BACKGROUND

Tempol is a permeant nitroxide superoxide dismutase (SOD) mimetic that lowers mean arterial pressure (MAP) in spontaneously hypertensive rats (SHRs). We investigated the hypothesis that the antihypertensive response entails a negative salt balance, blunting of plasma renin activity (PRA), endothelin-1 (ET-1), or catecholamines or correction of oxidative stress as indexed by 8-isoprostane prostaglandin F(2alpha) (PGF(2alpha)) (8-Iso).

METHODS

Groups (N= 6 to 8) of SHRs were infused for 2 weeks with vehicle or tempol (200 nmol/kg/min) or given tempol (2 mmol/L) in drinking water.

RESULTS

Tempol infusion reduced the MAP of anesthetized SHRs (150 +/- 5 vs. 126 +/- 6 mm Hg) (P < 0.005). Oral tempol did not change the heart rate but reduced the MAP of conscious SHRs (-23 +/- 6 mm Hg) (P < 0.01) but not Wistar-Kyoto (WKY) rats. Tempol infusion increased the PRA (2.2 +/- 0.2 vs. 5.0 +/- 0.9 ng/mL/hour) (P < 0.005), did not change excretion of nitric oxide (NO) [NO(2)+ NO(3) (NOx)], ET-1, or catecholamines but reduced excretion of 8-Iso (13.2 +/- 1.4 vs. 9.6 +/- 0.9 ng/24 hours; P < 0.01). Cumulative Na(+) balance and gain in body weight were unaltered by tempol infusion. Tempol prevented a rise in MAP with high salt intake.

CONCLUSIONS

Tempol corrects hypertension without a compensatory sympathoadrenal activation or salt retention. The response is independent of nitric oxide, endothelin, or catecholamines and occurs despite increased PRA. It is accompanied by a reduction in oxidative stress and is maintained during increased salt intake.

OBJECTIVE

To investigate maternal and neonatal outcomes after antioxidant supplementation relatively early in pregnancy (8 to 12 weeks) for pregnant women with low antioxidant status.

METHODS

A randomized, double-blind, placebo-controlled trial of daily antioxidant supplementation was performed on pregnant women screening positive for low antioxidant status at 8 to 12 weeks of gestation. Low antioxidant status was defined as a superoxidedismutase (SOD) level below 1102 U/g Hb or 164 U/mL. The supplementation group received the following antioxidants daily: vitamins A (1000 IU), B6 (2.2 mg), B12 (2.2 microg), C (200 mg), and E (400 IU), folic acid (400 microg), N-acetylcysteine (200 mg), Cu (2 mg), Zn (15 mg), Mn (0.5 mg), Fe (30 mg), calcium (800 mg), and selenium (100 microg). The control group received Fe (30 mg) and folic acid (400 microg). Maternal (preeclampsia, abortion, and hypertension) and perinatal outcomes were assessed.

RESULTS

In the supplementation group (29 subjects), we observed 2 cases of preeclampsia (6.8%, 1 mild and 1 severe), 1 of IUGR (birth weight 2300 g at 38 weeks), and 1 preterm delivery. In the control group (31 subjects), there were 8 abortions, 9 cases of preeclampsia (29%, 6 mild and 3 severe) with perinatal outcome: 3 preterm delivery cases and 1 IUGR (birth weight 2030 g at 39 weeks). Preeclampsia was significantly less frequent in the supplementation group when compared to the control group (2 vs. 9 cases, p = 0.043, OR = 0.18 [95% CI: 0.03, 0.92]). Finally we focused on the prediction of preeclampsia at 8 to 12 weeks. Combined sensitivity of markers of antioxidant status (SOD slutathione peroxidase, [GPx], and total anti-oxidant status [TAS]) was 33% (false-positive rate of 4.5%).

CONCLUSIONS

Antioxidant supplementation was associated with better maternal and perinatal outcome in pregnant women with low antioxidant status than control supplementation with iron and folate alone. In a selected population already screened positive for low SOD, preeclampsia can be detected in 33% of asymptomatic cases in the first trimester using SOD, GPx, and TAS. It seems feasible that panels of both biochemical and molecular markers may be clinically useful in the prediction of this disease.

The antifatigue effect of bacoside extract (BME) from Bacopa monniera (L.) Wettst. was investigated. Rats were subjected to weight-loaded forced swim test (WFST) every alternate day for <em>3</em> weeks. The BME at a dosage of 10 mg/kg body weight was administered orally to rats for 2 weeks in order to evaluate the following biomarkers of physical fatigue: swimming time, change in body weight, lipid peroxidation, lactic acid (LA), glycogen, antioxidant enzyme activities such as superoxide dismutase (<em>SOD</em>) and catalase (CAT) and blood parameters, namely blood urea nitrogen (BUN) and creatine kinase (CK). The exhaustive swimming time was increased by <em>3</em>-fold in the BME supplemented group compared with that of the control group on day 1<em>3</em>. The BME treatment lowered malondialdehyde (MDA) levels in brain, liver and muscle tissues by 11.2%, 16.2% and <em>3</em>7.7%, respectively, compared with the control exercised group (p < 0.05). The BME also reduced the LA, serum BUN and CK activities significantly compared with that of the control. Administration of BME significantly protected the depletion of <em>SOD</em> and CAT activities. The HSP-70 expression studies by western blot also confirmed the antifatigue property of BME. The present study thus indicates that BME ameliorates the various impairments associated with physical fatigue.

