1. The aim of this work was to study potential mechanisms participating in postischemic protection of selectively vulnerable CA1 neurons in the hippocampus. Experiments were focused on measuring changes in endogenous antioxidant enzyme activity. 2. Forebrain cerebral ischemia was induced in a rat by four-vessel occlusion. Ten minutes of ischemia induces so-called delayed neuronal death in selectively vulnerable CA1 region <em>3</em> days later. After 7 days of reperfusion, 71.6% of neurons succumb to neurodegeneration. When 5 min of ischemia was used as postconditioning, 2 days after 10 min of cerebral ischemia, delayed neuronal death in CA1 was almost completely (89.9%) prevented. <em>3</em>. Searching for mechanisms of protection, we measured the activity of endogenous antioxidant enzymes. Activities of the antioxidant enzymes superoxide dismutase (<em>SOD</em>) and catalase (CAT) were measured in the hippocampus, striatum and cortex by spectrophotometric methods after 10 min of ischemia used as the preconditioning. Two days after the preconditioning or the sham operation, second ischemia was induced for 5 min. We observed significant increase of total <em>SOD</em> activity in all studied regions of the brain 5 h after postconditioning (5 min of ischemia). <em>SOD</em> activity decreased to control values after 24 h. 4. In some experiments, we used intraperitoneal injections of norepinephrine (<em>3</em>.1 microM/kg) or <em>3</em>-nitropropionic acid (20 mg/kg) as postconditioning, instead of ischemia. All three treatments resulted in significant increase of <em>SOD</em> activity, but norepinephrine was the most effective. The same effect as was seen for total <em>SOD</em> activity could be observed for CuZn-<em>SOD</em> as well as Mn-<em>SOD</em> activity. Similarly, considerable increase in the activity of catalase was detected 5 h after postconditioning (5 min of ischemia). It is interesting that the greatest changes were established in selectively vulnerable hippocampus and striatum. As in the case of <em>SOD</em>, the highest levels of CAT activity were induced by norepinephrine, while lower but significant increase in CAT activity was induced by <em>3</em>-nitropropionic acid.5. Our results suggest that endogenous antioxidants <em>SOD</em> and CAT could play considerable neuroprotective role after postconditioning.

Free radical-initiated lipid peroxidation (LP) following intestinal ischemia/reperfusion (I/R) may disrupt mucosal integrity. It is unknown if inhibition of LP prevents this injury. We analyzed rat ileum, subjected to I/R, for evidence of LP inhibition and structural damage following treatment with the 21-aminosteroid, U74<em>3</em>89F, a potent LP inhibitor. Four groups of Lewis rats were studied after superior mesenteric artery occlusion with ligation of collateral arcades: (i) no ischemia, (ii) 10 min ischemia, (iii) 10 min ischemia + 1 hr reperfusion, (iv) 10 min ischemia + 1 hr reperfusion + U74<em>3</em>89F (6 mg/kg i.v. prior to clamp removal and reperfusion). Ileal mucosa was analyzed for: 9i0 superoxide dismutase (<em>SOD</em>; U/mg protein), a key antioxidant enzyme, (ii) myeloperoxidase (MPO; U/mg protein), an index of PMN stimulation, (iii) malondialdehyde (MDA; pmole/mg), an end product of LP, and (iv) routine histology. MDA rose from 2.09 +/- 0.44 (mean +/- SE) in Group 1 to 15.10 +/- 2.22 in Group <em>3</em> following I/R (P < 0.01). In Group 2 and Group 4, MDA remained unchanged at <em>3</em>.25 +/- 1.<em>3</em>8 and 1.7<em>3</em> +/- 0.15, respectively. MPO, likewise, rose during I/R from 0.59 +/- 0.17 in Group 1 to 1.10 +/- 0.1<em>3</em> in Group <em>3</em> (P = 0.08) and 1.49 +/- 0.24 in Group 4 (P < 0.05). <em>SOD</em> did not vary significantly in the four groups studied. Despite PMN stimulation indicated by increased MPO with reperfusion, no PMN infiltration was seen histologically. U74<em>3</em>89F normalized MDA, indicating effective inhibition of LP; however, similar epithelial sloughing and edema and hemorrhage in the lamina propria were seen in treated and untreated rats. These data implicate MDA-independent or possibly LP-independent pathways in intestinal morphologic damage occurring with I/R.

BACKGROUND

Vitamin E supplements containing tocotrienols are now being recommended for optimum health but its effects are scarcely known. The objective was to determine the effects of Tocotrienol Rich Fraction (TRF) supplementation on lipid profile and oxidative status in healthy older individuals at a dose of 160 mg/day for 6 months.

METHODS

Sixty-two subjects were recruited from two age groups: <em>3</em>5-49 years (n = <em>3</em>1) and above 50 years (n = <em>3</em>1), and randomly assigned to receive either TRF or placebo capsules for six months. Blood samples were obtained at 0, <em>3</em>rd and 6th months.

