Cigarette smoke is the primary risk factor for chronic obstructive pulmonary disease (COPD). Alterations in the balance between apoptosis and proliferation are involved in the etiology of COPD. Fibroblasts and epithelial cells are sensitive to the oxidative properties of cigarette smoke, and whose loss may precipitate the development of COPD. Fibroblasts express the aryl hydrocarbon receptor (AhR), a transcription factor that attenuates pulmonary inflammation and may also regulate apoptosis. We hypothesized the AhR would prevent apoptosis caused by cigarette smoke. Using genetically deleted in vitro AhR expression models and an established method of cigarette smoke exposure, we report that AhR expression regulates fibroblasts proliferation and prevents morphological features of apoptosis, including membrane blebbing and chromatin condensation caused by cigarette smoke extract (CSE). Absence of AhR expression results in cleavage of PARP, lamin, and caspase-<em>3</em>. Mitochondrial dysfunction, including cytochrome c release, was associated with loss of AhR expression, indicating activation of the intrinsic apoptotic cascade. Heightened sensitivity of AhR-deficient fibroblasts was not the result of alterations in GSH, Nrf2, or HO-1 expression. Instead, AhR(-/-) cells had significantly less Mn<em>SOD</em> and CuZn-<em>SOD</em> expression, enzymes that protects against oxidative stress. The ability of the AhR to suppress apoptosis was not restricted to fibroblasts, as siRNA-mediated knockdown of the AhR in lung epithelial cells also increased sensitivity to smoke-induced apoptosis. Collectively, these results suggest that cigarette smoke induced loss of lung structural support (i.e. fibroblasts, epithelial cells) caused by aberrations in AhR expression may explain why some smokers develop lung diseases such as COPD.

Oxidative stress and apoptosis are implicated in the pathogenesis of diabetic embryopathy. The proapoptotic c-Jun NH(2)-terminal kinases (JNK)1/2 activation is associated with diabetic embryopathy. We sought to determine whether 1) hyperglycemia-induced oxidative stress is responsible for the activation of JNK1/2 signaling, 2) JNK1 contributes to the teratogenicity of hyperglycemia, and <em>3</em>) both JNK1 and JNK2 activation cause activation of downstream transcription factors, caspase activation, and apoptosis, resulting in neural tube defects (NTDs). Wild-type (WT) embryos from nondiabetic WT dams and WT, superoxide dismutase (<em>SOD</em>)1-overexpressing, jnk1(+/-), jnk1(-/-), and jnk2(-/-) embryos exposed to maternal hyperglycemia were used to assess JNK1/2 activation, NTDs, activation of transcription factors downstream of JNK1/2, caspase cascade, and apoptosis. <em>SOD</em>1 overexpression abolished diabetes-induced activation of JNK1/2 and their downstream effectors: phosphorylation of c-Jun, activating transcription factor 2, and E twenty-six-like transcription factor 1 and dephosphorylation of forkhead box class O<em>3</em>a. jnk1(-/-) embryos had significantly lower incidences of NTDs than those of WT or jnk1(+/-) embryos. Either jnk1 or jnk2 gene deletion blocked diabetes-induced activation of JNK1/2 signaling, caspases <em>3</em> and 8, and apoptosis in Sox1(+) neural progenitors of the developing neural tube. Our results show that JNK1 and JNK2 are equally involved in diabetic embryopathy and that the oxidative stress-JNK1/2-caspase pathway mediates the proapoptotic signals and the teratogenicity of maternal diabetes.

OBJECTIVE

Oxidative damage induced by H(2)O(2) treatment can irreversibly damage the lens epithelium, resulting in cell death and cataract. Whether the effects of oxidative stress could be attenuated in cultured human lens epithelial cells by incubation with resveratrol (RES) is still unknown. In the present study, we examined the function of resveratrol in protecting human lens epithelial B-<em>3</em> (HLEB-<em>3</em>) cells against H(2)O(2) induced cell death and cell apoptosis, its role in reducing H(2)O(2) induced intracellular reactive oxygen species (ROS) accumulation, and investigated the mechanism by which resveratrol underlies the effect.

METHODS

HLEB-<em>3</em> cells, a human lens epithelial cell line, were exposed to 100 muM H(2)O(2) with or without RES pre-treatment at different concentrations for different time duration. Cell viabilities were monitored by 4-[<em>3</em>-[4-iodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,<em>3</em>-benzene disulfonate] (WST-1) assay. The apoptosis rate and ROS generation were detected by flow cytometric analysis. Expression levels of superoxide dismutases-1 (SOD-1), catalase, and heme oxygenase-1 (HO-1) proteins were measured by western-blotting analysis. p<em>3</em>8 and c-jun N terminal kinase (JNK) activation was also evaluated by western-blotting analysis.

RESULTS

Resveratrol clearly reduced H(2)O(2) induced cell apoptosis and ROS accumulation; protected HLEB-<em>3</em> cells from H(2)O(2) induced oxidative damage, and increased the expression levels of SOD-1, catalase, and HO-1. Further studies showed that RES also inhibited H(2)O(2) induced p<em>3</em>8 and JNK phosphorylation.