Mustard gas exposure causes inflammatory lung diseases. Many inflammatory lung diseases are associated with oxidative stress. Reactive oxygen species (ROS) are involved in the maintenance of physiological functions. In tissues, it is therefore essential to maintain a steady-state level of antioxidant activity to allow both for the physiological functions of ROS to proceed and at the same time preventing tissue damage. We have recently reported that mustard gas exposure decreases the overall activity of superoxide dismutase (<em>SOD</em>). In the present study, we investigated the effects of mustard gas on each of the three isozymes: <em>SOD</em>-1 (Cu/Zn), <em>SOD</em>-2 (Mn), and <em>SOD</em>-<em>3</em> (extracellular). Adult guinea pigs were intratracheally injected single doses of 2-chloroethyl ethyl sulfide (CEES) (2 mg/kg body weight) in ethanol. Control animals were injected with vehicle in the same way. The animals were sacrificed after 7 days, and lungs were removed after perfusion with physiological saline. Lung injury was established by measuring the leakage of iodinated-BSA into lung tissue. Mustard gas exposure caused a significant increase in the activity of <em>SOD</em>-1 (<em>3</em>5%). However, the <em>SOD</em>-<em>3</em> activity which is the predominant type in lung was significantly decreased (62%), whereas no change was observed in <em>SOD</em>-2 activity. Thus the decrease in the total activity of <em>SOD</em> was primarily due to the <em>SOD</em>-<em>3</em> isozyme. Northern blot analysis indicated <em>3</em>.5-fold increased expression of <em>SOD</em>-1 in mustard gas exposed lung, but no significant change in the expression of <em>SOD</em>-2 and <em>SOD</em>-<em>3</em> was observed. Mustard gas exposure did not cause mutation in the coding region of <em>SOD</em>-1 gene while causing modulation in expression levels. The protein levels of <em>SOD</em>-1, <em>SOD</em>-2, and <em>SOD</em>-<em>3</em> were not altered significantly in the mustard gas exposed lung. Our results indicate that the overall decrease in the activity of <em>SOD</em> by mustard gas exposure is probably mediated by direct inactivation of the <em>SOD</em>-<em>3</em> gene or the enzyme itself. This decrease in the activity of <em>SOD</em>-<em>3</em> may be due to the cleavage of active form of the protein to an inactive form. The existence of active and inactive forms of <em>SOD</em>-<em>3</em> as a result of shifts in Cys-Cys disulfide bonding has been described in human, recently. Studies are underway in our laboratory to investigate whether mustard gas induced inactivation of <em>SOD</em>-<em>3</em> in lung is similarly mediated by a change in Cys-Cys disulfide bonding.

BACKGROUND

Administration of Cu/Zn superoxide dismutase (SOD) without catalase fails to alleviate myocardial stunning, but extracellular SOD (Ec-SOD) may be more effective because it binds to heparan sulfate proteoglycans on the cellular glycocalyx. We therefore used in vivo gene transfer to increase systemic levels of Ec-SOD and determined whether this gene therapy protects against myocardial stunning.

RESULTS

The cDNA for human Ec-SOD was cloned behind the cytomegalovirus (CMV) promoter and incorporated into a replication-deficient adenovirus (Ad5/CMV/Ec-SOD). Injection of this virus (2x10(8) pfu/kg IV) produced high levels of Ec-SOD in the liver, which could be redistributed to the heart and other organs by injection of heparin. Conscious rabbits underwent a sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles for 3 consecutive days starting 3 days after intravenous injection of Ad5/CMV/Ec-SOD or Ad5/CMV/nls/LacZ (negative control). Both groups were given heparin (2000 U/kg IV) 2 hours before the first sequence of occlusions. The severity of myocardial stunning was measured as the total deficit of LV wall thickening after the last reperfusion. On day 1, the total deficit of wall thickening was markedly decreased in Ad5/CMV/Ec-SOD rabbits versus controls and similar to that seen on days 2 and 3 in controls.

CONCLUSIONS

The results demonstrate that in vivo gene transfer of the cDNA encoding Ec-SOD provides the heart with substantial protection against myocardial stunning without the need for concomitant administration of catalase. The present observations provide the basis for controlling gene therapy at the posttranslational level and for simultaneously protecting multiple organs from oxidant stress.