RESULTS

HDL-cholesterol in the TRF-supplemented group was elevated after 6 months (p < 0.01). Protein carbonyl contents were markedly decreased (p < 0.001), whereas AGE levels were lowered in the>> 50 year-old group (p < 0.05). Plasma levels of total vitamin E particularly tocopherols were significantly increased in the TRF-supplemented group after <em>3</em> months (p < 0.01). Plasma total tocotrienols were only increased in the>> 50 year-old group after receiving 6 months of TRF supplementation. Changes in enzyme activities were only observed in the>> 50 year-old group. <em>SOD</em> activity was decreased after <em>3</em> (p < 0.05) and 6 (p < 0.05) months of TRF supplementation whereas CAT activity was decreased after <em>3</em> (p < 0.01) and 6 (p < 0.05) months in the placebo group. GPx activity was increased at 6 months for both treatment and placebo groups (p < 0.05).

CONCLUSIONS

The observed improvement of plasma cholesterol, AGE and antioxidant vitamin levels as well as the reduced protein damage may indicate a restoration of redox balance after TRF supplementation, particularly in individuals over 50 years of age.

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP-sensitive K+ (K ATP) channels and generation of reactive oxygen species (ROS). Insulin resistance accompanying obesity causes mitochondrial dysfunction, but the consequences on the cerebral circulation have not been fully identified. We evaluated the mitochondrial effects of diazoxide, a putative mitochondrial K ATP channel activator, on cerebral arteries of Zucker obese (ZO) rats with insulin resistance and lean (ZL) controls. Diameter measurements showed diminished diazoxide-induced vasodilation in ZO compared with ZL rats. Maximal relaxation was <em>3</em>8 +/- <em>3</em>% in ZL vs. 21 +/- 4% in ZO rats (P < 0.05). Iberiotoxin, a Ca2+-activated K+ channel inhibitor, or manganese(III) tetrakis(4-benzoic acid)porphyrin chloride, an <em>SOD</em> mimetic, or endothelial denudation diminished vasodilation to diazoxide, implicating Ca2+-activated K+ channels, ROS, and endothelial factors in vasodilation. Inhibition of nitric oxide synthase (NOS) in ZL rats diminished diazoxide-induced vasodilation in intact arteries, but vasodilation was unaffected in endothelium-denuded arteries. In contrast, NOS inhibition in ZO rats enhanced vasodilation in endothelium-denuded arteries, but intact arteries were unaffected, suggesting that activity of endothelial NOS was abolished, whereas factors derived from nonendothelial NOS promoted vasoconstriction. Fluorescence microscopy showed decreased mitochondrial depolarization, ROS production, and nitric oxide generation in response to diazoxide in ZO arteries. Protein and mRNA measurements revealed increased expression of endothelial NOS and <em>SODs</em> in ZO arteries. Thus, cerebrovascular dilation to mitochondria-derived factors involves integration of endothelial and smooth muscle mechanisms. Furthermore, mitochondria-mediated vasodilation was diminished in ZO rats due to impaired mitochondrial K(ATP) channel activation, diminished mitochondrial ROS generation, increased ROS scavenging, and abnormal NOS activity.

During the development of sepsis, the complication in central nervous system (CNS), appearing early and frequently relative to other systems, can obviously increase the mortality of sepsis. Moreover, sepsis survivors also accompany long-term cognitive dysfunction, while the ultimate causes and effective therapeutic strategies of brain injury in sepsis are still not fully clear. We designed this study to investigate the effects of 2% hydrogen gas (H2) on brain injury in a mouse model of sepsis. Male ICR mice were underwent cecal ligation and puncture (CLP) or sham operation. 2% H2 was inhaled for 60min beginning at both 1 and 6h after sham or CLP operation, respectively. H2 concentration in arterial blood, venous blood and brain tissue was detected after H2 inhalation separately. The survival rate was observed and recorded within 7 days after sham or CLP operation. The histopathologic changes and neuronal apoptosis were observed in hippocampus by Nissl staining and TUNEL assay. The permeability of brain-blood barrier (BBB), brain water content, inflammatory cytokines, activities of antioxidant enzymes (<em>SOD</em> and CAT) and oxidative products (MDA and 8-iso-PGF2α) in serum and hippocampus were detected at 24h after sham or CLP operation. The expressions of nucleus and total nuclear factor erythroid 2-related factor 2 (Nrf2) and cytoplasmic heme oxygenase-1(HO-1) in hippocampus were measured at 24h after sham or CLP operation. We assessed their cognitive function via Y-maze and Fear Conditioning test on day <em>3</em>, 5, 7 and 14 after operation. H2 treatment markedly improved the survival rate and cognitive dysfunction of septic mice. CLP mice showed obvious brain injury characterized by aggravated pathological damage, BBB disruption and brain edema at 24h after CLP operation, which was markedly alleviated by 2% H2 treatment. Furthermore, we found that the beneficial effects of H2 on brain injury in septic mice were linked to the decreased levels of inflammatory cytokines and oxidative products and the increased activities of antioxidant enzymes in serum and hippocampus. In addition, 2% H2 inhalation promoted the expression and transposition of Nrf2 and the expression of HO-1 to mitigate brain injury in sepsis. Thus, the inhalation of hydrogen gas may be a promising therapeutic strategy to relieve brain injury in sepsis.