CONCLUSIONS

These findings suggested that RES protected HLEB-<em>3</em> cells from H(2)O(2) induced oxidative damage, presumably by inducing three antioxidative enzymes including catalase, SOD-1, and HO-1.

OBJECTIVE

Hyperglycemia is considered a primary cause of diabetic vascular complications and is associated with oxidative stress, impaired trace element and lipid metabolism as well as pancreatic enzyme abnormalities. The role of trace elements in some of the metabolic dysfunctions and their contributions in the development of vascular complications is not clear. Therefore, the present study investigates the relationship among diabetes mellitus, trace elements status, advanced glycation end products (AGEs), advanced oxidation protein products (AOPP), lipid profiles, antioxidant status, nitric oxide and pancreatic amylase activity in the sera of 55 non-insulin-dependent diabetes mellitus (NIDDM; <em>3</em>5 with microvascular complications and 20 without vascular complications), 40 insulin-dependent diabetes mellitus (IDDM; 25 with microvascular and 15 without microvascular complications), and 20 nondiabetic healthy control subjects. The mean age of the diabetic patients was similar to that of control. The mean duration of the disease was 11.8 +/- 6.8 years (<em>3</em>-27 years) in IDDM and 7.1 +/- 4.7 years (1-15 years) in NIDDM.

METHODS

Plasma Cu, Zn, Mg, Ca, thiobarbituric acid-reactive substance (i.e. malondialdehyde; MDA), nitric oxide (NO), glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), ceruloplasmin (Cp) and amylase activities as well as AOPP were assessed spectrophotometrically whereas AGEs were estimated spectrofluorometrically in two types of diabetes mellitus (DM) as well as control subjects of matched sex and ages.

RESULTS

SOD, CAT and Cp activities were decreased whereas serum alpha-amylase activity was increased in two types of DM in comparison to the corresponding activities of the control subjects. The plasma levels of MDA, NO and Cu were increased but GSH, Zn, Mg and Ca levels were significantly diminished in diabetic patients as compared to the controls. The averages of total cholesterol (CHOL), triglyceride (TG) and low-density lipoprotein-cholesterol (LDLc) were higher in both types of diabetes mellitus in comparison to the control subjects. The mean value of high-density lipoprotein-cholesterol (HDLc) was lower in both types of diabetes mellitus. Further, the mean values of AGEs and AOPP were elevated in diabetic patients vs. control. These parameters are significantly higher in NIDDM patients when compared to the IDDM subjects. Slight but not significant differences in these parameters were observed in patients with diabetic complications when compared to that of without diabetic complications.

CONCLUSIONS

These findings may explain the role of impaired trace element status, defect of antioxidants and increased of AGE and AOPP in the pathogenesis of pancreas and the vascular complications of diabetes mellitus. Oxidative stress is increased in both types of DM, but it is more in NIDDM patients than in IDDM subjects. In addition, oxidative stress also plays an important role in the formation of AGEs and AOPP in DM.

Effects of cadmium (Cd(2+)) on photosynthetic and antioxidant activities of maize (Zea mays L.) cultivars (<em>3</em>22<em>3</em> and <em>3</em>2D99) were investigated. Fourteen-day-old cultivar seedlings were exposed to different Cd concentrations [0, 0.<em>3</em>, 0.6 and 0.9mM Cd(NO(<em>3</em>))(2) x 4H(2)O] for 8 days. The results of chlorophyll fluorescence indicated that different levels of Cd affected photochemical efficiency in <em>3</em>22<em>3</em> much more than that in <em>3</em>2D99. In parallel, the level of Cd at 0.9mM caused oxidative damage but did not indicate cessation of PSII activity of the cultivars; plant death was not observed at highly toxic Cd levels. Additionally, the increase in Cd concentration caused loss of chlorophylls and carotenoid and membrane damage in both cultivars, but greater membrane damage was observed in <em>3</em>22<em>3</em> than in <em>3</em>2D99. Depending on Cd accumulation, a significant reduction in dry biomass was observed in both cultivars at all Cd concentrations. The accumulation of Cd was higher in roots than in leaves for both cultivars. Nevertheless, cultivar <em>3</em>22<em>3</em> transferred more Cd from roots to leaves than did <em>3</em>2D99. On the other hand, our results suggest that there were similar responses in <em>SOD</em>, APX and GR activities with increasing Cd concentrations for both cultivars. However, POD activity significantly increased at highly toxic Cd levels in <em>3</em>2D99. This result may be regarded as an indication of better tolerance of the Z. mays L. cultivar <em>3</em>2D99 to Cd contamination.