To investigate the role of superoxide in the toxicity of nitric oxide (NO), we examined the effect of nitric oxide synthase (NOS) inhibition on brain infarction in transgenic mice overexpressing CuZn-superoxide dismutase (<em>SOD</em>-1). Male <em>SOD</em>-transgenic mice and non-transgenic littermates (<em>3</em>0-<em>3</em>5 g) were subjected to 60 min of middle cerebral artery occlusion followed by 24 h of reperfusion. Either NG-nitro-L-arginine methyl ester (L-NAME; <em>3</em> mg/kg), a mixed neuronal and endothelial NOS inhibitor, or 7-nitroindazole (7-NI; 25 mg/kg), a selective neuronal NOS inhibitor, was administered intraperitoneally 5 min after the onset of ischemia. At 24 h of reperfusion, the mice were decapitated and the infarct volume was evaluated in each group. In the nontransgenic mice, L-NAME significantly increased the infarct volume as compared with the vehicle, while 7-NI significantly decreased it. In the <em>SOD</em>-transgenic mice, L-NAME-treated animals showed a significantly larger infarct volume than vehicle-treated ones, whereas there were no significant differences between 7-NI- and vehicle-treated mice. Our findings suggest that selective inhibition of neuronal NOS ameliorates ischemic brain injury and that both neuronal and endothelial NOS inhibition may result in the deterioration of ischemic injury due to vasoconstriction of the brain. Since L-NAME increased infarct volume even in <em>SOD</em>-transgenic mice, the protective effect of <em>SOD</em> could result from the vasodilation by increased endothelial NO as well as the reduction of neuronal injury due to less production of peroxynitrite compared to wild-type mice. Moreover, the neurotoxic role of NO might not be dependent on NO itself, but the reaction with superoxide to form peroxynitrite, because of no additive effects of <em>SOD</em> and a neuronal NOS inhibitor.

The mechanisms leading to microangiopathy in diabetes mellitus have still not been clearly elucidated. We hypothesized that type I diabetes mellitus affects the endothelium and alters flow-dependent dilation of arterioles, an important mechanism involved in local regulation of blood flow. Isolated, pressurized gracilis muscle arterioles (inside diameter approximately 150 microm at 80 mm Hg) from rats with streptozotocin (STZ)-induced diabetes mellitus exhibited reduced dilations induced by increases in perfusate flow compared to those of normal rats (plasma glucose: 25.7 +/- 0.7 vs. 6.4 +/- 0.5 mmol/l; maximum increase in diameter: 15 +/- 4 vs. <em>3</em>1+/- <em>3</em> microm, p < 0.05). In control arterioles, both nitric oxide (NO) and prostaglandins mediated the flow-dependent dilation, whereas flow-induced dilations of diabetic arterioles were unaffected by N(omega)-nitro-L-arginine methyl ester (L-NAME) and were abolished by indomethacin. Sepiapterin - precursor of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin (BH(4)) - restored the L-NAME-sensitive portion of flow-dependent dilations of diabetic arterioles. Furthermore, depletion of BH(4) by 2,4-diamino-6-hydroxypyrimidine (DAHP) in control arterioles also resulted in reduced flow-dependent dilations, which were restored by intraluminal sepiapterin [but not with superoxide dismutase (<em>SOD</em>) plus catalase (CAT) (<em>SOD</em>+CAT)] and then could be inhibited by L-NAME. Dilations induced by the NO donor sodium nitroprusside (SNP) were unaffected by L-NAME in diabetes mellitus arterioles or when eNOS was activated by intraluminal flow in DAHP-treated arterioles (with or without <em>SOD</em>+CAT). In contrast, pyrogallol (known to produce reactive oxygen species) substantially reduced acetylcholine- and SNP-induced dilation in a <em>SOD</em>+CAT-reversible manner. Collectively, these findings suggest that in diabetic arterioles, due to the reduced bioavailability of BH(4), the synthesis of NO by eNOS is limited, resulting in a reduced flow-induced dilation, a mechanism that may also be responsible for the development of diabetic microangiopathy and exacerbation of other vascular diseases.

Mechanisms of burn-related cardiac dysfunction may involve defects in mitochondria. This study determined 1) whether burn injury alters myocardial mitochondrial integrity and function; and 2) whether an antioxidant vitamin therapy prevented changes in cardiac mitochondrial function after burn. Sprague-Dawley rats were given a <em>3</em> degrees burn over 40% total body surface area and fluid resuscitated. Antioxidant vitamins or vehicle were given to sham and burn rats. Mitochondrial and cytosolic fractions were prepared from heart tissues at several times postburn. In mitochondria, lipid peroxidation was measured to assess oxidative stress, mitochondrial outer membrane damage and cytochrome-c translocation were determined to estimate mitochondrial integrity, and activities of <em>SOD</em> and glutathione peroxidase were examined to evaluate mitochondrial antioxidant defense. Cardiac function was measured by Langendorff model in sham and burn rats given either vitamins or vehicle. Twenty-four hours postburn, mitochondrial outer membrane damage was progressively increased to approximately 50%, and cytosolic cytochrome-c gradually accumulated to approximately three times more than that measured in shams, indicating impaired mitochondrial integrity. Maximal decrease of mitochondrial <em>SOD</em> activity occurred 8 h postburn ( approximately 6<em>3</em>.5% of shams), whereas maximal decrease in glutathione peroxidase activity persisted 2-24 h postburn ( approximately 60% of shams). In burn animals, lipid peroxidation in cardiac mitochondria increased <em>3</em>0-50%, suggesting burn-induced oxidative stress. Antioxidant vitamin therapy prevented burn-related loss of membrane integrity and antioxidant defense in myocardial mitochondria and prevented cardiac dysfunction. These data suggest that burn-mediated mitochondrial dysfunction and loss of reactive oxygen species defense may play a role in postburn cardiac dysfunction.