There is evidence that reactive nitrogen species are implicated in diabetic vascular complications, but their sources and targets remain largely unidentified. In the present study, we aimed to study the roles of endothelial nitric oxide synthase (eNOS) in diabetes. Exposure of isolated bovine coronary arteries to high glucose (<em>3</em>0 mmol/l d-glucose) but not to osmotic control mannitol (<em>3</em>0 mmol/l) switched angiotensin II-stimulated prostacyclin (PGI(2))-dependent relaxation into a persistent vasoconstriction that was sensitive to either indomethacin, a cyclooxygenase inhibitor, or SQ29548, a selective thromboxane receptor antagonist. In parallel, high glucose, but not mannitol, significantly increased superoxide and <em>3</em>-nitrotyrosine in PGI(2) synthase (PGIS). Concurrent administration of polyethylene-glycolated superoxide dismutase (<em>SOD</em>), l-nitroarginine methyl ester, or sepiapterin not only reversed the effects of high glucose on both angiotensin II-induced relaxation and PGI(2) release but also abolished high-glucose-enhanced PGIS nitration, as well as its association with eNOS. Furthermore, diabetes significantly suppressed PGIS activity in parallel with increased superoxide and PGIS nitration in the aortas of diabetic C57BL6 mice but had less effect in diabetic mice either lacking eNOS or overexpressing human <em>SOD</em> (h<em>SOD</em>(+/+)), suggesting an eNOS-dependent PGIS nitration in vivo. We conclude that diabetes increases PGIS nitration in vivo, likely via dysfunctional eNOS.

Our ex vivo study revealed that BRE had significantly stronger ability to inhibit LDL oxidation than white rice extract (WRE). The purpose of this study was to investigate whether black rice extract (BRE) supplementation might ameliorate oxidative stress and enhance antioxidant enzyme activities in HepG2 cells and in C57BL/6 mice. In the cellular study, superoxide anions (O2*-) and reactive oxygen species (ROS) in the BRE group were significantly suppressed. The BRE group also showed significant increases in superoxide dismutase (<em>SOD</em>) and catalase (CAT) activities by 161.6% and 7<em>3</em>.4%, respectively. The major components responsible for the free-radical-scavenging and antioxidative properties might be cyanidin-<em>3</em>-O-glucoside chloride and peonidin-<em>3</em>-O-glucuside chloride. In the animal study, male C57BL/6 mice were divided into three groups (control, BRE, and WRE). Plasma HDL-cholesterol was significantly higher, and thiobarbituric, acid-reactive substances were significantly lower in the BRE group, whereas plasma levels of total cholesterol and triglyceride were not affected by BRE supplementation. Increased hepatic <em>SOD</em> and CAT activities were observed in BRE-treated mice as compared to the control mice. However, no changes were detected for the protein expression of antioxidant enzymes by Western blot analysis. Our data suggest that antioxidative effects exerted by BRE are mediated through decreases in free-radical generation as well as increases in <em>SOD</em> and CAT activities both in vitro and in vivo.

OBJECTIVE

Acute liver failure (ALF) of viral origin results from massive hepatocyte apoptosis induced by the interaction between Fas expressed on hepatocytes and Fas ligand on activated T lymphocytes. Because Fas-induced apoptosis of hepatocytes involves mitochondrial damages and potential reactive oxygen species (ROS) overproduction, we investigated whether manganese III tetrakis (5,10,15,20 benzoic acid) (MnTBAP), a nonpeptidyl mimic of superoxide dismutase (SOD), can inhibit Fas-induced ALF.

METHODS

An agonist anti-Fas monoclonal antibody was used to induce hepatocyte apoptosis in vitro and ALF in vivo.

RESULTS

Preventive and curative treatments by MnTBAP significantly increased survival rates and significantly reduced aspartate aminotransferase levels and parenchymal lesions. ROS generation was suggested by those beneficial effects and significant increases in SOD and Gpx activities after anti-Fas injection. Flow cytometry of isolated hepatocytes incubated with anti-Fas monoclonal antibody showed that ROS production was associated with the collapse of transmembrane potential and loss of cardiolipin content. After injection of anti-Fas monoclonal antibody, mitochondrial Bcl-2 was decreased, cytochrome c released, and caspase-3 activated. Mitochondrial alterations and their consequences were abrogated by MnTBAP.

CONCLUSIONS

ROS are key executioners in Fas-induced hepatocyte apoptosis. This finding explains why a nonpeptidyl mimic of SOD can cure ALF in a model of viral hepatitis, pointing out the potential interest of this molecule in humans.

