DCFH-DA Oxidation Research Articles

Studies related to the enhanced the effect of 5-aminolevulinic acid-based photodynamic therapy combined with tirapazamine

Objective5-aminolevulinic acid (5-ALA) is a precursor of the photosensitizer Protoporphyrin IX (PpIX) and photodynamic therapy (PDT) with 5-ALA has been used in clinical practice. However, tumor cellular hypoxia severely affects the efficiency of photodynamic therapy. In this study, photodynamic therapy was combined with tirapazamine to investigate the effects of the combined intervention and the related mechanisms it may involve. MethodsColony formation assays were used to demonstrate cell proliferation. Transwell assays were performed to observe the effect on cell invasion and metastasis after the corresponding intervention. DCFH-DA probe was used to detect the reactive oxygen species content. Flow cytometry was used to detect the effects of the interventions on apoptosis and cell cycle. The relevant pathways that may be involved are explored by examining the expression levels of the relevant proteins and genes. ResultsColony formation assays indicated that the combined intervention inhibited cell proliferation. Transwell assays demonstrated that PDT combined with TPZ effectively inhibited tumor cell invasion and metastasis. In addition, fluorescence intensity generated by DCFH-DA oxidation was detected indicating that the combined intervention increased the formation of reactive oxygen species. Flow cytometry clearly showed that the combination of PDT and TPZ further increased apoptosis and cell cycle arrest. The results of western blotting and qRT-PCR experiments confirmed that the combination therapy inhibited HIF-1α/VEGF axis and the PI3K/Akt/mTOR pathway activation. Conclusion5-ALA-PDT combined with TPZ can inhibit cell proliferation, increase apoptosis, and inhibit the PI3K/Akt/mTOR pathway, thus inhibiting tumor growth and metastasis and improving anti-cancer effects.

Ethyl Acetate Fraction of Harpagophytum procumbens Prevents Oxidative Stress In Vitro and Amphetamine-Induced Alterations in Mice Behavior.

Microglial activation has been associated to the physiopathology of neurodegenerative diseases, such as schizophrenia, and can occur during inflammation and oxidative stress. Pharmacological treatment is associated with severe side effects, and studies for use of plant extracts may offer alternatives with lower toxicity. Harpagophytum procumbens (HP) is a plant known for its anti-inflammatory properties. In the present study, we characterized the ethyl acetate fraction of HP (EAF HP) by ESI-ToF-MS and investigated the effects EAF HP in a lipopolysaccharide (LPS) induced inflammation model on microglial cells (BV-2 lineage). MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DCFH-DA (2',7'-dichlorofluorescein diacetate) and cell cycle flow cytometer analysis were performed. In vivo was investigated the amphetamine-induced psychosis model through behavioral (locomotor and exploratory activities, stereotypies and working memory) and biochemical (DCFH-DA oxidation and protein thiols) parameters in cortex and striatum of mice. EAF HP reduced activation and proliferation of microglial cells in 48h (300µg/mL) and in 72h after treatments (50-500µg/mL). Reactive oxygen species levels were lower at the concentration of 100µg/mL EAF HP. We detected a modulatory effect on the cell cycle, with reduction of cells in S and G2/M phases. In mice, the pre-treatment with EAF HP, for 7days, protected against positive and cognitive symptoms, as well as stereotypies induced by amphetamine. No oxidative stress was observed in this amphetamine-induced model of psychosis. Such findings suggest that EAF HP can modulate the dopaminergic neurotransmission and be a promising adjuvant in the treatment of locomotor alterations, cognitive deficits, and neuropsychiatric disorders.

Identification of Potential Artefacts in In Vitro Measurement of Vanadium-Induced Reactive Oxygen Species (ROS) Production.