Hygrophila auriculata (K. Schum.) Heine (Family: Acanthaceae) is a wild herb widely used in 'Ayurveda' as 'Rasayana' drug for treatment of various disorders. Treatment of diabetic rats with aerial parts of Hygrophila auriculata extract (HAEt, 100 and 250 mg/kg body weight) for <em>3</em> weeks showed significant reduction in blood glucose, thiobarbituric acid reactive substances (TBARS) and hydroperoxide in both liver and kidney. The treatment with HAEt significantly increased the glutathione (GSH), glutathione peroxidase (GPx), glutathione S-transferase (GST) and catalase (CAT) in the drug-treated group, which is comparable to the control group. HAEt and glibenclamide-treated rats also showed decreased lipid peroxidation that is associated with increased activity of superoxide dismutase (<em>SOD</em>) and catalase. The ability of HAEt on tissue lipid peroxidation and antioxidant status in diabetic animals has not been studied before. The result of this study thus shows that HAEt possesses significant antidiabetic activity along with potent antioxidant potential in diabetic conditions.

This experimental study was designed to investigate the effects of vitamin E supplementation, especially on lipid peroxidation and antioxidant status elements <em>3</em>/4 namely, glutathione (GSH), CuZn superoxide dismutase (CuZn <em>SOD</em>), and glutathione peroxidase (GSH Px), both in blood and liver tissues of streptozotocin (STZ) diabetic rats. The extent to which blood can be used to reflect the oxidative stress of the liver is also investigated. In diabetic rats, plasma lipid peroxide values were not significantly different,from control,whereas erythrocyte CuZn <em>SOD</em> (p < 0.01), GSH Px (p < 0.001) activities and plasma vitamin E levels (p < 0.001), were significantly more elevated than controls. Vitamin E supplementation caused significant decreases of erythrocyte GSH level (p < 0.01) in control rats and of erythrocyte GSH Px activity (p < 0.05) in diabetic rats. Liver findings revealed significantly higher lipid peroxide (p < 0.001) and vitamin E (p < 0.01) levels and lower GSH (p < 0.001), CuZn <em>SOD</em> (p < 0.001) and GSH Px (p < 0.01) levels in diabetic rats. A decreased hepatic lipid peroxide level (p < 0.01) and increased vitamin E/lipid peroxide ratio (p < 0.001) were observed in vitamin E supplemented, diabetic rats. A vitamin E supplementation level which did not cause any increase in the concentration of the vitamin in the liver or blood, was sufficient to lower lipid peroxidation in the liver. Vitamin E/lipid peroxide ratio is suggested as an appropriate index to evaluate the efficiency of vitamin E activity,independent of tissue lipid values. Further, the antioxidant components GSH, GSH Px and CuZn <em>SOD</em> and the relationships among them, were affected differently in the liver and blood by diabetes or vitamin E supplementation.

BACKGROUND

Porphyromonas gingivalis is strongly implicated in the etiology of adult periodontitis by inducing inflammatory cytokines, resulting in gingival and periodontal tissue inflammation and alveolar bone resorption. This study tested the hypothesis that supplementing the diet with omega-<em>3</em> fatty acid (omega-<em>3</em> FA; i.e. fish oil) would exert anti-inflammatory effects in the gingival tissues of P. gingivalis-infected rats.

METHODS

Rats were fed either fish oil or corn oil diets ad libitum for 22 weeks and infected with P. gingivalis strain <em>3</em>81 or strain A7A1-28. After sacrifice, rat gingival tissues were excised and the RNA was isolated and analyzed for proinflammatory mediators [interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), IL-6], T helper type 1 and type 2 cytokines [interferon-gamma (IFN-gamma), IL-4, IL-10), antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD)], and genes critical for eicosanoid mediator production [cyclo-oxygenase-2 (COX-2), 5-lipoxygenase (5-LO)] by reverse transcription-polymerase chain reaction using rat-specific primers.

RESULTS

Rats on the omega-<em>3</em> FA diet exhibited decreased proinflammatory cytokine gene expression (IL-1beta, TNF-alpha) and enhanced IFN-gamma, CAT and SOD messenger RNA expression compared to rats fed a corn oil diet, supporting a diet-induced modulation of host inflammatory reactions. Analyses of alveolar bone resorption in the rats related to gene expression profiles demonstrated significant positive correlations with IL-1beta, IL-6 and COX-2 and negative correlations with CAT and SOD.

CONCLUSIONS

These findings suggest that diets enriched for omega-<em>3</em> FA modulate the local gingival inflammatory milieu of the host following oral P. gingivalis infection, which impacts on alveolar bone resorption in rats.