OBJECTIVE

The protective effect of N-acetylcysteine (NAC) on nephrotoxicity due to contrast nephropathy and reperfusion-induced ischemia has been reported in experimental models. However, its efficacy on colistin-induced nephrotoxicity has not been elucidated yet. The primary aim of this study was to evaluate the nephrotoxic effect of colistin and to investigate the possible protective effect of NAC on colistin-induced nephrotoxicity. The secondary aim was to research the systemic effects of nephrotoxicity-induced oxidative stress on the lung.

METHODS

Eighteen female Sprague-Dawley rats were randomly assigned and were given (a) 1 ml/kg sterile saline, (b) <em>3</em>00,000 IU/kg/day colistin, and (c) <em>3</em>00,000 IU/kg/day colistin and 150 mg/kg NAC for six consecutive days.

RESULTS

Plasma blood urea nitrogen (BUN), creatinine, urinary creatinine, urinary protein, plasma TNF-alpha levels, renal tissue superoxide dismutase (<em>SOD</em>) and malondialdehyde (MDA) activity and immunocytochemical findings were evaluated. Colistin exerted nephrotoxicity and achieved a significant increase in plasma BUN and creatinine levels and renal tissue <em>SOD</em> levels. NAC exhibited no significant effect on biochemical parameters but reduced renal tissue <em>SOD</em> level and reversed immunocytochemical staining of inducible nitric oxide synthase (i-NOS) and neurotrophin-<em>3</em>. Increased oxidative stress in the lung tissue of the rats treated with colistin has also been documented. Additionally, NAC significantly reduced the immunostaining of endothelial NOS (e-NOS) and i-NOS in the lung tissue.

CONCLUSIONS

Colistin-induced renal toxicity may be attributable to oxidative damage. The combined treatment of colistin plus NAC seems to have a beneficial role in restoration of the oxidant injury which may be related to its antioxidant effect.

Several studies associate ethanol hepatic toxicity to the generation of reactive oxygen species. Ethanol metabolism by alcohol dehydrogenase (ADH) originates acetaldehyde and NADH, with the subsequent increase of the NADH/NAD+ ratio. Some authors have suggested that the oxidation of acetaldehyde by aldehyde oxidase (AO) may be responsible for oxyradical generation during ethanol metabolism. In this study we demonstrated that AO acts not only upon acetaldehyde but also upon NADH, with superoxide anion radical (O2.-) formation. The apparent Km of NADH for AO was approximately 28 microM, a much smaller value than the one reported for acetaldehyde (1 mM). The NADH oxidation by AO promoted the O2.- generation and the ADP-Fe(<em>3</em>+)-dependent microsomal lipid peroxidation in a NADH and AO concentration-dependent manner. If in these experiments NADH is substituted by ethanol, NAD+, and ADH, a higher level of lipid peroxidation will be obtained. To explain this observation a vicious cycle which increases the oxyradical production is suggested: ADH reduces NAD+ to NADH, which is oxidized by AO, generating reactive oxidative species plus NAD+ available again for reduction by ADH. From the studies which were done in the presence of some antioxidants it was observed that the addition of <em>SOD</em> and/or catalase did not inhibit lipid peroxidation, but these results do not exclude the participation of reactive oxygen species. Our studies indicate that the NADH oxidation by AO may play a role in ethanol-induced generation of reactive oxygen species, contributing to its hepatotoxicity.

Oxidative stress may be involved in the development of vascular complications associated with diabetes; however, the molecular mechanism responsible for increased production of free radicals in diabetes remains uncertain. Therefore, we examined whether acute hyperinsulinemia increases the production of free radicals and whether this condition affects proliferative extracellular signal-regulated kinase (ERK-1 and -2) signaling in human fibroblasts in vitro. Insulin treatment significantly increased intracellular superoxide anion (O(2)(-)) production, an effect completely abolished by Tiron, a cell-permeable superoxide dismutase (<em>SOD</em>) mimetic and by polyethylene glycol (PEG)-<em>SOD</em>, but not by PEG catalase. Furthermore, insulin-induced O(2)(-) production was attenuated by the NAD(P)H inhibitor apocynin, but not by rotenone or oxypurinol. Inhibition of the phosphatidylinositol <em>3</em>'-kinase (PI <em>3</em>'-kinase) pathway with LY294002 blocked insulin-stimulated O(2)(-) production, suggesting a direct involvement of PI <em>3</em>'-kinase in the activation of NAD(P)H oxidase. The insulin-induced free radical production led to membranous translocation of p47phox and markedly enhanced ERK-1 and -2 activation in human fibroblasts. In conclusion, these findings provided direct evidence that elevated insulin levels generate O(2)(-) by an NAD(P)H-dependent mechanism that involves the activation of PI <em>3</em>'-kinase and stimulates ERK-1- and ERK-2-dependent pathways. This effect of insulin may contribute to the pathogenesis and progression of cardiovascular disease in the insulin resistance syndrome.

