t-BOOH or hyperthermia alone. This decrease in cell numbers was associated with retardation in the S phase transit and not through apoptosis. Cell death was noted to be accompanied by specific features characteristic of autophagy: the presence of cytoplasmic autophagic vacuoles; autophagosome membrane association of microtubule-associated protein light chain 3; accumulation of acidic vesicular organelles; and increased incorporation of MDC in the autophagosome. Thermal sensitization through modulation of cellular ROS may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.     

The effects of hexavalent chromium on thioredoxin reductase and peroxiredoxins in human bronchial epithelial cells

Inhalational exposure to hexavalent chromium (Cr(VI)) compounds (e.g., chromates) is of concern in many Cr-related industries and their surrounding environments. The bronchial epithelium is directly exposed to inhaled Cr(VI). Cr(VI) species gain easy access inside cells, where they are reduced to reactive Cr species, which may also contribute to the generation of reactive oxygen species. The thioredoxin (Trx) system promotes cell survival and has a major role in maintaining intracellular thiol redox balance. Previous studies with normal human bronchial epithelial cells (BEAS-2B) demonstrated that chromates cause dose- and time-dependent oxidation of Trx1 and Trx2. The Trx’s keep many intracellular proteins reduced, including the peroxiredoxins (Prx’s). Prx1 (cytosolic) and Prx3 (mitochondrial) were oxidized by Cr(VI) treatments that oxidized all, or nearly all, of the respective Trx’s. Prx oxidation is therefore probably the result of a lack of reducing equivalents from Trx. Trx reductases (TrxR’s) keep the Trx’s largely in the reduced state. Cr(VI) caused pronounced inhibition of TrxR, but the levels of TrxR protein remained unchanged. The inhibition of TrxR was not reversed by removal of residual Cr(VI) or by NADPH, the endogenous electron donor for TrxR. In contrast, the oxidation of Trx1, Trx2, and Prx3 was reversible by disulfide reductants. Prolonged inhibition of TrxR in Cr(VI)-treated cells might contribute to the sustained oxidation of Trx’s and Prx’s. Reduced Trx binds to an N-terminal domain of apoptosis signaling kinase (ASK1), keeping ASK1 inactive. Cr(VI) treatments that significantly oxidized Trx1 resulted in pronounced dissociation of Trx1 from ASK1. Overall, the effects of Cr(VI) on the redox state and function of the Trx’s, Prx’s, and TrxR in the bronchial epithelium could have important implications for redox-sensitive cell signaling and tolerance of oxidant insults.     

Oxidation of glutathione and cysteine in human plasma associated with smoking

Cigarette smoking contributes to the development or progression of numerous chronic and age-related disease processes, but detailed mechanisms remain elusive. In the present study, we examined the redox states of the GSH/GSSG and Cys/CySS couples in plasma of smokers and nonsmokers between the ages of 44 and 85 years (n = 78 nonsmokers, n = 43 smokers). The Cys/CySS redox in smokers (−64 ± 16 mV) was more oxidized than nonsmokers (− 76 ± 11 mV; p < .001), with decreased Cys in smokers (9 ± 5 μM) compared to nonsmokers (13 ± 6 μM; p < .001). The GSH/GSSG redox was also more oxidized in smokers (−128 ± 18 mV) than in nonsmokers (−137 ± 17 mV; p = .01) and GSH was lower in smokers (1.8 ± 1.3 μM) than in nonsmokers (2.4 ± 1.0; p < .005). Although the oxidation of GSH/GSSG can be explained by the role of GSH in detoxification of reactive species in smoke, the more extensive oxidation of the Cys pool shows that smoking has additional effects on sulfur amino acid metabolism. Cys availability and Cys/CySS redox are known to affect cell proliferation, immune function, and expression of death receptor systems for apoptosis, suggesting that oxidation of Cys/CySS redox or other perturbations of cysteine metabolism may have a key role in chronic diseases associated with cigarette smoking.     