Manganese superoxide dismutase (Mn-<em>SOD</em>) is a mitochondrial enzyme that converts toxic O(2)(-) to H(2)O(2). Previous studies have reported that a systemic deficiency in Mn-<em>SOD</em> causes neonatal lethality in mice. Therefore, no mouse model is available for the analysis of the pathological role of O(2)(-) injuries in adult tissues. To explore an adult-type mouse model, we generated tissue-specific Mn-<em>SOD</em> conditional knockout mice using a Cre-loxp system. First, we generated liver-specific Mn-<em>SOD</em>-deficient mice by crossbreeding with albumin-Cre transgenic mice. Mn-<em>SOD</em> proteins were significantly downregulated in the liver of liver-specific Mn-<em>SOD</em> knockout mice. Interestingly, the mutant mice showed no obvious morphological abnormalities or biochemical alterations in the liver, suggesting a redundant or less important physiological role for Mn-<em>SOD</em> in the liver than previously thought. Next, we generated heart/muscle-specific Mn-<em>SOD</em>-deficient mice by crossbreeding muscle creatine kinase-Cre transgenic mice. The mutant mice developed progressive dilated cardiomyopathy with specific molecular defects in mitochondrial respiration. Furthermore, brain-specific Mn-<em>SOD</em>-deficient mice that had been developed by crossbreeding with nestin-Cre transgenic mice developed a spongiform encephalopathy-like pathology associated with gliosis and died within <em>3</em> weeks of birth. These results imply that the superoxide generated in mitochondria plays a pivotal role in the development and progression of pathologies in the heart and brain, but not in the liver. In conclusion, we successfully generated various tissue-specific Mn-<em>SOD</em> conditional knockout mice that provide useful tools for the analysis of various oxidative stress-associated diseases.

In this study, serum antioxidant and oxygen derived free radical status of patients with ankylosing spondylitis (AS) was investigated and compared with that of age- and sex-matched healthy controls. The relationship of these parameters to disease activity indices was also examined. Thirty patients with AS not currently under disease-modifying antirheumatic drug (DMARD) treatment (e.g., sulfasalazine or methotrexate) (15 active and 15 inactive) and 16 age- and sex-matched healthy controls were included in the study. Catalase (EC 1.11.1.6), total (Cu-Zn and Mn) superoxide dismutase (<em>SOD</em>) (EC 1.15.1.1) activities, and malondialdehyde (MDA), nitrite (NO(2)(-)), and nitrate (NO(<em>3</em>)(-)) levels as indices of nitric oxide (NO) production were evaluated using appropriate methods. There was no statistically significant difference found in <em>SOD</em> activity or NO and MDA levels between active and inactive patients. Inactive patients showed no significant difference in all the measured oxidant/antioxidant parameters when compared to healthy controls. Active patients had significantly higher levels of MDA and catalase enzyme activity ( P=0.002 and P=0.007, respectively). There was no significant correlation between oxidant/antioxidant parameters and disease activity, C-reactive protein, erythrocyte sedimentation rate, or Bath Ankylosing Spondylitis Disease Activity Index (CRP, ESR, or BASDAI) in either group, except catalase enzyme activity, which had a significant correlation with CRP and ESR levels in active patients ( r=0.69 and P=0.004, r=0.52 and P=0.04, respectively). Our results indicate that oxidative stress and lipid peroxidation are accelerated in untreated patients with active AS. Serum catalase activity may be closely related to disease activity. In this regard, we underscore the likely benefit of some therapeutic interventions including high-potential antioxidants that will potentiate the antioxidant defense mechanism and reduce peroxidation in the management of AS.

Puerarin (PU), a natural flavonoid, has been reported to have many benefits and medicinal properties. In this study, we valuated the protective effect of puerarin against lead-induced oxidative DNA damage and apoptosis in rat liver. A total of forty male Wistar rats (8-week-old) was divided into 4 groups: control group; lead-treated group (500 mg Pb/l as the only drinking fluid); lead+puerarin treated group (500 mg Pb/l as the only drinking fluid plus 400 mg PU/kg bwt intra-gastrically once daily); and puerarin-treated group (400 mg PU/kg bwt intra-gastrically once daily). The experimental period was lasted for 75 successive days. Our data showed that puerarin significantly effectively improved the lead-induced histology changes in rat liver and decreased the serum ALT and AST activities in lead-treated rats. Puerarin markedly restored Cu/Zn-<em>SOD</em>, CAT and GPx activities and the GSH/GSSG ratio in the liver of lead-treated rat. Furthermore, the increase of 8-hydroxydeoxyguanosine induced by lead was effectively suppressed by puerarin. The enhanced caspase-<em>3</em> activity in the rat liver induced by lead was also inhibited by puerarin. TUNEL assay showed that lead-induced apoptosis in rat liver was significantly inhibited by puerarin, which might be attributed to its antioxidant property. In conclusion, these results suggested that puerarin could protect the rat liver against lead-induced injury by reducing ROS production, renewing the activities of antioxidant enzymes and decreasing DNA oxidative damage.

Urolithins were the metabolites of ellagic acid by intestinal flora in gastrointestinal tract. In previous research, it was found that urolithins could mainly inhibit prostate cancer and colon cancer cell growth. However, there is no report about bladder cancer therapy of urolithins. In this paper, three urolithin-type compounds (urolithin A, urolithin B, 8-OMe-urolithin A) and ellagic acid were evaluated for antiproliferative activity in vitro against human bladder cancer cell lines T24. The IC₅₀ values for T24 cell inhibition were 4<em>3</em>.9, <em>3</em>5.2, 46.<em>3</em> and <em>3</em><em>3</em>.7 μM for urolithin A, urolithin B, 8-OMe-urolithin A and ellagic acid, respectively. After the administration of urolithins and ellagic acid, we found these compounds could increase mRNA and protein expression of Phospho-p<em>3</em>8 MAPK, and decrease mRNA and protein expression of MEKK1 and Phospho-c-Jun in T24 cells. Caspase-<em>3</em> was also activated and PPAR-γ protein expression increased in drug-induced apoptosis. And what's more, the antioxidant assay afforded by three urolithins and EA treatments were associated with decreases in the intracellular ROS and MDA levels, and increased <em>SOD</em> activity in H₂O₂-treated T24 cells. The results suggested that these compounds could inhibit cell proliferation by p<em>3</em>8-MAPK and/or c-Jun medicated caspase-<em>3</em> activation and reduce the oxidative stress status in bladder cancer.