Ethanol is known to cause both tolerance and sensitization to endotoxin (lipopolysaccharide). It is also known that ethanol modulates the expression and activity of several intracellular signaling molecules and transcription factors in monocytes and Kupffer cells, the resident hepatic macrophages. Expression of CD14, the endotoxin receptor, is up-regulated following chronic exposure to endotoxin and ethanol. Ethanol-induced oxidative stress is important in the regulation of transcription factor activation and cytokine production by Kupffer cells. Thus, it was hypothesized that acute ethanol increases CD14 expression through a mechanism dependent upon oxidant production. This hypothesis was tested by overexpression of superoxide dismutase via recombinant adenovirus. Mice were infected with adenovirus (<em>3</em> x 10(9) plaque-forming units, intravenously) containing either Cu,Zn superoxide dismutase (Ad.<em>SOD</em>1) or beta-galactosidase (Ad.lacZ), which caused significant expression of Cu,Zn-<em>SOD</em> in hepatocytes and Kupffer cells. Three days post-infection, mice were given saline or ethanol (5 g/kg, intragastrically). A significant increase in CD14 mRNA was observed <em>3</em> h after ethanol, and this increase was almost completely blocked in mice overexpressing Cu,Zn-<em>SOD</em>. Additionally, overexpression of <em>SOD</em> also blunted ethanol-induced activation of redox-sensitive transcription factors NFkappaB and AP-1 and production of cytokines. However, only inhibition of AP-1 with dominant-negative TAK1 but not NFkappaB by dominant-negative IkappaBalpha significantly blunted ethanol-induced increases in CD14, suggesting that AP-1 is important for CD14 transcriptional regulation. It is also shown here that NADPH oxidase is important in the increase in CD14 due to ethanol. Moreover, these data suggest that acute ethanol causes sensitization to endotoxin through mechanisms dependent upon oxidative stress.

Hyperbaric oxygen (HBO) therapy is an effective adjunct in treating ischemia-reperfusion (I/R) injury of brain, small intestine, testis, and crushing extremities. This study was designed to test the hypotheses that preconditioning the rats with HBO could protect the liver against subsequent I/R injury. Daily treatment with one-dose HBO (90 min, 2.5 ATA) was brought about for male Sprague Dawley rats for 1 to <em>3</em> days before an I/R injury of liver. Hepatic expression of heat-shock protein 70 (Hsp70), total concentration of glutathione (GSH), activity of catalase, superoxide dismutase (<em>SOD</em>), and serum AST and ALT were estimated before and after HBO, as well as after I/R injury. The results showed that activity of hepatic catalase was decreased by one dose, but not three doses, of HBO as compared with baseline data. However, hepatic Hsp70 expression fluctuated insignificantly. AST and ALT increase less in rats preconditioned with one-dose HBO as compared with those without HBO or with three-dose HBO. Our results showed preconditioning by one-dose HBO protects rat liver against subsequent ischemia-reperfusion injury.

1. The aim of the present study was to investigate the effect and mechanism of berberine, an alkaloid extracted from the traditional Chinese medicine coptis, on rat liver fibrosis induced by multiple hepatotoxic factors. 2. Male Wistar rats were separated into five groups, a normal control group, a fibrotic control group and fibrotic groups treated with three different doses of berberine. The fibrotic models were established by introduction of multiple hepatotoxic factors, including CCl(4), ethanol and high cholesterol. Rats in the treatment groups were administered 50, 100 or 200 mg/kg berberine, intragastrically, daily for 4 weeks. Serum levels of alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST), hepatic activity of superoxide dismutase (<em>SOD</em>) and hepatic malondialdehyde (MDA) and hepatic hydroxyproline (Hyp) content were determined. Liver biopsies were obtained for histological and immunohistochemical studies to detect the expressions of alpha-smooth muscle actin (SMA) and transforming growth factor (TGF)-beta1. <em>3</em>. The results showed that, compared with the fibrotic control group, serum levels of ALT and AST and hepatic content of MDA and Hyp were markedly decreased, but the activity of hepatic <em>SOD</em> was significantly increased in berberine-treated groups in a dose-dependent manner. In addition, histopathological changes, such as steatosis, necrosis and myofibroblast proliferation, were reduced and the expression of a-SMA and TGF-b1 was significantly downregulated in the berberine-treated groups (P < 0.01). 4. These results suggest that berberine could be used to prevent experimental liver fibrosis through regulation of the anti-oxidant system and lipid peroxidation.

BACKGROUND

Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction.

METHODS

IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 12<em>3</em> (Rh12<em>3</em>) and propidium iodide. Free radicals were trapped with alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone and measured using electron spin resonance.

RESULTS

After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2'-deoxyuridine labeling and increases of graft weight were only approximately <em>3</em>% and 0.2% in quarter-size grafts without IP, respectively, but increased to <em>3</em>2% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10, 27, <em>3</em>2, 60, 70, 72, 75 and Cu/Zn-superoxide dismutase (<em>SOD</em>) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-<em>SOD</em>.