Elevated generation of reactive oxygen species (ROS) by endothelial enzymes, including NADPH-oxidase, is implicated in vascular oxidative stress and endothelial proinflammatory activation involving exposure of vascular cell adhesion molecule-1 (VCAM-1). Catalase and superoxide dismutase (<em>SOD</em>) conjugated with antibodies to platelet/endothelial cell adhesion molecule 1 (PECAM-1) bind specifically to endothelium and inhibit effects of corresponding ROS, H(2)O(2), and superoxide anion. In this study, anti-PECAM/<em>SOD</em>, but not anti-PECAM/catalase or nontargeted enzymes, including polyethylene glycol (PEG)-<em>SOD</em>, inhibited 2- to <em>3</em>-fold VCAM expression caused by tumor necrosis factor (TNF), interleukin-1β, and lipopolysaccharide. Anti- PECAM/<em>SOD</em>, but not nontargeted counterparts, accumulated in vascular endothelium after intravenous injection, localized in endothelial endosomes, and inhibited by 70% lipopolysaccharide-caused VCAM-1 expression in mice. Anti-PECAM/<em>SOD</em> colocalized with EEA-1-positive endothelial vesicles and quenched ROS produced in response to TNF. Inhibitors of NADPH oxidase and anion channel ClC<em>3</em> blocked TNF-induced VCAM expression, affirming that superoxide produced and transported by these proteins, respectively, mediates inflammatory signaling. Anti-PECAM/<em>SOD</em> abolished VCAM expression caused by poly(I:C)-induced activation of toll-like receptor <em>3</em> localized in intracellular vesicles. These results directly implicate endosomal influx of superoxide in endothelial inflammatory response and suggest that site-specific interception of this signal attained by targeted delivery of anti-PECAM/<em>SOD</em> into endothelial endosomes may have anti-inflammatory effects.

MnTE-2-PyP(5+) is a potent catalytic scavenger of reactive oxygen and nitrogen species, primarily superoxide and peroxynitrite. It therefore not only attenuates primary oxidative damage, but was found to modulate redox-based signaling pathways (HIF-1alpha, NF-kappaB, SP-1, and AP-1) and thus, in turn, secondary oxidative injury also. Cancer has been widely considered an oxidative stress condition. The goal of this study was to prove if and why a catalytic <em>SOD</em> mimic/peroxynitrite scavenger would exert anti-cancer effects, i.e., to evaluate whether the attenuation of the oxidative stress by MnTE-2-PyP(5+) could suppress tumor growth in a 4T1 mouse breast tumor model. Tumor cells were implanted into Balb/C mouse flanks. Three groups of mice (n=25) were studied: control (PBS) and 2 and 15 mg/kg/day of MnTE-2-PyP(5+) given subcutaneously twice daily starting when the tumors averaged 200 mm(<em>3</em>) (until they reached approximately 5-fold the initial volume). Intratumoral hypoxia (pimonidazole, carbonic anhydrase), HIF-1alpha, VEGF, proliferating capillary index (CD105), microvessel density (CD<em>3</em>1), protein nitration, DNA oxidation (8-OHdG), NADPH oxidase (Nox-4), apoptosis (CD<em>3</em>1), macrophage infiltration (CD68), and tumor drug levels were assessed. With 2 mg/kg/day a trend toward tumor growth delay was observed, and a significant trend was observed with 15 mg/kg/day. The 7.5-fold increase in drug dose was accompanied by a similar (6-fold) increase in tumor drug levels. Oxidative stress was largely attenuated as observed through the decreased levels of DNA damage, protein <em>3</em>-nitrotyrosine, macrophage infiltration, and NADPH oxidase. Further, hypoxia was significantly decreased as were the levels of HIF-1alpha and VEGF. Consequently, suppression of angiogenesis was observed; both the microvessel density and the endothelial cell proliferation were markedly decreased. Our study indicates for the first time that MnTE-2-PyP(5+) has anti-cancer activity in its own right. The anti-cancer activity via HIF/VEGF pathways probably arises from the impact of the drug on the oxidative stress. Therefore, the catalytic scavenging of ROS/RNS by antioxidants, which in turn suppresses cellular transcriptional activity, could be an appropriate strategy for anti-cancer therapy. Enhancement of the anti-cancer effects may be achieved by optimizing the dosing regime, utilizing more bioavailable Mn porphyrins (MnP), and combining MnP treatment with irradiation, hyperthermia, and chemotherapy. Mn porphyrins may be advantageous compared to other anti-cancer drugs, owing to their radioprotection of normal tissue and the ability to afford pain management in cancer patients via prevention of chronic morphine tolerance.

Carrageenan produces both inflammation and pain when injected in rat paws via enhancement of the formation of reactive oxygen species. We have tested the effect of 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPOL), a membrane-permeable superoxide dismutase (<em>SOD</em>) mimetic in carrageenan-induced rat paw edema. Treatment of rats with TEMPOL (15, <em>3</em>0, and 60 mg/kg, 15 min prior to carrageenan) inhibited the paw edema. Furthermore, treatment of rats with the <em>SOD</em> inhibitor diethylthiocarbamate (DETCA, 100 mg/kg, 1 h before carrageenan) enhanced the carrageenan-induced paw edema. Co-administration of peroxynitrite with carrageenan produced a similar fortification of the carrageenan-induced edema. Prior treatment of rats with TEMPOL (<em>3</em>0 mg/kg) inhibited the enhancement produced by DETCA treatment (endogenous superoxide anion stress) as well as that produced by the peroxynitrite stress. The effect of TEMPOL as well as the influence of superoxide anion and peroxynitrite stresses was also tested in carrageenan-induced hyperalgesia model. Carrageenan (500 mug/paw) produced significant hyperalgesia presented as shortening of withdrawal latency times using hot plate (52 degrees C) starting <em>3</em>0 min after carrageenan and lasting for <em>3</em> h. TEMPOL (60 mg/kg, injected 15 min before carrageenan) ameliorated this hyperalgesia during the first 2 h. Concurrent administration of peroxynitrite promptly intensified the carrageenan hyperalgesia. TEMPOL (60 mg/kg, 15 min before peroxynitrite-carrageenan) inhibited the peroxynitrite enhancement of carrageenan hyperalgesia when tested at 60 min after injection of the cocktail. The present investigation gives the proof for the effectiveness of TEMPOL as anti-inflammation and analgesic agents in carrageenan-induced model of inflammation and hyperalgesia. It further indicated the importance of superoxide anion and peroxynitrite in acute inflammation and inflammatory pain. This raises the chances for considering pharmacologic interventions that interrupt superoxide anion and peroxynitrite stress for putative alternative agents as anti-inflammatory analgesic new medical strategies.