Vitamin B12 protects against superoxide-induced cell injury in human aortic endothelial cells

Superoxide (O₂^•−) is implicated in inflammatory states including arteriosclerosis and ischemia-reperfusion injury. Cobalamin (Cbl) supplementation is beneficial for treating many inflammatory diseases and also provides protection in oxidative-stress-associated pathologies. Reduced Cbl reacts with O₂^•− at rates approaching that of superoxide dismutase (SOD), suggesting a plausible mechanism for its anti-inflammatory properties. Elevated homocysteine (Hcy) is an independent risk factor for cardiovascular disease and endothelial dysfunction. Hcy increases O₂^•− levels in human aortic endothelial cells (HAEC). Here, we explore the protective effects of Cbl in HAEC exposed to various O₂^•− sources, including increased Hcy levels. Hcy increased O₂^•− levels (1.6-fold) in HAEC, concomitant with a 20% reduction in cell viability and a 1.5-fold increase in apoptotic death. Pretreatment of HAEC with physiologically relevant concentrations of cyanocobalamin (CNCbl) (10-50 nM) prevented Hcy-induced increases in O₂^•− and cell death. CNCbl inhibited both Hcy and rotenone-induced mitochondrial O₂^•− production. Similarly, HAEC challenged with paraquat showed a 1.5-fold increase in O₂^•− levels and a 30% decrease in cell viability, both of which were prevented with CNCbl pretreatment. CNCbl also attenuated elevated O₂^•− levels after exposure of cells to a Cu/Zn-SOD inhibitor. Our data suggest that Cbl acts as an efficient intracellular O₂^•− scavenger.     

Exercise training combined with antioxidant supplementation prevents the antiproliferative activity of their single treatment in prostate cancer through inhibition of redox adaptation

In preclinical models, exercise training (ET) or pomegranate juice (PJ) prevents prostate cancer progression. Here, we hypothesized that physical exercise combined with antioxidants could induce synergistic effects through oxidative stress modulation. Forty male Copenhagen rats with prostate tumors were divided into four groups: control, PJ, ET, and PJ+ET. Rats from the PJ group consumed 750 µl of PJ daily, rats from the ET group ran on a treadmill 5 days per week, and PJ+ET rats received the combined treatment. Each week, tumor growth was evaluated. After 4 weeks of treatment, the rats were euthanized and blood, muscles, and tumors were collected. Tumor Ki67, extracellular signal-regulated kinase (ERK) activation, Bcl-2 expression, and enzymatic and nonenzymatic antioxidant defenses, as well as oxidative stress markers (oxidized base, lipid peroxidation, protein carbonylation), were measured. PJ or ET significantly decreased prostate tumor proliferation (Ki67 staining, p<0.05) through the modulation of ERK phosphorylation, whereas the combination of treatments did not limit cancer progression. PJ significantly reduced Bcl-2 expression in tumors (p<0.05) and the combination of PJ and ET prevented this effect. PJ or ET increased enzymatic antioxidant defenses in muscle, PJ increased nonenzymatic antioxidant defenses in plasma and whole blood. In addition, PJ reduced TBARS and 8-oxodGuo levels in tumors as well as ET (p<0.05), whereas protein carbonyl levels were not affected by these two strategies. Paradoxically, association of PJ+ET did not increase antioxidant defenses and no reduction in oxidative stress markers was induced. Loading cancer cells with antioxidants blunts the positive effects of ET and interferes with important reactive oxygen species-mediated physiological processes such as antioxidant adaptations.     

Roundup disrupts male reproductive functions by triggering calcium-mediated cell death in rat testis and Sertoli cells