CONCLUSIONS

Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.

To determine if oxygen free radical scavengers administered before coronary artery reperfusion can limit reperfusion arrhythmias, increase the return of regional function in ischemic myocardium, and reduce tissue necrosis at 1 week after 90-minute coronary artery occlusion and reperfusion, conscious dogs were treated with superoxide dismutase (<em>SOD</em>) and catalase before and for 1 hour after coronary artery reperfusion. Another group was treated with recombinant <em>SOD</em> (r<em>SOD</em>) because the commercially available <em>SOD</em> and catalase contained endotoxin. The conscious dogs were studied <em>3</em>-4 weeks after implanting left ventricular pressure gauges, ultrasonic wall thickness gauges in the posterior left ventricular wall, left atrial catheters, and arterial catheters, Doppler flow transducers, and hydraulic occluders on the left circumflex coronary artery. The only beneficial effect observed was that the number of arrhythmic beats per minute in the r<em>SOD</em>-treated group was significantly lower (p less than 0.05) when compared with a control group after coronary artery reperfusion. Treatment neither increased the amount of recovery of wall thickening in the ischemic zone nor reduced infarct size when expressed either as a percentage of the area at risk or as a function of collateral blood flow in the ischemic zone. For example, infarct size as a percentage of the area at risk was <em>3</em>2.6 +/- 5.8%, <em>3</em>7.4 +/- 6.4%, 28.<em>3</em> +/- 5.1% in the control, <em>SOD</em> and catalase-, and r<em>SOD</em>-treated groups, respectively. Thus, although treatment with oxygen free radical scavengers invoked a transient reduction in the number of reperfusion arrhythmias, this treatment in conscious dogs failed to improve regional myocardial dysfunction or reduce the amount of necrosis when compared with a control group. The lack of a sustained salutary effect may indicate that longer periods of treatment with free radical scavengers are required in chronic preparations.

Formation of the oxygen radical superoxide anion is one of the final events of several metabolic pathways in the cascade that leads to delayed neuronal death after traumatic or ischemic brain injury. In the laboratory, scavenging of the superoxide anion with native superoxide dismutase (<em>SOD</em>) or polyethylene glycol (PEG)-conjugated <em>SOD</em> (PEG-<em>SOD</em>) has been shown to be beneficial in several types of traumatic and ischemic injury. Accordingly, PEG-<em>SOD</em> was utilized in a randomized controlled Phase II trial to evaluate its safety and efficacy in severely head-injured patients with a Glasgow Coma Scale score of 8 or less. At two institutions, 104 patients were randomly assigned to receive either placebo or PEG-<em>SOD</em> (2000, 5000, or 10,000 U/kg) intravenously as a bolus, an average of 4 hours after injury. Prognostic factors were evenly distributed in the four groups, except for mean age which was significantly higher in the group receiving 10,000 U/kg than in the placebo group (mean age <em>3</em>4 years vs. 25 years). No complications attributed to the study medication were noted. The average intracranial pressure (ICP) was similar in the four groups, but the percentage of time during which ICP was above 20 mm Hg was less in the groups receiving 5000 or 10,000 U/kg of PEG-<em>SOD</em>. Patients in the group receiving 10,000 U/kg also required less mannitol for ICP control than the placebo group. Outcome was assessed using the Glasgow Outcome Scale at <em>3</em> and 6 months postinjury in 91 and 9<em>3</em> patients, respectively, by blinded observers not involved in the clinical management of the patients. At <em>3</em> months, 44% of patients in the placebo group were vegetative or had died, while only 20% of patients in the group receiving 10,000 U/kg of PEG-<em>SOD</em> were in these outcome categories (p < 0.0<em>3</em>, multiple logistic regression test); at 6 months, these figures were <em>3</em>6% and 21%, respectively (p = 0.04). Differences in outcome between the placebo group and either of the other two dosage groups were not statistically significant. It is concluded that PEG-<em>SOD</em> was generally well tolerated and appears promising in improving outcome after severe head injury. A larger, multicenter, Phase III trial, using a higher dose (20,000 U/kg) compared to placebo and to 10,000 U/kg of PEG-<em>SOD</em> is planned.