We investigated vanadium, i.e., a redox-active heavy metal widely known for the generation of oxidative stress in cultured mammalian cells, to determine its ability to interfere with common oxidative stress-related bioassays in cell-free conditions. We first assessed the prooxidant abilities (H2O2 level, oxidation of DHR 123, and DCFH-DA dyes) and antioxidant capacity (ABTS, RP, OH, and DPPH methods) of popular mammalian cell culture media, i.e., Minimal Essential Medium (MEM), Dulbecco's Minimal Essential Medium (DMEM), Dulbecco's Minimal Essential Medium-F12 (DMEM/F12), and RPMI 1640. Out of the four media studied, DMEM has the highest prooxidant and antioxidant properties, which is associated with the highest concentration of prooxidant and antioxidant nutrients in its formulation. The studied vanadium compounds, vanadyl sulphate (VOSO4), or sodium metavanadate (NaVO3) (100, 500, and 1000 µM), either slightly increased or decreased the level of H2O2 in the studied culture media. However, these changes were in the range of a few micromoles, and they should rather not interfere with the cytotoxic effect of vanadium on cells. However, the tested vanadium compounds significantly stimulated the oxidation of DCFH-DA and DHR123 in a cell-independent manner. The type of the culture media and their pro-oxidant and antioxidant abilities did not affect the intensity of oxidation of these dyes by vanadium, whereas the vanadium compound type was important, as VOSO4 stimulated DCFH-DA and DHR oxidation much more potently than NaVO3. Such interactions of vanadium with these probes may artefactually contribute to the oxidation of these dyes by reactive oxygen species induced by vanadium in cells.

Oxidative effects of glyphosate on the lipophobic intracellular environment in the microalgae

The studied hypothesis is that the herbicide glyphosate (GLY) can affect the oxidative balance in the hydrophobic intracellular medium in non-target Chlorella vulgaris cells. Analytical GLY and RoundUp (RUP) supplementation, affected the growth profile. A significant 42% decrease in the cellular biomass in stationary (St) phase was observed in cultures supplemented with either 5 µM of GLY or RUP, as compared to control cultures. The treatment with 0.3 µM of GLY generated non-significant effects on the oxidation rate of 2’, 7’ dichlorofluorescein diacetate (DCFH-DA), neither in exponential (Exp) nor in St phase of development, as compared to control cultures. However, the treatment with either 5 µM GLY or 0.3 and 5 µM RUP lead to a significant decrease in the DCFH-DA oxidation rate, as compared to control cultures. The lipid radical (LR●) generation rate, detected by Paramagnetic Resonance Spectroscopy (EPR), was significantly increased in the presence of RUP, in Lag and Exp phase of growth. The non-enzymatic antioxidants, α-Tocopherol (α-T) and β-Carotene (β-C), are aimed to protect membranes against the damage produced by the radical reactions. The content of β-C was not significantly affected, as compared to control cultures, by any of the treatments, in both growth phases of cellular development. The content of α-T was significantly decreased by the supplementation with either 0.3 or 5 µM of RUP or 5 µM GLY. The LR●/α-T ratio, used as indicator of the oxidative balance in the hydrophobic cellular media, was significantly different between samples obtained from control and RUP-exposed microalgae in both, Exp and St phase of development, with either 0.3 or 5 μM RUP. The data presented here showed evidence that suggested that oxidative balance in the hydrophobic environment was affected by either GLY or RUP.

148 - Pharmacological ascorbate enhances expression of dual oxidase which mediates sustained toxicity via alterations in cellular metabolism

Pancreatic Ductal Adenocarcinoma is a leading cause of cancer related death in the United States that is in critical need of adjuvant therapies to enhance patient survival. One such adjuvant is pharmacological ascorbate treatment (P-AscH-, high-dose, intravenous vitamin C). P-AscH- treatment results in a short-term increased flux of H2O2 that is preferentially cytotoxic to cancer cells vs. normal cells. Treatment with P-AscH- results in sustained increases in oxygen consumption (25-30 attomoles cell-1 sec-1) and reactive oxygen species production (1.5 fold as DCFH-DA oxidation), with no change in glucose metabolism in tumor cells compared to non-tumorigenic pancreatic ductal epithelial cells. These results lead to the hypothesis that there is a sustained effect (> 24 h) of P-AscH- that contributes to cytotoxicity. Seahorse XF96 mito stress test (48 h) demonstrated that P-AscH- results in a significant and sustained increase in non-mitochondrial OCR (~15 attomoles cell-1 sec-1). A potential source of this sustained non-mitochondrial OCR is the NADPH oxidase family of enzymes Dual Oxidase 1 and 2 (DUOX) which are up-regulated after P-AscH-. P-AscH- treatment of PDAC cell lines resulted in a dose (1-10 mM; 4-6 fold) and duration (24-72 h; 5-20 fold) dependent increase in DUOX. Catalase pretreatment reverses the P-AscH--induced increases in DUOX, while DUOX inhibition partially rescues P-AscH- toxicity. Additionally, nutritional ascorbate (100 mm; Ascorbate-2-Phosphate) is unable to mediate an increase in DUOX expression. P-AscH- also induces DUOX protein expression in pancreatic tumor xenografts. These results suggest that P-AscH--induced toxicity may be enhanced by a sustained metabolic shift in tumor cells that is related to the H2O2 produced from P-AscH-. We suggest that P-AscH- results in a feed-forward mechanism of H2O2 generation and induction of metabolic stress via enhanced DUOX expression and OCR.