Glyphosate is the primary active constituent of the commercial pesticide Roundup. The present results show that acute Roundup exposure at low doses (36 ppm, 0.036 g/L) for 30 min induces oxidative stress and activates multiple stress-response pathways leading to Sertoli cell death in prepubertal rat testis. The pesticide increased intracellular Ca²⁺ concentration by opening L-type voltage-dependent Ca²⁺ channels as well as endoplasmic reticulum IP₃ and ryanodine receptors, leading to Ca²⁺ overload within the cells, which set off oxidative stress and necrotic cell death. Similarly, 30 min incubation of testis with glyphosate alone (36 ppm) also increased ⁴⁵Ca²⁺ uptake. These events were prevented by the antioxidants Trolox and ascorbic acid. Activated protein kinase C, phosphatidylinositol 3-kinase, and the mitogen-activated protein kinases such as ERK1/2 and p38MAPK play a role in eliciting Ca²⁺ influx and cell death. Roundup decreased the levels of reduced glutathione (GSH) and increased the amounts of thiobarbituric acid-reactive species (TBARS) and protein carbonyls. Also, exposure to glyphosate–Roundup stimulated the activity of glutathione peroxidase, glutathione reductase, glutathione S-transferase, γ-glutamyltransferase, catalase, superoxide dismutase, and glucose-6-phosphate dehydrogenase, supporting downregulated GSH levels. Glyphosate has been described as an endocrine disruptor affecting the male reproductive system; however, the molecular basis of its toxicity remains to be clarified. We propose that Roundup toxicity, implicated in Ca²⁺ overload, cell signaling misregulation, stress response of the endoplasmic reticulum, and/or depleted antioxidant defenses, could contribute to Sertoli cell disruption in spermatogenesis that could have an impact on male fertility.     

Formation of DNA-protein cross-links in cultured mammalian cells upon treatment with iron ions

Formation of DNA-protein crosslinks (DPCs) in mammalian cells upon treatment with iron or copper ions was investigated. Cultured murine hybridoma cells were treated with Fe(II) or Cu(II) ions by addition to the culture medium at various concentrations. Subsequently, chromatin samples were isolated from treated and control cells. Analyses of chromatin samples by gas chromatography/mass spectrometry after hydrolysis and derivatization revealed a significant increase over the background amount of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)-methyl]- -tyrosine (Thy-Tyr crosslink) in cells treated with Fe(II) ions in the concentration range of 0.01 to 1 mM. In contrast, Cu(II) ions at the same concentrations did not produce this DPC in cells. No DNA base damage was observed in cells treated with Cu(II) ions, either. Preincubation of cells with ascorbic acid or coincubation with dimethyl sulfoxide did not significantly alleviate the Fe(II) ion-mediated formation of DPCs. In addition, a modified fluorometric analysis of DNA unwinding assay was used to detect DPCs formed in cells. Fe(II) ions caused significant formation of DPCs, but Cu(II) ions did not. The nature of the Fe(II)-mediated DPCs suggests the involvement of the hydroxyl radical in their formation. The Thy-Tyr crosslink may contribute to pathological processes associated with free radical reactions.     

Mechanism of cell death induced by an antioxidant extract of Cratoxylum cochinchinense (YCT) in Jurkat T cells: the role of reactive oxygen species and calcium

YCT is a semipurified extract from Cratoxylum cochinchinense that has antioxidant properties and contains mostly mangiferin. We show here that YCT is selectively toxic to certain cell types and investigate the mechanisms of this toxicity in Jurkat T cells. By flow cytometric analyses, we show that YCT causes intense oxidative stress and a rise in cytosolic Ca²⁺. This is followed by a rise in mitochondrial Ca²⁺, release of cytochrome c, collapse of Δψ_m, a fall in ATP levels, and eventually cell death. The mechanism(s) of intense oxidative stress may involve a plasma membrane redox system, as cell death is inhibited by potassium ferricyanide. Cell death has some features of apoptosis (propidium iodide staining, externalization of phosphatidylserine, limited caspase-3 and -9 activities), but there was no internucleosomal DNA fragmentation.     