Mussels are used as sentinel organisms and bioindicators to evaluate the toxic effects of chemical pollutants in marine organisms, especially heavy metals, representing an important tool for biomonitoring environmental pollution in coastal areas. Antioxidant defence enzymes play an important role in cellular antioxidant defence systems and protect from oxidative damage by reactive oxygen species (ROS). Indigenous mussels Mytilus galloprovincialis of the Saronikos Gulf of Greece were used for monitoring heavy metal pollution in three polluted sites in the area and in one unpolluted site. Seasonal variations of the activity of antioxidant defence enzymes, superoxide dismutase (<em>SOD</em>) and catalase (CAT), as well as lipid peroxidation (LP) were measured as biomarkers in a period of three years in relation to concentrations of trace metals in their gills and mantle and compared to mussels from an unpolluted sampling site. <em>SOD</em> activity increased at least 2 fold at the polluted sites when compared to the control site (the high activity was recorded in the spring time). CAT activity was increased 2-<em>3</em> times at the polluted sites, with high activity in the winter and spring time, compared to the control site. LP concentration was twice higher at the polluted sites, following the same seasonal pattern. Trace metals contents in mussels collected at polluted sites were <em>3</em>-4 fold higher compared to the control site and showed moderate variations along the months, with a winter maximum followed by a summer pre-spawning minimum matching the seasonal trends of temperature and salinity. Our results showed that metal pollution in the Elefsis Bay (the most polluted coastal area) causes relatively medium levels of oxidative stress in tissues of mussels due to cellular oxy-radical generation. This study, which is the first in the area, showed that seasonal variations of the activity of antioxidant defence enzymes and LP concentrations in mussels can be used as potential biomarkers of toxicity for long-term monitoring in marine coastal ecosystems.

Mutation in any of the four clock genes (clk-1, clk-2, clk-<em>3</em>, gro-1) causes an average slowing down of many temporal processes, and an increase of mean life span. The latter effect has been linked to the slow phenotype, and it has been reasoned that any reduction of the rate of living would reduce the load of oxidative damage, which is thought to drive the ageing process. To test this model we measured several parameters describing metabolic output in wild type worms and all four Clk mutants. We found no gross changes in metabolic output, as assessed from oxygen consumption and heat production rates, lucigenin-mediated light production capacity, ATP content, and lipofuscin autofluorescence. Catalase and superoxide dismutase (<em>SOD</em>) were variably altered, but not cooperatively, as would be expected to enhance reactive oxygen species (ROS) scavenging activity. Thus we conclude that the prolonged life span of Clk mutants cannot be attributed to reduced metabolic rate or an increased activity of the major antioxidant enzymes catalase and <em>SOD</em>.

Indoleamine 2,<em>3</em>-dioxygenase (IDO) is the rate-limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme-free globin (Apo Hb), induced IDO expression in bone marrow-derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of I kappaB alpha. Hb-induced IDO expression was inhibited by inhibitors of PI<em>3</em>-kinase (PI<em>3</em>K), PKC and nuclear factor (NF)-kappaB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb-induced IDO expression was inhibited by anti-oxidant N-acetyl-L-cysteine (NAC) or mixtures of <em>SOD</em> and catalase, however, IDO expression was enhanced by <em>3</em>-amino-1,2,4-triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O(2) (-), H(2)O(2), and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI<em>3</em>K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI<em>3</em>K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF-kappaB through the PI<em>3</em>K-PKC-ROS and PI<em>3</em>K-Akt pathways is required for the Hb-induced IDO expression in BMDCs.

The mechanisms of sodium selenite-induced cell death in cervical carcinoma cells were studied during 24 h of exposure in the HeLa Hep-2 cell line. Selenite at the employed concentrations of 5 and 50 micromol/L produced time- and dose-dependent suppression of DNA synthesis and induced DNA damage which resulted in phosphorylation of histone H2A.X. These effects were influenced by pretreatment of cells with the <em>SOD</em>/catalase mimetic MnTMPyP or glutathione-depleting buthionine sulfoximine, suggesting the significant role of selenite-generated oxidative stress. Following the DNA damage, selenite activated p5<em>3</em>-dependent pathway as evidenced by the appearance of phosphorylated p5<em>3</em> and accumulation of p21 in the treated cells. Concomitantly, selenite activated p<em>3</em>8 pathway but its effect on JNK was very weak. p5<em>3</em>- and p<em>3</em>8-dependent signaling led to the accumulation of Bax protein, which was preventable by specific inhibitors of p<em>3</em>8 (SB 20<em>3</em>580) and p5<em>3</em> (Pifithrin-alpha). Mitochondria in selenite-treated cells changed their dynamics (shape and localization) and released AIF and Smac/Diablo, which initiated caspase-independent apoptosis as confirmed by the caspase-<em>3</em> activity assay and the low effect of caspase inhibitors z-DEVD-fmk and z-VAD-fmk on cell death. We conclude that selenite induces caspase-independent apoptosis in cervical carcinoma cells mostly by oxidative stress-mediated activation of p5<em>3</em> and p<em>3</em>8 pathways, but other selenite-mediated effects, in particular mitochondria-specific ones, are also involved.