Matrine, an active constituent of the Chinese herb, Sophora flavescens Ait., and it is known for its antioxidant, anti-inflammatory and antitumor activities. It has been demonstrated that matrine exerts protective effects against heart failure by decreasing the expression of caspase-<em>3</em> and Bax, and increasing Bcl‑2 levels. In this study, we aimed to determine whether these protective effects of matrine can be applied to cerebral ischemia. Following 7 successive days of treatment with matrine (7.5, 15 and <em>3</em>0 mg/kg) and nimodipine (1 mg/kg) by intraperitoneal injection, male Institute of Cancer Research (ICR) mice were subjected to middle cerebral artery occlusion (MCAO). Following reperfusion, the neurobehavioral score and brain infarct volume were estimated, and morphological changes were analyzed by hematoxylin and eosin (H&E) staining and electron microscopy. The percentage of apoptotic neurons was determined by flow cytometry. The levels of oxidative stress were assessed by measuring the levels of malondialdehyde (MDA), superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GSH-Px) and catalase (CAT), and the total antioxidant capacity (T-AOC). Western blot analysis and immunofluorescence staining were used to examine the expression of the apoptosis-related proteins, caspase-<em>3</em>, Bax and Bcl-2. Our results revealed that pre-treatment with matrine significantly decreased the infarct volume and improved the neurological scores. Matrine also reduced the percentage of apoptotic neurons and relieved neuronal morphological damage. Furthermore, matrine markedly decreased the MDA levels, and increased <em>SOD</em>, GSH-Px and CAT activity, and T-AOC. Western blot analysis and immunofluorescence staining revealed a marked decrease in caspase-<em>3</em> expression and an increase in the Bcl-2/Bax ratio in the group pre-treated with matrine (<em>3</em>0 mg/kg) as compared with the vehicle-treated group. The findings of the present study demonstrate that matrine exerts neuroprotective effects against cerebral ischemic injury and that these effects are associated with its antioxidant and anti-apoptotic properties.

Endothelial cell injury caused by reactive oxygen species (ROS) plays a critical role in the pathogenesis of atherosclerosis. Therefore, phytochemicals or antioxidants that inhibit the production of ROS have clinical value for the treatment of atherosclerosis. Rhein is one of the most important active components of rhubarb (Rheum officinale), a famous traditional Chinese remedy that possesses potent antioxidant properties through undefined mechanism(s). The aim of the present study was to determine whether rhein inhibits hydrogen peroxide (H2O2)-induced injury in human umbilical vein endothelial cells (HUVECs). The oxidative injury model was established with H2O2. HUVECs were treated with different concentrations of rhein in the presence/absence of H2O2. The protective effects of rhein against the injury caused by H2O2 were evaluated. HUVECs incubated with 200 µmol/l H2O2 had significantly decreased cell viability, which was accompanied by cell apoptosis and upregulated Bid and caspase-<em>3</em>, -8 and -9 mRNA expression. Meanwhile, H2O2 treatment induced a marked increase in malondialdehyde (MDA) and lactate dehydrogenase (LDH) content and decreased the nitric oxide (NO) content and nitrogen oxide synthase (NOS), superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GSH-PX) activity. However, pre-treatment with different rhein concentrations (2, 4, 8 and 16 µmol/l) significantly increased the viability of H2O2-injured HUVECs, decreased the MDA and LDH content, increased the NO content and NOS, <em>SOD</em> and GSH-PX activity in a dose-dependent manner and resulted in significant recovery from H2O2-induced cell apoptosis. In addition, the results of the qRT-PCR indicated that pre‑treatment with rhein downregulates the expression of Bid and caspase-<em>3</em>, -8 and -9 mRNA, which plays a key role in H2O2-induced cell apoptosis. The present study shows that rhein protects endothelial cells against oxidative injury induced by H2O2, suggesting that rhein is a potential compound for the prevention and treatment of atherosclerosis.