Biochemical changes accompanying apoptotic cell death in retinoblastoma cancer cells treated with lipogenic enzyme inhibitors

Retinoblastoma (RB) is a malignant intra-ocular neoplasm that affects children (usually below the age of 5 years). In addition to conventional chemotherapy, novel therapeutic strategies that target metabolic pathways such as glycolysis and lipid metabolism are emerging. Fatty acid synthase (FASN), a lipogenic multi-enzyme complex, is over-expressed in retinoblastoma cancer. The present study evaluated the biochemical basis of FASN inhibition induced apoptosis in cultured Y79 RB cells. FASN inhibitors (cerulenin, triclosan and orlistat) significantly inhibited FASN enzyme activity (P < 0.05) in Y79 RB cells. This was accompanied by a decrease in palmitate synthesis (end-product depletion), and increased malonyl CoA levels (substrate accumulation). Differential lipid profile was biochemically estimated in neoplastic (Y79 RB) and non-neoplastic (3T3) cells subjected to FASN inhibition. The relative proportion of phosphatidyl choline to neutral lipids (triglyceride + total cholesterol) in Y79 RB cancer cells was found to be higher than the non-neoplastic cells, indicative of altered lipid distribution and utilization in tumor cells. FASN inhibitor treated Y79 RB and fibroblast cells showed decrease in the cellular lipids (triglyceride, cholesterol and phosphatidyl choline) levels. Apoptotic DNA damage induced by FASN inhibitors was accompanied by enhanced lipid peroxidation.     

Involvement of an aldo-keto reductase (AKR1C3) in redox cycling of 9,10-phenanthrenequinone leading to apoptosis in human endothelial cells

9,10-Phenanthrenequinone (9,10-PQ), a major quinone found in diesel exhaust particles, is considered to generate reactive oxygen species (ROS) through its redox cycling. Here, we show that 9,10-PQ evokes apoptosis in human aortic endothelial cells (HAECs) and its apoptotic signaling includes ROS generation and caspase activation. The 9,10-PQ-induced cytotoxicity was inhibited by ROS scavengers, indicating that intracellular ROS generation is responsible for the 9,10-PQ-induced apoptosis. Comparison of mRNA expression levels and kinetic constants in the 9,10-PQ reduction among 10 human reductases suggests that aldo-keto reductase 1C3 (AKR1C3) is a 9,10-PQ reductase in HAECs. In in vitro 9,10-PQ reduction by AKR1C3, the reduced product 9,10-dihydroxyphenanthrene and superoxide anions were formed, suggesting the enzymatic two-electron reduction of 9,10-PQ that thereby causes oxidative stress through its redox cycling. In addition, the participation of AKR1C3 in 9,10-PQ-redox cycling was confirmed by the data that AKR1C3 overexpression in endothelial cells augmented the ROS generation and cytotoxicity by 9,10-PQ, and the ROS scavengers inhibited the toxic effects. Pretreatment of the overexpressing cells with AKR1C3 inhibitors, flufenamic acid and indomethacin, suppressed the 9,10-PQ-induced GSH depletion. These results suggest that AKR1C3 is a key enzyme in the initial step of 9,10-PQ-induced cytotoxicity in HAECs.     

Role of Auf1 in elimination of oxidatively damaged messenger RNA in human cells

In aerobically growing cells, in which reactive oxygen species are produced, the guanine base of RNA is oxidized to 8-oxo-7,8-dihydroguanine, which induces alterations in gene expression. Here we show that the human Auf1 protein, also called HNRNPD, binds specifically to RNA containing this oxidized base and may be involved in cellular processes associated with managing the problems caused by RNA oxidation. Auf1-deficient cells were constructed from human HeLa and Nalm-6 lines using two different targeting procedures. Both types of Auf1-deficient cells are viable, but exhibit growth retardation. The stability of messenger RNA for four different housekeeping genes was determined in Auf1-deficient and -proficient cells, treated with or without hydrogen peroxide. The level of oxidized messenger RNA was considerably higher in Auf1-deficient cells than in Auf1-proficient cells. Auf1 may play a role in the elimination of oxidized RNA, which is required for the maintenance of proper gene expression under conditions of oxidative stress.     