Two modern fungicides, a strobilurin, azoxystrobin (AZO), and a triazole, epoxiconazole (EPO), applied as foliar spray on spring barley (Hordeum vulgare L. cv. Scarlett) <em>3</em> days prior to fumigation with injurious doses of ozone (150-250 ppb; 5 days; 7 h/day) induced a 50-60% protection against ozone injury on leaves. Fungicide treatments of barley plants at growth stage (GS) <em>3</em>2 significantly increased the total leaf soluble protein content. Additionally, activities of the antioxidative enzymes superoxide dismutase (<em>SOD</em>), catalase (CAT), ascorbate-peroxidase (APX) and glutathione reductase (GR) were increased by both fungicides at maximal rates of 16, 75, 51 and 144%, respectively. Guiacol-peroxidase (POX) activity was elevated by 50-110% only in AZO treated plants, while this effect was lacking after treatments with EPO. This coincided with elevated levels of hydrogen peroxide (H2O2) only in EPO and not in AZO treated plants. The enhancement of the plant antioxidative system by the two fungicides significantly and considerably reduced the level of superoxide (O2*-) in leaves. Fumigation of barley plants for 4 days with non-injurious ozone doses (120-150 ppb, 7 h/day) markedly and immediately stimulated O2*- accumulation in leaves, while H2O2 was increased only after the third day of fumigation. Therefore, O2*- itself or as precursor of even more toxic oxyradicals appears to be more indicative for ozone-induced leaf damage than H2O2. Ozone also induced significant increases in the activity of antioxidant enzymes (<em>SOD</em>, POX and CAT) after 2 days of fumigation in fungicide untreated plants, while after 4 days of fumigation these enzymes declined to a level lower than in unfumigated plants, due to the oxidative degradation of leaf proteins. This is the first report demonstrating the marked enhancement of plant antioxidative enzymes and the enhanced scavenging of potentially harmful O2*- by fungicides as a mechanism of protecting plants against noxious oxidative stress from the environment. The antioxidant effect of modern fungicides widely used in intense cereal production in many countries represents an important factor when evaluating potential air pollution effects in agriculture.

Idiopathic pulmonary arterial hypertension (IPAH) is associated with lower levels of the pulmonary vasodilator nitric oxide (NO) and its biochemical reaction products (nitrite [NO(2) (-)], nitrate [NO(<em>3</em>) (-)]), in part, due to the reduction in pulmonary endothelial NO synthesis. However, NO levels are also determined by consumptive reactions, such as with superoxide to form peroxynitrite, which subsequently may generate stable products of nitrotyrosine (Tyr-NO(2)) and/or NO(<em>3</em>) (-). In this context, superoxide dismutase (<em>SOD</em>) preserves NO in vivo by scavenging superoxide and preventing the consumptive reactions. Here, we hypothesized that reactive oxygen species (ROS) consumption of NO may contribute to the low NO level and development of pulmonary hypertension. To test this, nitrotyrosine and antioxidants glutathione (GSH), glutathione peroxidase (GPx), catalase, and <em>SOD</em> were evaluated in IPAH patients and healthy controls. <em>SOD</em> and GPx activities were decreased in IPAH lungs (all p < 0.05), while catalase and GSH activities were similar among the groups (all p>> 0.2). <em>SOD</em> activity was directly related to exhaled NO (eNO) (R(2)= 0.72, p= 0.002), and inversely related to bronchoalveolar lavage (BAL) NO(<em>3</em>) (-) (R(2)=-0.7<em>3</em>, p= 0.04). Pulmonary artery pressure (PAP) could be predicted by a regression model incorporating <em>SOD</em>, GPx, and NO(<em>3</em>) values (R(2)= 0.96, p= 0.01). These findings suggest that <em>SOD</em> and GPx are associated with alterations in NO and PAP in IPAH.

BACKGROUND

In acute pancreatitis (AP) administration of n-<em>3</em> polyunsaturated fatty acids (PUFAs) might change the course of the disease through modulation of eicosanoid synthesis.

METHODS

In a prospective, randomized clinical trial from 28 patients with moderate-severe AP, 14 received n-<em>3</em> PUFAs (fish oil) enterally (<em>3</em>.<em>3</em>g/day for 5-7 days). Measurement of erythrocyte superoxide-dysmutase (SOD) activity, serum total antioxidant status (TAS), vitamin A and E, fatty acids, C-reactive protein, transthyretin concentrations were performed at admission, day <em>3</em>, 7 and 14.

RESULTS

The n-<em>3</em> to n-6 LCPUFA ratios increased significantly in serum lipids of the patients receiving n-<em>3</em> PUFA supplementation, whereas remained unchanged in the controls. Supplementation resulted in significant decrease in length of hospitalization (1<em>3</em>.07+/-6.70 vs. 19.28+/-7.18 days, P<0.05) and jejunal feeding (10.57+/-6.70 vs. 17.57+/-10.52, P<0.05). Complications developed in 6/14 (42%) of treated and 9/14 (64%) of control patients. The SOD activity was significantly higher at day <em>3</em> in the supplemented group (P<0.05), but there were no significant differences between the two groups in other antioxidants and acute phase reactants.

CONCLUSIONS

The use of enteral formula enriched with n-<em>3</em> PUFAs in the treatment of AP seems to have clinical benefits based upon the shortened time of jejunal feeding and hospital stay.