Irisin, a newly identified hormone, is critical to modulating body metabolism, thermogenesis and reducing oxidative stresses. However, whether irisin protects the heart against myocardial ischemia and reperfusion (I/R) injury remains unknown. In this study, we determine the effect of irisin on myocardial I/R injury in the Langendorff perfused heart and cultured myocytes. Adult C57/BL6 mice were treated with irisin (100 mg/kg) or vehicle for <em>3</em>0 min to elicit preconditioning. The isolated hearts were subjected to <em>3</em>0 min ischemia followed by <em>3</em>0 min reperfusion. Left ventricular function was measured and infarction size were determined using by tetrazolium staining. Western blot was employed to determine myocardial <em>SOD</em>-1, active-caspase <em>3</em>, annexin V, p<em>3</em>8, and phospho-p<em>3</em>8. H9c2 cardiomyoblasts were exposed to hypoxia and reoxygenation for assessment of the effects of irisin on mitochondrial respiration and mitochondrial permeability transition pore (mPTP). Irisin treatment produced remarkable improvements in ventricular functional recovery, as evident by the increase in RPP and attenuation in LVEDP. As compared to the vehicle treatment, irisin resulted in a marked reduction of myocardial infarct size. Notably, irisin treatment increased <em>SOD</em>-1 and p<em>3</em>8 phosphorylation, but suppressed levels of active-caspase <em>3</em>, cleaved PARP, and annexin V. In cardiomyoblasts exposed to hypoxia/reoxygenation, irisin treatment significantly attenuated hypoxia/reoxygenation (H/R), as indicated by the reduction of lactate dehydrogenase (LDH) leakage and apoptotic cardiomyocytes. Furthermore, irisin treatments suppressed the opening of mPTP, mitochondrial swelling, and protected mitochondria function. Our results indicate that irisin serves as a novel approach to eliciting cardioprotection, which is associated with the improvement of mitochondrial function.

We have studied the photochemical quantum yields of singlet oxygen production (using the RNO bleaching method) and superoxide production (using the EPR-spin trapping method and the <em>SOD</em>-inhibitable ferricytochrome c reduction spectral assay) of kynurenine (Ky), N-formylkynurenine (NFK), <em>3</em>-hydroxykynurenine (<em>3</em>HK), kynurenic acid (KUA), and the flavins, riboflavin (RF) and flavin mononucleotide (FMN). Such a study of the photodynamic efficiencies is important since these compounds appear endogenously in the eye. The singlet oxygen quantum yields of the flavins and KUA are high, while Ky and <em>3</em>HK generate no detectable amounts of singlet oxygen. The superoxide quantum yields of the sensitizers are low compared to their singlet oxygen, and Ky and <em>3</em>HK produce no detectable amounts of superoxide. The production of the superoxide radical is enhanced in the presence of electron donor molecules such as EDTA and NADH. These results suggest that the production of oxyradicals in the lens may be modulated by the presence of endogenous electron donor molecules such as the coenzymes NADH and NADPH, which are present in significant amounts in some lenses. They also suggest that Ky and <em>3</em>HK, which are known to be present in aged lenses, might play a protective rather than a deleterious role in the eye.

Recent studies indicate that sepsis is associated with enhanced generation of several free radical species (nitric oxide, superoxide, hydrogen peroxide) in skeletal muscle. While studies suggest that free radical generation causes uncoupling of oxidative phosphorylation in sepsis, no previous report has examined the role of free radicals in modulating skeletal muscle oxygen consumption during State <em>3</em> respiration or inhibiting the electron transport chain in sepsis. The purpose of the present study was to examine the effects of endotoxin-induced sepsis on State <em>3</em> diaphragm mitochondrial oxygen utilization and to determine if inhibitors/scavengers of various free radical species would protect against these effects. We also examined mitochondrial protein electrophoretic patterns to determine if observed endotoxin-related physiological derangements were accompanied by overt alterations in protein composition. Studies were performed on: (a) control animals, (b) endotoxin-treated animals, (c) animals given endotoxin plus PEG-<em>SOD</em>, a superoxide scavenger, (d) animals given endotoxin plus L-NAME, a nitric oxide synthase inhibitor, (e) animals given only PEG-<em>SOD</em> or L-NAME, (f) animals given endotoxin plus D-NAME, and (g) animals given endotoxin plus denatured PEG-<em>SOD</em>. We found: (a) no alteration in maximal State <em>3</em> mitochondrial oxygen consumption rate at 24 h after endotoxin administration, but (b) a significant reduction in oxygen consumption rate at 48 h after endotoxin, (c) no effect of endotoxin to induce uncoupling of oxidative phosphorylation, (d) either PEG-<em>SOD</em> or L-NAME (but neither denatured PEG-<em>SOD</em> nor D-NAME) prevented endotoxin-mediated reductions in State <em>3</em> respiration rates, (e) some mitochondrial proteins underwent tyrosine nitrosylation at 24 h after endotoxin administration, and (f) SDS-page electrophoresis of mitochondria from endotoxin-treated animals revealed a selective depletion of several proteins at 48 h after endotoxin administration (but not at 24 h); (g) administration of L-NAME or PEG-<em>SOD</em> prevented this protein depletion. These data provide the first evidence that endotoxin-induced reductions in State <em>3</em> mitochondrial oxygen consumption are free radical-mediated.