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-<em>3</em> activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-<em>3</em> activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-<em>3</em> activation and extensive <em>SOD</em>/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.

This study was conducted to investigate the neuroprotective effects of 20(S)-ginsenoside Rg<em>3</em> on focal cerebral ischemia in rats. Middle cerebral artery occlusion (MCAO) model in male Wistar-Kyoto (WKY) rats was employed. The behavioral tests were used to evaluate the damage to central nervous system. The infarct area of brain was assessed in the brain slices stained with 2,<em>3</em>,5-triphenyltetrazolium chloride (TTC). Hydrogen clearance techniques were used to monitor regional cerebral blood flow (rCBF), spectrophotometric assay methods were used to determine the activities of superoxide dismutase (<em>SOD</em>) and glutathione-peroxidase (GSH-Px), contents of malondialdehyde (MDA) and adenosine triphosphate (ATP) of the brain. Furthermore, the respiratory control ratio (RCR=State <em>3</em>/State 4) was assessed in the brain mitochondria. The results showed that sublingual vein injection of 20(S)-ginsenoside Rg<em>3</em> at doses of 10 and 5 mg kg(-1), but not 2.5 mg kg(-1) exhibited significant neuroprotective effects on rats against focal cerebral ischemic injury by markedly decreasing neurological deficit scores, reducing the infarct area and enhancing the rCBF compared with the control group. At the same time, 20(S)-ginsenoside Rg<em>3</em> significantly improved mitochondrial energy metabolism, antagonized decreases in <em>SOD</em> and GSH-Px activities and increase in MDA level induced by cerebral ischemia. All these findings suggest that 20(S)-ginsenoside Rg<em>3</em> might provide neuroprotection against the cerebral ischemia-induced injury in rat brain through reducing lipid peroxides, scavenging free radicals and improving the energy metabolism.

Recent studies have indicated that sepsis is associated with enhanced generation of several free-radical species (nitric oxide [NO], superoxide, hydrogen peroxide) in skeletal muscle. It is also known that this enhanced free-radical generation results in reductions in skeletal muscle force-generating capacity, but the precise mechanism(s) by which free radicals exert this effect in sepsis has not been determined. We postulated that free radicals might react directly with the contractile proteins in this condition, altering contractile protein force-generating capacity. To test this theory, we compared the force generation of single Triton-skinned diaphragmatic fibers (Triton skinning exposes the contractile apparatus, permitting direct assessment of contractile protein function) from the following groups of rats: (1) control animals; (2) endotoxin-treated animal; (<em>3</em>) animals given endotoxin plus polyethylene glycol- superoxide dismutase (PEG-<em>SOD</em>), a superoxide scavenger; (4) animals given endotoxin plus N(omega)-nitro-L-arginine methylester (L-NAME), a NO synthase inhibitor; (5 ) animals given only PEG-<em>SOD</em> or L-NAME; and (6 ) animals given endotoxin plus denatured PEG-<em>SOD</em>. We found that endotoxin administration produced both a reduction in the maximum force-generating capacity (Fmax) (i.e., a decrease in Fmax) of muscle fibers and a reduction in fiber calcium sensitivity (i.e., an increase in the Ca2+ concentration required to produce half-maximal activation [Ca50]). L-NAME and PEG-<em>SOD</em> administration preserved Fmax and Ca50 in endotoxin-treated animals; neither drug affected these parameters in non-endotoxin treated animals. Denatured PEG-<em>SOD</em> failed to inhibit endotoxin-related alterations in contractile protein function. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of skinned fibers from endotoxin-treated animals revealed a selective depletion of several proteins; administration of L-NAME or PEG-<em>SOD</em> to endotoxin-treated animals prevented this protein depletion, paralleling the effect of these two agents to prevent a reduction in contractile protein force-generating capacity. These data indicate that free radicals (superoxide, NO, or daughter species of these radicals) play a central role in altering skeletal muscle contractile protein force-generating capacity in endotoxin-induced sepsis.

BACKGROUND

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in children, for which no specific treatment or vaccine is currently available. We have previously shown that RSV induces reactive oxygen species in cultured cells and oxidative injury in the lungs of experimentally infected mice. The mechanism(s) of RSV-induced oxidative stress in vivo is not known.

OBJECTIVE

To measure changes of lung antioxidant enzymes expression/activity and activation of NF-E2-related factor 2 (Nrf2), a transcription factor that regulates detoxifying and antioxidant enzyme gene expression, in mice and in infants with naturally acquired RSV infection.