β-Carotene and its biological oxidation products: are they good modulators of pro-oxidant reactive species in human blood and neutrophils assay systems?

β-carotene is the most abundant carotenoid found in the diet and human tissues [1]. High ingestion of carotenoids has been associated with low incidence of degenerative diseases and it has been mainly attributed to their ability to scavenge physiologically relevant reactive species. In humans, β-carotene is metabolized by oxidative cleavage into trans-β-apo-8’-carotenal and β-ionone [2]. Therefore, it is crucial to disclose the potential antioxidant effects of β-carotene and its oxidation products. In this sense, the main aim of this work was to elucidate the potential antioxidant effects of β-carotene, trans-β-apo-8’-carotenal and β-ionone, at relevant physiologic concentrations. The two selected biological systems were human blood, a complex matrix that most approximately mimic the in vivo biologic environment; and human neutrophils, the first line of defense cells utterly implicated in the immune and inflammatory responses. Two fluorescent probes were used in both matrices: 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) that unspecifically detects hydrogen peroxide (H2O2), hydroxyl radical, nitric oxide and peroxynitrite anion, and amplex red (AR) that specifically detects H2O2. In both matrices, none of the tested carotenoids showed any ability to stimulate reactive species production. As so, they do not seem to be pro-oxidants. For the study of the antioxidant potential, phorbol 12-myristate 13-acetate was used to stimulate reactive species production by human blood cells. Interestingly, in what concerns the human blood matrix, none of the tested carotenoids (0.3 - 49 µM) showed antioxidant activity. In what respects human neutrophils, also none of the tested compounds (0.3 - 100 µM) was able to inhibit DCFH-DA oxidation; trans-β-apo-8’-carotenal was the only one that showed a slight ability to inhibit AR oxidation, suggesting some modulation of H2O2 production. The obtained results show that β-carotene and its oxidation products, at biological relevant concentrations, did not provide acute pro- or antioxidant activity, in the studied experimental conditions. These results indicate that carotenoids and/or its oxidation products probably require different types of exposure on biological systems to enable a better incorporation in the lipidic system opening a window to new studies with this kind of compounds.

Seasonality and toxins effects on oxidative/nitrosative metabolism in digestive glands of the bivalve Mytilus edulis platensis

The hypothesis presented here is that oxidative and/or nitrosative metabolism in the bivalve Mytilus edulis platensis is altered by the presence of planktonic toxins. Digestive glands (DG) were isolated from specimens collected in the Argentinean Sea during summer, winter and spring (in the presence of harmful planktonic toxins). The labile iron pool content was not significantly different in DG from animals collected in summer and winter, but was 2.3-fold increased in samples from spring compared to summer collected mollusks. The 2′,7′ dichlorofluorescein diacetate (DCFH-DA) oxidation, ascorbyl radical/ascorbate and lipid radical/α-tocopherol content ratios showed no significant differences between samples collected in winter and summer. However, spring collected samples showed significantly higher DCFH-DA oxidation rate and oxidative ratios in comparison to DG from mollusks collected in summer. Superoxide dismutase activity decreased by 75% in winter, and 93% in spring, compared to samples collected in summer. Glutathione S-transferase activity decreased by 89% in winter, and 30% in spring, compared to samples collected in summer. Catalase activity in winter animals increased by 3.8-fold in comparison to summer values, with no differences between spring and summer collected mollusks. Nitrite plus nitrate content was not significantly different among samples collected in the three seasons, but nitric oxide content was 8.5- and 2.7-fold higher in samples from winter and spring collected mollusks than values obtained in summer, respectively. These results showed the lack of effects of climatic changes on the integrative oxidative indexes; however, under exposure to toxins, both oxidative and nitrosative metabolisms were affected.