Mitochondrial free radical overproduction due to respiratory chain impairment in the brain of a mouse model of Rett syndrome: protective effect of CNF1

Rett syndrome (RTT) is a pervasive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene associated with severe intellectual disability, movement disorders, and autistic-like behaviors. Its pathogenesis remains mostly not understood and no effective therapy is available. High circulating levels of oxidative stress markers in patients and the occurrence of oxidative brain damage in MeCP2-deficient mouse models suggest the involvement of oxidative stress in RTT pathogenesis. However, the molecular mechanism and the origin of the oxidative stress have not been elucidated. Here we demonstrate that a redox imbalance arises from aberrant mitochondrial functionality in the brain of MeCP2-308 heterozygous female mice, a condition that more closely recapitulates that of RTT patients. The marked increase in the rate of hydrogen peroxide generation in the brain of RTT mice seems mainly produced by the dysfunctional complex II of the mitochondrial respiratory chain. In addition, both membrane potential generation and mitochondrial ATP synthesis are decreased in RTT mouse brains when succinate, the complex II respiratory substrate, is used as an energy source. Respiratory chain impairment is brain area specific, owing to a decrease in either cAMP-dependent phosphorylation or protein levels of specific complex subunits. Further, we investigated whether the treatment of RTT mice with the bacterial protein CNF1, previously reported to ameliorate the neurobehavioral phenotype and brain bioenergetic markers in an RTT mouse model, exerts specific effects on brain mitochondrial function and consequently on hydrogen peroxide production. In RTT brains treated with CNF1, we observed the reactivation of respiratory chain complexes, the rescue of mitochondrial functionality, and the prevention of brain hydrogen peroxide overproduction. These results provide definitive evidence of mitochondrial reactive oxygen species overproduction in RTT mouse brain and highlight CNF1 efficacy in counteracting RTT-related mitochondrial defects.     

Absence of MGST1 mRNA and protein expression in human neuroblastoma cell lines and primary tissue

A recent study identified a haplotype on a small region of chromosome 12, between markers D12S1725 and D12S1596, shared by all patients with familial neuroblastoma (NB). We previously localized the human MGST1 gene, whose gene product protects against oxidative stress, to this very same chromosomal region (12p112.1–p13.33). Owing to the chromosomal location of MGST1; its roles in tumorigenesis, drug resistance, and oxidative stress; and the known sensitivity of NB cell lines to oxidative stress, we considered a role for MGST1 in NB development. Surprisingly there was no detectable MGST1 mRNA or protein in either NB cell lines or NB primary tumor tissue, although all other human tissues, cell lines, and primary tumor tissue examined to date express MGST1 at high levels. The mechanism behind the failure of NB cells and tissue to express MGST1 mRNA is unknown and involves the failure of MGST1 pre-mRNA expression, but does not involve chromosomal rearrangement or nucleotide variation in the promoter, exons, or 3' untranslated region of MGST1. MGST1 provides significant protection against oxidative stress and constitutes 4 to 6% of all protein in the outer membrane of the mitochondria. As NB cells are extremely sensitive to oxidative stress, and often used as a model system to investigate mitochondrial response to endogenous and exogenous stress, these findings may be due to the lack of expression MGST1 protein in NB. The significance of this finding to the development of neuroblastoma (familial or otherwise), however, is unknown and may even be incidental. Although our studies provide a molecular basis for previous work on the sensitivity of NB cells to oxidative stress, and possibly marked variations in NB mitochondrial homeostasis, they also imply that the results of these earlier studies using NB cells are not transferable to other tumor and cell types that express MGST1 at high concentrations.     

Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells

Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.     

Down-regulation of aldose reductase renders J774A.1 cells more susceptible to acrolein- or hydrogen peroxide-induced cell death

Aldose reductase (AR) is abundantly expressed in a variety of cell lineages and has been implicated in the cellular response against oxidative stress. However, the exact functional role of AR against oxidative stress remains relatively unclear. This study investigated the role of AR in acrolein- or hydrogen peroxide-induced apoptosis using the J774.A.1 macrophage cell line. Ablation of AR with a small interference RNA or inhibition of AR activity significantly enhanced the acrolein- or hydrogen peroxide-induced generation of reactive oxygen species and aldehydes, leading to increased apoptotic cell death. Blockade of AR activity in J774A.1 cells markedly augmented the acrolein- or hydrogen peroxide-induced translocation of Bax to mitochondria along with reduced Bcl-2 and increased release of cytochrome c from the mitochodria. Taken together, these findings indicate that AR plays an important role in the cellular response against oxidative stress, by sequestering the reactive molecules generated in cells exposed to toxic substances.     