METHODS

Superoxide dismutase 1 (<em>SOD</em> 1), <em>SOD</em> 2, <em>SOD</em> <em>3</em>, catalase, glutathione peroxidase, and glutathione S-transferase, as well as Nrf2 expression, were measured in murine bronchoalveolar lavage, cell extracts of conductive airways, and/or in human nasopharyngeal secretions by Western blot and two-dimensional gel electrophoresis. Antioxidant enzyme activity and markers of oxidative cell injury were measured in either murine bronchoalveolar lavage or nasopharyngeal secretions by colorimetric/immunoassays.

RESULTS

RSV infection induced a significant decrease in the expression and/or activity of SOD, catalase, glutathione S-transferase, and glutathione peroxidase in murine lungs and in the airways of children with severe bronchiolitis. Markers of oxidative damage correlated with severity of clinical illness in RSV-infected infants. Nrf2 expression was also significantly reduced in the lungs of viral-infected mice.

CONCLUSIONS

RSV infection induces significant down-regulation of the airway antioxidant system in vivo, likely resulting in lung oxidative damage. Modulation of oxidative stress may pave the way toward important advances in the therapeutic approach of RSV-induced acute lung disease.

A new spectrophotometric assay of superoxide dismutase (<em>SOD</em>) activity is described. The assay is based on the <em>SOD</em>-mediated increase in the rate of autoxidation of 5,6,6a,11b-tetrahydro-<em>3</em>,9,10-trihydroxybenzo[c]fluorene (BXT-01050) in aqueous alkaline solution. This autoxidation yields a chromophore with a maximal absorbance wavelength of 525 nm. The optimized assay of <em>SOD</em> activity is performed at pH 8.8, <em>3</em>7 degrees C, in 50 mM air-saturated 2-amino-2-methyl-1,<em>3</em>- propanediol buffer containing <em>3</em> mM boric acid and 0.1 mM diethylenetriaminepentaacetic acid. The kinetic measurement of 525-nm absorbance is performed for 1 min upon addition of BXT-01050. BXT-01050 is stabilized in stock solution acidified at pH 1.5. The <em>SOD</em> activity is determined from the Vs/Vc ratio of the autoxidation rates measured in the presence (Vs) and in the absence (Vc) of sample. One <em>SOD</em> activity unit (U-525) has been defined as the activity that doubles the autoxidation background (Vs/Vc = 2). The equation that fits the standard curve is the same with all studied <em>SODs</em>. Another reagent, 1,4,6-trimethyl-2-vinylpyridinium trifluoromethanesulfonate, directly eliminates interference due to sample mercaptans such as glutathione by means of a very fast alkylation reaction. A fast and reproducible measurement of <em>SOD</em> activity requires only a single determination per sample. At pH 8.8, an optimal assay sensitivity is achieved without strongly affecting the activity of known <em>SODs</em> such as Cu/Zn-, Mn-, or Fe-<em>SOD</em>.

Previously, we have shown in an experimental model of Trypanosoma cruzi infection that increased oxidative stress and antioxidant insufficiency are associated with myocardial (cellular and mitochondrial) oxidative damage and mitochondrial functional decline and might be of pathological significance in Chagas disease. In the present study, we investigated whether enhanced oxidative stress and mitochondrial functional decline are found in human chagasic patients. Our data show substantially higher plasma (two-four-fold) and mitochondrial (67%) malonylaldehyde (MDA) levels in chagasic (n = 80, group 2) compared to healthy (n = 50, group 1) subjects. Moreover, antioxidant defense was compromised in chagasic patients. Hence, we noted a 50% decline in glutathione content and losses of <em>3</em>1, 60, and 68% in glutathione peroxidase, superoxide dismutase (<em>SOD</em>), and Mn<em>SOD</em> activities, respectively, relative to the findings in healthy controls. Further, chagasic subjects exhibited decreased mitochondrial respiratory complex (CI: 72%; CIII: 71%) activities. Nonchagasic cardiomyopathy subjects (n = 20, group <em>3</em>) exhibited marginally higher plasma MDA levels compared to gp1 subjects and were not compromised in plasma antioxidant defense capacity. These data suggest that human chagasic patients sustain an antioxidant/oxidant imbalance and a mitochondrial decline of respiratory complex activities in the circulatory system. A positive correlation between increased MDA levels, Mn<em>SOD</em> decline, and inhibition of respiratory complexes suggests that oxidative stress may contribute to mitochondrial dysfunction in chagasic patients.

Enrichment of diet with omega-<em>3</em> lipid rich-menhaden fish oil (FO) when fed ad libitum to autoimmune lupus-prone NZB/NZW F1 (B/W) female mice delayed the onset and slowed progression of renal disease while significantly extending life-span compared to omega-6 lipid rich-corn oil (CO)-fed mice. Northern blot analysis of kidneys from FO-fed mice revealed no detectable levels of IL-1 beta, IL-6 and TNF alpha mRNA contrasted to levels that were easily detected in CO-fed mice. In contrast to the cytokines, FO-fed mice showed higher renal levels of the antioxidant enzymes-catalase, glutathione peroxidase (GSH-Px), superoxide dismutase (<em>SOD</em>)-mRNAs compared to CO-fed mice. The results suggest that dietary supplementation with FO, as compared to CO, inhibits the production of pro-inflammatory cytokines and ameliorates immune-complex-mediated kidney injury possibly by enhancing the ability of cells to dispose of harmful reactive oxygen intermediates.