Effects of diphenyl diselenide on behavioral and biochemical changes induced by amphetamine in mice.

Diphenyl diselenide (PhSe)2, an organoselenium compound, has been studied as a potential pharmacological agent in different in vitro and in vivo models, mainly due to its antioxidant properties. However, there are few studies concerning the effects of (PhSe)2 on dopaminergic system. Thus, the purpose of the present study was to evaluate the effects of acute and sub-chronic treatment of (PhSe)2 on amphetamine-induced behavioral and biochemical parameters. In acute protocol, mice were pre-treated with 5 or 10 mg/kg of (PhSe)2 and 30 min after, amphetamine was administered. In sub-chronic protocol, mice were pre-treated with 5 or 10 mg/kg of (PhSe)2 during 7 days and 24 h after, amphetamine was administered. Twenty-five minutes after amphetamine administration, behavioral (crossing, rearing, time of stereotypy and immobility) and biochemical (MAO activity, DCFH-DA oxidation, protein and non-protein thiol groups) parameters were analyzed. Amphetamine increased the number of crossing and rearing and (PhSe)2 prevented only the increase in the number of crossings when acutely administered to mice. Furthermore, amphetamine increased stereotypy and time of immobility in mice. (PhSe)2, at 10 mg/kg, increased per se the stereotypy and time of immobility when sub-chronically administered. (PhSe)2, at 10 mg/kg, potentiated the stereotypy caused by amphetamine in both protocols. Sub-chronic treatment with (PhSe)2 either alone (5 and 10 mg/kg) or in combination (10 mg/kg) with amphetamine decreased brain MAO-B activity. Oxidative stress parameters were not modified by (PhSe)2 and/or amphetamine treatments. In conclusion, sub-chronic administration of (PhSe)2 can promote a behavioral sensitization that seems to be, at least in part, dependent of MAO-B inhibition.

Antioxidant activity elicited by low dose of caffeine attenuates pentylenetetrazol-induced seizures and oxidative damage in rats

Although caffeine supplementation has a beneficial effect on people with neurological disorders, its implications for oxidative damage related to seizures are not well documented. Thus the aim of this study was to investigate the effects of two weeks caffeine supplementation (6mg/kg; p.o.) on seizures and neurochemical alterations induced by pentylenetetrazol (PTZ 60mg/kg i.p.). Statistical analyses showed that long-term rather than single dose caffeine administration decreased the duration of PTZ-induced seizures in adult male Wistar rats as recorded by cortical electroencephalographic (EEG) and behavioral analysis. The quantification of EEG recordings also revealed that caffeine supplementation protected against a wave increase induced by PTZ. Neurochemical analyses revealed that caffeine supplementation increased glutathione (GSH) content per se and protected against the increase in the levels of thiobarbituric acid reactive substances (TBARS) and oxidized diclorofluoresceine diacetate (DCFH-DA). Also, caffeine prevent the decrease in GSH content and Na+, K+-ATPase activity induced by PTZ. Our data also showed that the infusion of L-buthionine sulfoximine (BSO; 3.2μmol/site i.c.v), an inhibitor of GSH synthesis, two days before injecting PTZ reversed the anticonvulsant effect caused by caffeine. BSO infusion also decreased GSH content and Na+, K+-ATPase activity. However, it increased DCFH-DA oxidation and TBARS per se and reversed the protective effect of caffeine. Results presented in this paper support the neuroprotective effects of low long-term caffeine exposure to epileptic damage and suggest that the increase in the cerebral GSH content caused by caffeine supplementation may provide a new therapeutic approach to the control of seizure.