The neuroprotective properties of the superoxide dismutase mimetic tempol correlate with its ability to reduce pathological glutamate release in a rodent model of stroke

The contribution of oxidative stress to ischemic brain damage is well established. Nevertheless, for unknown reasons, several clinically tested antioxidant therapies have failed to show benefits in human stroke. Based on our previous in vitro work, we hypothesized that the neuroprotective potency of antioxidants is related to their ability to limit the release of the excitotoxic amino acids glutamate and aspartate. We explored the effects of two antioxidants, tempol and edaravone, on amino acid release in the brain cortex, in a rat model of transient occlusion of the middle cerebral artery (MCAo). Amino acid levels were quantified using a microdialysis approach, with the probe positioned in the ischemic penumbra as verified by a laser Doppler technique. Two-hour MCAo triggered a dramatic increase in the levels of glutamate, aspartate, taurine, and alanine. Microdialysate delivery of 10 mM tempol reduced the amino acid release by 60–80%, whereas matching levels of edaravone had no effect. In line with these data, an intracerebroventricular injection of tempol but not edaravone (500 nmol each, 15 min before MCAo) reduced infarction volumes by ~50% and improved neurobehavioral outcomes. In vitro assays showed that tempol was superior at removing superoxide anion, whereas edaravone was more potent at scavenging hydrogen peroxide, hydroxyl radical, and peroxynitrite. Overall, our data suggest that the neuroprotective properties of tempol are probably related to its ability to reduce tissue levels of the superoxide anion and pathological glutamate release and, in such a way, limit progression of brain infarction within ischemic penumbra. These new findings may be instrumental in developing new antioxidant therapies for treatment of stroke.     

Biomarkers of diabetes-associated oxidative stress and antioxidant status in young diabetic patients with or without subclinical complications

The aims of the study were to ascertain the potential role of oxidative stress in the onset of disease-related pathophysiological complications in young type 1 diabetes patients. Indicative parameters of lipoperoxidation, protein oxidation, and changes in antioxidant defense system status were measured in blood samples from 26 young diabetic patients with recently diagnosed (< 6 months) microangiopathy (+DC), 28 diabetic patients without complications (−DC), and 40 healthy age-matched controls (CR). Both diabetic groups presented similar fructosamine and glycated hemoglobin (HbA1c) values. Results showed erythrocyte glutathione peroxidase activity, glutathione content, and plasma β-carotene to be significantly lower in diabetic patients compared with control subjects, but with no significant differences between −DC and +DC groups. Antioxidant enzyme superoxide dismutase activity was significantly higher in the erythrocytes of diabetic patients independently of the presence of microvascular complications. However, the plasma -tocopherol/total lipids ratio was significantly diminished in +DC group compared with −DC (p = .008). Lipid peroxidation indices measured in plasma included malondialdehyde, lipid hydroperoxides, and lipoperoxides, which were significantly elevated in our diabetic patients regardless of the presence of complications. Evidence of oxidative damage to proteins was shown both through the quantification of plasma protein carbonyl levels, which were significantly higher in −DC (0.61 ± 0.09 mmol/mg prot), and higher still in the +DC patients (0.75 ± 0.09 mmol/mg prot) compared with those of controls (0.32 ± 0.03 mmol/mg prot; p < .01) and immunoblot analysis of protein-bound carbonyls. Additionally, a marked increase in protein oxidation was observed in +DC patients through assessment of advanced oxidation protein products (AOPP) considered to be an oxidized albumin index; AOPP values were significantly higher in +DC than in −DC patients (p < .01) and CR (p < .0001). These results point to oxidatively modified proteins as a differential factor possibly related to the pathogenesis of diabetic complications.