High altitude exposure results in decreased partial pressure of oxygen and an increased formation of reactive oxygen and nitrogen species (RONS), which causes oxidative damage to lipids, proteins and DNA. Exposure to high altitude appears to decrease the activity and effectiveness of antioxidant enzyme system. The antioxidant system is very less in brain tissue and is very much susceptible to hypoxic stress. The aim of the present study was to investigate the time dependent and region specific changes in cortex, hippocampus and striatum on oxidative stress markers on chronic exposure to hypobaric hypoxia. The rats were exposed to simulated high altitude equivalent to 6100 m in animal decompression chamber for <em>3</em> and 7 days. Results indicate an increase in oxidative stress as seen by increase in free radical production, nitric oxide level, lipid peroxidation and lactate dehydrogenase levels. The magnitude of increase in oxidative stress was more in 7 days exposure group as compared to <em>3</em> days exposure group. The antioxidant defence system such as reduced glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GR), superoxide dismutase (<em>SOD</em>) and reduced/oxidized glutathione (GSH/GSSG) levels were significantly decreased in all the three regions. The observation suggests that the hippocampus is more susceptible to hypoxia than the cortex and striatum. It may be concluded that hypoxia differentially affects the antioxidant status in the cortex, hippocampus and striatum.

Pyrethroid pesticides are used preferably over organochlorines and organophosphates due to their high effectiveness, low toxicity to non-target organisms and easy biodegradibility. However, it is possible that during the pyrethroid metabolism, there is generation of reactive oxygen species (ROS) and pyrethroids may produce oxidative stress in intoxicated rats. The present study was therefore, undertaken to determine pyrethroid-induced lipid peroxidation (LPO) and to show whether pyrethroid intoxication alters the antioxidant system in erythrocytes. A single dose of cypermethrin and/or fenvalerate (0.001% LD50) was administered orally to rats and the animals were sacrificed at 0, 1, <em>3</em>, 7 and 14 days of treatment. The results showed that lipid peroxidation (LPO) in erythrocytes increased within <em>3</em> days of pyrethroid treatment. The increased oxidative stress resulted in an increase in the activity of antioxidant enzymes such as superoxide dismutase (<em>SOD</em>) and catalase (CAT). The increase in reduced glutathione (GSH) content in erythrocytes may probably be an initial adaptive response to increased oxidative stress in pyrethroid intoxicated rats. Erythrocytes and serum acetylcholinesterase (AChE) activity was measured in pyrethroid-induced oxidative stress as it may mimic inhibition in target tissues such as muscle and brain. The inhibition in erythrocytes and serum AChE activity was partially relieved over a period of time indicating recovery from pyrethroid intoxication. The increase in erythrocyte LPO correlated with the inhibition in erythrocyte AChE activity and so erythrocyte AChE can be a marker enzyme in pyrethroid toxicity. The results show oxidative stress and alteration in antioxidant enzymes in erythrocytes of pyrethroid intoxicated rats.

Cognitive and neuropsychological functions have been impaired at high altitude and the effects depend on altitude and duration of stay. However, the neurobiological mechanism of this impairment is poorly understood especially exposure to different duration. Aim of the present study was to investigate the changes of behavior, biochemistry and morphology after exposure to different duration of hypobaric hypoxia. The rats were exposed continuously to a simulated high altitude of 6100m for <em>3</em>, 7, 14 and 21 days in an animal decompression chamber. Spatial reference memory was tested by Morris water maze. The oxidative stress markers like free radicals, NO, lipid peroxidation, LDH activity and antioxidant systems like GSH, GSSG, GPx, GR, <em>SOD</em> were estimated from cortex, hippocampus and striatum. The morphological changes, neurodegeneration, DNA fragmentation and mode of cell death have also been studied. It was observed that the spatial reference memory was significantly affected after exposure to hypobaric hypoxia. Increased oxidative stress markers along with decreased effectiveness of antioxidant system were also observed in hypoxia-exposed animals. Further pyknotic, shrunken, tangle-like neurons were observed in all these regions after hypoxia and neurodegeneration, DNA fragmentation and apoptosis were also observed in all the three regions. But after 21 days of exposure, the spatial memory was improved along with improvement of antioxidant activities. Our result suggests that the apoptotic death may be involved in HA-induced memory impairment and after 7 days of exposure the effect was more pronounced but after 21 days of exposure recovery was observed.