Gallic acid decreases vacuous chewing movements induced by reserpine in rats

Involuntary oral movements are present in several diseases and pharmacological conditions; however, their etiology and efficient treatments remain unclear. Gallic acid is a natural polyphenolic acid found in gall nuts, sumac, oak bark, tea leaves, grapes and wine, with potent antioxidant and antiapoptotic activity. Thus, the present study investigated the effects of gallic acid on vacuous chewing movements (VCMs) in an animal model induced by reserpine. Rats received either vehicle or reserpine (1mg/kg/day, s.c.) during three days, followed by treatment with water or different doses of gallic acid (4.5, 13.5 or 40.5mg/kg/day, p.o.) for three more days. As result, reserpine increased the number of VCMs in rats, and this effect was maintained for at least three days after its withdrawal. Gallic acid at two different doses (13.5 and 40.5mg/kg/day) has reduced VCMs in rats previously treated with reserpine. Furthermore, we investigated oxidative stress parameters (DCFH-DA oxidation, TBARS and thiol levels) and Na+,K+-ATPase activity in striatum and cerebral cortex, however, no changes were observed. These findings show that gallic acid may have promissory use in the treatment of involuntary oral movements.

Antioxidant effect of organic purple grape juice on exhaustive exercise

This study aimed to assess the potential protective effect of organic purple grape juice (PGJ) on oxidative stress produced by an exhaustive exercise bout in rats. To test this hypothesis, rats were acutely treated with organic PGJ (Vitis labrusca) and subsequently submitted to an exhaustive exercise bout. Parameters of oxidative stress, such as thiobarbituric acid reactive species (TBARS) levels, 2',7',-dichlorofluorescein diacetate (DCFH-DA) oxidation, and nonprotein sulfhydryl levels (NP-SH) in the brain, skeletal muscle, and blood, were evaluated. Enzyme activity of Na(+),K(+)-ATPase, Ca(2+)-ATPase, and δ-aminolevulinate dehydratase (δ-ALA-D) in the brain, skeletal muscle, and blood were also assayed. Statistical analysis showed that the exhaustive exercise bout increased TBARS levels and DCFH-DA oxidation, and decreased NP-SH levels in rat tissue. Ca(2+)-ATPase activity was increased in groups exposed to both exercise and PGJ treatment. The results indicate that organic PGJ intake was able to protect against the oxidative damage caused by an exhaustive exercise bout in different rat tissues.

Effect of garlic-derived organosulfur compounds on mitochondrial function and integrity in isolated mouse liver mitochondria

The objectives of this work were to evaluate the direct effects of diallysulfide (DAS) and diallyldisulfide (DADS), two major organosulfur compounds of garlic oil, on mitochondrial function and integrity, by using isolated mouse liver mitochondria in a cell-free system. DADS produced concentration-dependent mitochondrial swelling over the range 125–1000μM, while DAS was ineffective. Swelling experiments performed with de-energized or energized mitochondria showed similar maximal swelling amplitudes. Cyclosporin A (1μM), or ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA, 1mM) were ineffective in inhibiting DADS-induced mitochondrial swelling. DADS produced a minor (12%) decrease in mitochondrial membrane protein thiols, but did not induce clustering of mitochondrial membrane proteins. Incubation of mitochondria with DADS (but not DAS) produced an increase in the oxidation rate of 2′,7′ dichlorofluorescein diacetate (DCFH-DA), together with depletion of reduced glutathione (GSH) and increased lipid peroxidation. DADS (but not DAS) produced a concentration-dependent dissipation of the mitochondrial membrane potential, but did not induce cytochrome c release. DADS-dependent effects, including mitochondrial swelling, DCFH-DA oxidation, lipid peroxidation and loss of mitochondrial membrane potential, were inhibited by antioxidants and iron chelators. These results suggest that DADS causes direct impairment of mitochondrial function as the result of oxidation of the membrane lipid phase initiated by the GSH- and iron-dependent generation of oxidants.