Coontail (Ceratophyllum demersum L.) plants when exposed to various concentrations of Pb (1-100microM) for 1-7days, exhibited both phytotoxic and tolerance responses. The specific responses were function of concentration and duration. Plants accumulated 1748mugPbg(-1) dw after 7d which reflected its metal accumulation ability, however most of the metal (1222microgg(-1) dw, 70%) was accumulated after 1d exposure only. The toxic effect and oxidative stress caused by Pb were evident by the reduction in biomass and photosynthetic pigments and increase in malondialddehyde (MDA) content and electrical conductivity with increase in metal concentration and exposure duration. Morphological symptoms of senescence phenomena such as chlorosis and fragmentation of leaves were observed after 7d. The metal tolerance and detoxification strategy adopted by the plant was investigated with reference to antioxidant system and synthesis of phytochelatins. Protein and antioxidant enzymes viz., superoxide dismutase (<em>SOD</em>, EC 1.15.1.1), guaiacol peroxidase (GPX, EC 1.11.1.7) ascorbate peroxidase (APX, EC 1.11.1.11), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) showed induction at lower concentration and duration followed by decline. All enzymes except GPX showed maximum activity after 1d. An increase in cysteine, non-protein thiols (NP-SH) and glutathione (GSH) content was observed at moderate exposure conditions followed by decline. Phytochelatins (PC(2) and PC(<em>3</em>)) were synthesized to significant levels at 10 and 50microM Pb with concomitant decrease in GSH levels. Thus production of PCs seems important for the detoxification of metal, however it may lead to depletion of GSH and consequently oxidative stress. Results suggest that plants responded positively to moderate Pb concentrations and accumulated high amount of metal. Due to metal accumulation coupled with detoxification potential, the plant appears to have potential for its use as phytoremediator species in aquatic environments having moderate pollution of Pb.

The exercise-induced expression of heat shock proteins (HSPs) in rodent models is relatively well defined. In contrast, comparable data from human studies are limited and the exercise-induced stress response of human skeletal muscle is far from understood. This study has characterized the time course and magnitude of the HSP response in the skeletal muscles of a healthy active, but untrained, young male population following a running exercise protocol. Eight subjects performed 45 min of treadmill running at a speed corresponding to their lactate threshold (11.7 +/- 0.5 km/h; 69.8 +/- 4.8% maximum O2 uptake). Muscle biopsies were obtained from the vastus lateralis muscle immediately before and at 24 h, 48 h, 72 h, and 7 days postexercise. Exercise induced a significant (P < 0.05) but variable increase in HSP70, heat shock cognate (HSC) 70, and HSP60 expression with peak increases (typically occurring at 48 h postexercise) to 210, 170, and 1<em>3</em>9% of preexercise levels, respectively. In contrast, exercise did not induce a significant increase in either HSP27, alphaB-crystallin, <em>SOD</em> 2 (Mn<em>SOD</em>) protein content, or the activity of <em>SOD</em> and catalase. When examining baseline protein levels, HSC70, HSP27, and alphaB-crystallin appeared consistently expressed between subjects, whereas HSP70 and Mn<em>SOD</em> displayed marked individual variation of up to <em>3</em>- and 1.5-fold, respectively. These data are the first to define the time course and extent of HSP production in human skeletal muscle following a moderately demanding and nondamaging running exercise protocol. Data demonstrate a differential effect of aerobic exercise on specific HSPs.

BACKGROUND

Among the procedure-related factors associated with post-ERCP pancreatitis, selective cannulation of the common bile duct by insertion of a guidewire may be associated with fewer complications than conventional methods of cannulation with contrast injection to access the bile duct. However, the results of studies regarding the usefulness of wire-guided cannulation (WGC) are conflicting.

OBJECTIVE

This prospective randomized trial was designed to determine whether WGC reduces the rate of post-ERCP pancreatitis.

METHODS

A prospective randomized controlled trial.

METHODS

Tertiary-care academic medical center.

METHODS

A total of <em>3</em>00 consecutive patients with native papilla and pancreaticobiliary disease who were candidates for therapeutic ERCP were randomized from June 2006 to May 2007.

METHODS

WGC without contrast injection or conventional cannulation with contrast injection.

METHODS

Post-ERCP pancreatitis, risk factors, and procedure-related complications were evaluated prospectively.

RESULTS

A total of <em>3</em> patients (2%) in the WGC group and 17 patients (11.<em>3</em>%) in the conventional group had post-ERCP pancreatitis (P = .001). Among the cases of acute pancreatitis in the WGC group, 2 patients with suspected sphincter of Oddi dysfunction (<em>SOD</em>) and unintentional main pancreatic duct (PD) guidewire cannulation showed post-ERCP pancreatitis despite the use of WGC. In multivariate analysis, WGC was a protective factor (odds ratio 0.1; 95% CI, 0.024-0.490, P = .004), whereas female sex and <em>SOD</em> were risk factors for post-ERCP pancreatitis.

CONCLUSIONS

Our study population was a low-risk cohort.

CONCLUSIONS

WGC is associated with a lower rate of post-ERCP pancreatitis. However, WGC may not prevent post-ERCP pancreatitis in patients with suspected SOD and unintentional PD guidewire cannulation.