Antioxidant activity and low toxicity of (E)-1-(1-(methylthio)-1-(selenopheny) hept-1-en-2-yl) pyrrolidin-2-one

The aim of the present study was to evaluate the potential pharmacological and toxicological properties of (E)-1-(1-(methylthio)-1-(selenopheny) hept-1-en-2-yl) pyrrolidin-2-one (compound 1), an organoselenium compound. In vitro experiments showed that compound 1 presented a reduction in the lipid peroxidation induced by Fe²⁺ in thiobarbituric acid-reactive species (TBARS) production, and in the generation of reactive species caused by Fe²⁺/malonate in DCFH-DA oxidation. The high dose (500 mg/kg) induced an increase on ALT but not on AST activity. Hepatic, but not cerebral, δ-ALA-D activity from mice treated with 500 mg/kg presented a significant inhibition. Brain catalase activity was significantly inhibited by 100 mg/kg whereas hepatic catalase activity showed a significant increase at all doses. Hepatic lipid peroxidation was diminished only at lowest dose (100 mg/kg) whereas for brain tissue, all doses induced a significant reduction in TBARS levels. Brain and liver ascorbic acid contents were increased only at highest dose of compound 1. Urea and creatinine levels were not significantly altered by treatments. This is a promising compound with antioxidant activity and low toxicity, suggesting the potential beneficial activity of compound 1 against oxidative damage in many parameters studied in rats and mice.

Oxidative stress-mediated inhibition of brain creatine kinase activity by methylmercury

Methylmercury (MeHg), a potent neurotoxicant, easily passes through the blood–brain barrier and accumulates in brain causing severe irreversible damage. However, the underlying neurotoxic mechanisms elicited by MeHg are still not completed defined. In this study, we aimed to investigate the in vitro toxic effects elicited by crescent concentrations (0–1500 μM) of MeHg on creatine kinase (CK) activity, thiol content (NPSH) and protein carbonyl content (PCC) in mouse brain preparations. In addition, CK activity, MTT reduction and DCFH-DA oxidation (reactive oxygen species (ROS) formation) were also measured in C6 glioma cell linage. CK activity was severely reduced by MeHg treatment in mouse brain preparations. This inhibitory effect was positively correlated to the MeHg-induced reduction of NPSH levels and increment in PCC. Moreover, the positive correlation between brain CK activity and NPSH levels was observed at either 15 or 60 min of MeHg pre-incubation. In addition, MeHg-treated C6 cells showed also a significant inhibition of CK activity at MeHg concentrations, as low as, 50 μM in parallel to reduced mitochondrial function and increased ROS production. Taking together, these data demonstrate that MeHg severely affects CK activity, an essential enzyme for brain energy buffering to maintain cellular energy homeostasis. This effect appears to be mediated by oxidation of thiol groups that might cause subsequent oxidative stress.

Retinol and retinoic acid increase MMP-2 activity by different pathways in cultured Sertoli cells

Diseases such as atherosclerosis, arthritis and cancer have been related with imbalance in ROS production and failures in regulation of the MMPs. Authors suggested a relationship between MPP activity and ROS. Our research group has demonstrated that retinol 7µM induced changes in Sertoli cell metabolism linking retinol treatment and oxidative stress. We verified MMP activity in Sertoli cells treated with vitamin A using gelatin zymography. We found that retinol (7µM) and retinoic acid (1nM) induced MMP-2 activity in Sertoli cells. Antioxidants reversed retinol-induced but not retinoic acid-induced MMP-2 activity. Moreover, retinol but not retinoic acid increased ROS production quantified by DCFH-DA oxidation. We found that retinol and retinoic acid induced ERK1/2 phosphorylation, but only retinol-increased MMP-2 activity was inhibited by UO126, an ERK1/2 phosphorylation inhibitor. Our findings suggested that retinol-induced MMP-2 activity, but not retinoic acid-induced MMP-2 activity, was related to ERK1/2 phosphorylation and ROS production.

CD40 Ligand Influences Platelet Release of Reactive Oxygen Intermediates

Soluble CD40 ligand (sCD40L) has been recently implicated in the pathogenesis of atherosclerosis. Elevated levels of sCD40L in acute coronary syndrome patients suggests enhanced platelet function; however, the exact mechanism by which this occurs is unknown. In this study, we examined the effect of sCD40L on platelet function and reactive oxygen and nitrogen species (RONS) generation. Platelet stimulation in the presence of recombinant sCD40L (rsCD40L) led to enhanced generation of RONS as measured by DCFHDA oxidation and confocal microscopy. Incubation with rsCD40L led to enhanced platelet P-selectin expression, aggregation, and platelet-leukocyte conjugation. Platelets isolated from CD40L-deficient mice had decreased agonist-induced NO release as compared with wild-type mice. Incubation of platelets with rsCD40L enhanced stimulation-induced p38 MAP kinase and Akt phosphorylation. Soluble CD40L enhances platelet activation, aggregation, and platelet-leukocyte conjugation, as well as increases stimulation-induced platelet release of RONS through activation of Akt and p38 MAP kinase signaling pathways. These data suggest that sCD40L regulates platelet-dependent inflammatory and thrombotic responses that contribute to the pathogenesis of atherothrombosis.

Hindlimb unloading increases oxidative stress and disrupts antioxidant capacity in skeletal muscle.

Skeletal muscle disuse with space-flight and ground-based models (e.g., hindlimb unloading) results in dramatic skeletal muscle atrophy and weakness. Pathological conditions that cause muscle wasting (i.e., heart failure, muscular dystrophy, sepsis, COPD, cancer) are characterized by elevated "oxidative stress," where antioxidant defenses are overwhelmed by oxidant production. However, the existence, cellular mechanisms, and ramifications of oxidative stress in skeletal muscle subjected to hindlimb unloading are poorly understood. Thus we examined the effects of hindlimb unloading on hindlimb muscle antioxidant enzymes (e.g., superoxide dismutase, catalase, glutathione peroxidase), nonenzymatic antioxidant scavenging capacity (ASC), total hydroperoxides, and dichlorohydrofluorescein diacetate (DCFH-DA) oxidation, a direct indicator of oxidative stress. Twelve 6 month old Sprague Dawley rats were divided into two groups: 28 d of hindlimb unloading (n = 6) and controls (n = 6). Hindlimb unloading resulted in a small decrease in Mn-superoxide dismutase activity (10.1%) in the soleus muscle, while Cu,Zn-superoxide dismutase increased 71.2%. In contrast, catalase and glutathione peroxidase, antioxidant enzymes that remove hydroperoxides, were significantly reduced in the soleus with hindlimb unloading by 54.5 and 16.1%, respectively. Hindlimb unloading also significantly reduced ASC. Hindlimb unloading increased soleus lipid hydroperoxide levels by 21.6% and hindlimb muscle DCFH-DA oxidation by 162.1%. These results indicate that hindlimb unloading results in a disruption of antioxidant status, elevation of hydroperoxides, and an increase in oxidative stress.

Role of Nitric-Oxide Synthase, Free Radicals, and Protein Kinase C δ in Opioid-Induced Cardioprotection

Opioids generate free radicals that mediate protection in isolated cultured cardiomyocytes. We hypothesize that the nature of these radicals is nitric oxide, and that nitric oxide activates the protein kinase C (PKC) delta isoform. Through this signal transduction pathway, opiates protect cardiomyocytes during hypoxia and reoxygenation. Cell viability was quantified in chick embryonic ventricular myocytes with propidium iodide. Oxygen radicals were quantified using a molecular probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA). After a 10-min infusion of the opioid delta receptor agonist BW373U86 (BW; 2 or 20 pM) and a 10-min drug-free period, cells were subjected to hypoxia for 1 h followed by reoxygenation for 3 h. BW produced a concentration-dependent reduction in cardiomyocyte death (2 pM, 35.3 +/- 3.9%, n = 5; 20 pM, 21.5 +/- 4.0%, n = 8, p < 0.05 versus controls) and attenuated oxidant stress compared with controls (43.3 +/- 4.2%, n = 8). The increase in DCFH-DA oxidation with BW before hypoxia was abolished by the specific nitric-oxide synthase inhibitors nitro-L-arginine methyl ester (L-NAME) or N(G)-monomethyl-L-arginine (L-NMMA) (100 microM each). L-NAME or L-NMMA blocked the protective effects of BW. BW selectively increased the activity of PKC delta isoform in the particulate fraction, and its protection was abolished by the selective PKC delta inhibitor rottlerin (1 microM). Similar to BW, infusion with 5 microM of the nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) reduced cardiomyocyte death (24.6 +/- 3.7, n = 8), and this protection was blocked by chelerythrine or rottlerin. Chelerythrine and rottlerin had no effect on BW-generated oxygen radicals before hypoxia, but they abolished the protection of SNAP. The nature of DCFH oxidation produced by opioid delta receptor stimulation is nitric oxide. Nitric oxide mediates cardioprotection via activating PKC delta in isolated myocytes.