Researches on the pathology of spinal cord injury (SCI) have been recently focused on oxidative radicals stress and inflammation associated neuronal apoptosis. Resveratrol, a natural phenolic compound, has been extensively studied and shown a wide variety of health beneficial effects, including prevention of cardiovascular diseases and cancer and neuroprotective activities. However, the study of its potential role in neuroprotection and underlying mechanism in SCI model has been limited. In this study, we investigated the effect of resveratrol on neurologic functions and histopathologic changes after SCI and the mechanism underlying its neuroprotective effects. First, neuronal function after SCI was evaluated with Basso Beattle Bresnahan locomotor rating scale (BBB) and the result showed that injured animals treated with resveratrol showed a significant increase in BBB scores. Further, histopathological alternations were evaluated with HE and Nissl staining, showing a restored neural morphology and an increase of the number of neurons after resveratrol administration. To explore the underlying mechanism, anti-oxidation effect of resveratrol was assessed by measuring superoxide dismutase (<em>SOD</em>) activity and malondialdehyde (MDA) level after SCI. Resveratrol treatment reversed the decrease of <em>SOD</em> activity and increase of MDA level caused by SCI, suggesting its anti-oxidation role in response to the injury. In addition, resveratrol treatment suppressed immunoreactivity and expression of inflammatory cytokines including IL-1β, IL-10, TNF-α, and myeloperoxidase (MPO) after SCI, suggesting an anti-inflammation effect of resveratrol. Finally, resveratrol treatment inhibited injury-induced apoptosis as assessed by electrical microscopy and TUNEL staining and affected the expression level of apoptosis-related gene Bax, Bcl-2 and caspase-<em>3</em>, which indicated its anti-apoptosis role after SCI. Our data suggest that resveratrol significantly promotes the recovery of rat dorsal neuronal function after SCI, and this effect is related to its characteristics of anti-oxidation, anti-inflammation and anti-apoptosis.

BACKGROUND

Chronic consumption of a high-salt diet causes hypertension (HTN) and renal injury in Dahl salt-sensitive (SSR) but not salt-resistant rats (SRR). These events are, in part, mediated by oxidative stress and inflammation in the kidney and vascular tissues. Activation of the angiotensin II type 1 (AT(1)) receptor plays an important role in the pathogenesis of oxidative stress and inflammation in many hypertensive disorders. However, the systemic renin-angiotensin system (RAS) is typically suppressed in salt-sensitive HTN. This study was designed to test the hypothesis that differential response to a high-salt diet in SSR versus SRR may be related to upregulation of tissue RAS and pathways involved in inflammation and reactive oxygen species (ROS) production.

RESULTS

SSR and SRR were studied <em>3</em> weeks after consumption of high- (8%) or low-salt (0.07%) diets. The SSR consuming a low-salt diet exhibited significant increases in AT(1) receptor, cyclooxygenase (COX) 2, plasminogen activator inhibitor (PAI) and phospho-I kappaB in the kidney as compared to those found in SRR. The high-salt diet resulted in severe HTN and proteinuria (in SSR but not SRR) and marked elevations of renal tissue monocyte chemoattractant protein 1, p22(phox), NADPH oxidase subunit 4, angiotensin-II-positive cell count, infiltrating T cells and macrophages and further increases in AT(1) receptor, COX-2, PAI-1 and phospho-I kappaB in the SSR group. The high-salt diet significantly lowered plasma renin activity (PRA) in SRR but not in the SSR. COX-1 abundance was similar on the low-salt diet and rose equally with the high-salt diet in both groups. Among subgroups of animals fed the low-salt diet, kidney glutathione peroxidase (GPX) abundance was significantly lower in the SSR than SRR. The high-salt diet raised GPX and mitochondrial superoxide dismutase (<em>SOD</em>) abundance in the SRR kidneys but failed to do so in SSR. Cu/Zn-<em>SOD</em> abundance was similar in the subgroups of SSR and SRR fed the low-salt diet. The high-salt diet resulted in downregulation of Cu/Zn-<em>SOD</em> in SSR but not SRR.

CONCLUSIONS

Salt sensitivity in the SSR is associated with upregulations of the intrarenal angiotensin system, ROS-generating and proinflammatory/profibrotic proteins and an inability to raise antioxidant enzymes and maximally suppress PRA in response to high salt intake. These events can contribute to renal injury with high salt intake in SSR.

Stimulation of the macrophage-like cell line THP-1 with the phorbol ester phorbol 12-myristate 1<em>3</em>-acetate (PMA) resulted in differentiation into cells with many features of macrophages. This differentiation was accompanied by transcriptional activation of the lipoprotein lipase (LPL) and apo E genes and accumulation of their protein products in the media. PMA-induced differentiation of the HEL and HL-60 cell lines was not accompanied by induction of the gene for LPL, whereas the apo E gene was induced slightly in HL-60 cells. By contrast, the gene for superoxide dismutase (<em>SOD</em>-1) was either unaffected (THP-1) or down regulated (HL-60 or HEL cells) by PMA treatment. Induction of LPL mRNA in THP-1 cells was dependent upon the concentration of phorbol ester added. A minimal concentration of 1.6 x 10(-8) M PMA was necessary for macrophage differentiation, induction of LPL mRNA, and synthesis of the enzyme. LPL mRNA accumulates within <em>3</em> h after stimulation with PMA and attains a maximum concentration after 6 h, thereafter slowly decreasing over the next <em>3</em> days. In contrast, the steady-state level of apo E mRNA in the same THP-1 cultures increased gradually over a period of 48 h after induction. These studies thus demonstrate that THP-1 cells are of value as a model to study the quantitative and temporal expression of the LPL and apo E genes during macrophage differentiation.