Differential Role of CYP2E1 Binders and Isoniazid on CYP2E1 Protein Modification in NADPH-dependent Microsomal Oxidative Reactions: Free Radical Scavenging Ability of Isoniazid

We evaluated the effect of "weak" CYP2E1 binders (ethanol, acetone and glycerol) "tight" CYP2E1 binders (4-methylpyrazole, imidazole, isoniazid and pyridine) and CCl 4 (suicide substrate of CYP2E1) on the NADPH-dependent production of microsomal reactive oxygen species (ROS), lipid peroxidation (LPO), and subsequent modification of microsomal and CYP2E1 proteins. The oxidation of 2',7'-dichlorofluorescin diacetate (DCFHDA) was used as an index of formation of microsomal ROS and LPO-derived reactive species. Microsomal LPO was determined by malondialdehyde (MDA) HPLC measurement. Addition of NADPH to rat liver microsomes initiated DCFHDA oxidation and MDA formation, leading to further selective modification of microsomal proteins and proteases-independent degradation of CYP2E1 protein. Iron chelators prevented these processes whereas hydroxyl radical scavengers showed weak effects, suggesting an important role of LPO. Among the tested CYP2E1 binders, only isoniazid strongly inhibited NADPH-dependent DCFHDA oxidation, LPO and modification of microsomal proteins. Other CYP2E1 binders showed weak inhibitory effects of these processes. Concerning NADPH-dependent modification of CYP2E1 protein, all of the tested CYP2E1 binders, except glycerol, prevented this process with a different potency (isoniazid > 4-methylpyrazole=imidazole=pyridine d acetone > ethanol). "Tight" binders were more effective than "weak" binders. The CCl 4 stimulated the DCFHDA oxidation, LPO and CYP2E1 protein modification. Among the tested CYP2E1 binders, only isoniazid effectively scavenged 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals. In microsomes isolated from CYP2E1 transfected HepG2 cells, isoniazid inhibited the CYP2E1-dependent DCFHDA oxidation whereas other CYP2E1 binders did not inhibit this reaction although these compounds strongly inhibited CYP2E1 activity. The present study demonstrates that CYP2E1 binders and isoniazid differentially inhibit LPO-catalyzed oxidative modification of CYP2E1 protein in NADPH-dependent microsomal reactions. It seems that CYP2E1 binders protect CYP2E1 from the oxidative modification mainly by binding to the active site of the enzyme, rather than by blocking the reactive species production. The strong protective effect of isoniazid can be attributed to its ability to scavenge free radicals. These effects of CYP2E1 binders are considered to contribute to the regulation of hepatic CYP2E1 protein levels via stabilization of the protein.

Effect of Ascorbic Acid and Its Hydrophobic Derivative Palmitoyl Ascorbate on the Redox State of Primary Human Fibroblasts.

Ascorbic acid (AA) and its derivatives participate in vitro in oxidative-reductive reactions both as antioxidants and as prooxidants. The physiological relevance of these prooxidant effects of AA and its derivatives remains unclear. There is little evidence that AA can initiate formation of reactive oxygen species (ROS) or lipid peroxidation in cells or tissue. In order to examine the effect of AA and its derivative palmitoyl ascorbate on in situ intracellular ROS production and lipid peroxidation, 2('),7(')-dichlorofluorescin diacetate (DCFH-DA) and cis-parinaric acid were used as fluorescent probes in cultural neonatal foreskin fibroblasts. The results demonstrated that the effect of AA depended on the in vitro growth conditions. AA induced augmentation of the intracellular ROS concentration in newly plated (24 hours) cells. However, in cells cultured for 72 hours, AA had a different effect: it moderately reduced intracellular ROS concentration but stimulated lipid peroxidation in the cytoplasmic membrane. Palmitoyl ascorbate demonstrated significant inhibition of intracellular DCFH-DA oxidation presumably caused by inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.