To identify the transductional mechanisms responsible for the neuroprotective effect of nitric oxide (NO) during ischemic preconditioning (IPC), we investigated the effects of this gaseous mediator on mitochondrial Mn-superoxide dismutase (Mn-<em>SOD</em>) expression and activity. In addition, the possible involvement of Ras/extracellular-regulated kinase (ERK) ERK1/2 pathway in preserving cortical neurons exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation was also examined. Ischemic preconditioning was obtained by exposing neurons to a <em>3</em>0-min sublethal OGD (95% N(2) and 5% CO(2)). Then, after a 24-h interval, neurons were exposed to <em>3</em> h of OGD followed by 24 h of reoxygenation (OGD/Rx). Our results revealed that IPC reduced cytochrome c (cyt c) release into the cytosol, improved mitochondrial function, and decreased free radical production. Moreover, it induced an increase in nNOS expression and NO production and promoted ERK1/2 activation. These effects were paralleled by an increase in Mn-<em>SOD</em> expression and activity that persisted throughout the following OGD phase. When the neurons were treated with L-NAME, a well known NOS inhibitor, the increase in Mn-<em>SOD</em> expression occurring during IPC was reduced and, as a result, IPC-induced neuroprotection was prevented. Similarly, when ERK1/2 was inhibited by its selective inhibitor PD98059, the increase in Mn-<em>SOD</em> expression observed during IPC was almost completely abolished. As a result, its neuroprotective effect on cellular survival was thwarted. The present findings indicate that during IPC the increase in Mn-<em>SOD</em> expression and activity are paralleled by NO production. This suggests that NO neuroprotective role occurs through the stimulation of Mn-<em>SOD</em> expression and activity. In particular, NO via Ras activation stimulates downstream ERK1/2 cascade. This pathway, in turn, post-transcriptionally activates Mn-<em>SOD</em> expression and activity, thus promoting neuroprotection during preconditioning.

High dietary salt intake decreases the endothelium-dependent dilation of skeletal muscle arterioles by inhibiting local nitric oxide (NO) activity without changing vascular smooth muscle responsiveness to NO. Under these conditions, microvascular walls show evidence of oxidative stress, and scavengers of reactive oxygen species (ROS) abolish this oxidative stress and restore normal arteriolar responses to acetylcholine (ACh). We tested the hypothesis that the salt-dependent appearance of microvascular ROS, and accompanying reduction in endothelium-dependent dilation, is due to a decrease in antioxidant enzyme expression or activity. We studied spinotrapezius muscle microvessels in rats fed normal (NS) (0.45%) or high (HS) (7%) salt diets for 4-5 weeks. Western analysis of arteriolar and venular protein showed no difference between groups in the content of superoxide dismutase (Cu/Zn <em>SOD</em>), catalase, or glutathione peroxidase. The catalase inhibitor <em>3</em>-amino-1,2,4-triazole (<em>3</em>AT) increased arteriolar and venular oxidant activity (assessed by tetranitroblue tetrazolium reduction) by the same amount in both groups, suggesting similar levels of catalase activity. <em>3</em>AT did not affect arteriolar responses to ACh in either group. The Cu/Zn <em>SOD</em> inhibitor diethyldithiocarbamate increased arteriolar and venular oxidant activity to a lesser extent in HS rats, suggesting reduced Cu/Zn <em>SOD</em> activity in this group. Cu/Zn <em>SOD</em> inhibition decreased arteriolar responses to ACh only in NS rats. These findings suggest that endogenous Cu/Zn <em>SOD</em> preserves the endothelium-dependent control of arteriolar tone in NS rats, and that a reduction in Cu/Zn <em>SOD</em> activity contributes to the loss of arteriolar NO activity in HS rats.

It is claimed that WBC (whole-body cryotherapy) enhances the resistance of the human body, also thanks to the beneficial effect on the antioxidant system. Accordingly, this research aimed to evaluate the effect of a series of whole-body cryostimulations on the level of non-enzymatic antioxidants and the activity of antioxidant enzymes in healthy men. The study was carried out on <em>3</em>0 young and healthy men aged 27.8±6.1 years with average body mass index and peak oxygen consumption (46.<em>3</em>4±6.15 ml kg(-1) •min(-1)). The participants were daily exposed for <em>3</em> minutes to cryogenic temperatures (-1<em>3</em>0°C). Blood samples were obtained in the morning before cryostimulation, again <em>3</em>0 min after exposure and the following day in the morning, during the 1(st), 10(th) and 20(th) session. Analysis concerned changes in plasma concentrations of total protein, albumin, glucose, uric acid and ceruloplasmin, and the most important components of the antioxidant system in red blood cells: superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, reduced and oxidized glutathione. To assess the oxidative stress level the 8-isoprostane concentration in plasma was measured. The obtained results indicate that cryogenic temperatures in repeated daily treatments result in changes in the peroxidant and antioxidant status. These changes seem to depend on the number of cryostimulations. After 20 daily treatments there was an increase in <em>SOD</em>, <em>SOD</em>:CAT ratio, a decrease in the concentration of reduced and oxidized glutathione and in the activity of GPx. It could be possible that differences in the activity of GSSG-R after 20 treatments depended on the body mass index of participants.

Although melatonin has been established as a free radical scavenger and antioxidant, its effects in diabetes have not been thoroughly investigated. The purpose of this study, therefore, was to investigate the effects of melatonin administration on lipid peroxidation and antioxidant status in streptozotocin (STZ)-induced diabetes in rats. Concentrations of malondialdehyde (MDA) and reduced glutathione (GSH) in erythrocytes and activities of superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GSH-Px) were compared in <em>3</em> groups of 10 rats each [control non-diabetic rats (group I), untreated diabetic rats (group II) and diabetic rats treated with melatonin (group III)]. In the study groups, diabetes developed <em>3</em> days after intraperitoneal (i.p.) administration of a single 60-mg/kg dose of STZ. Thereafter, while the rats in group II received no treatment, the rats in group III began to receive a 10-mg/kg i.p. dose of melatonin per day. After 6 wk, the rats in groups II and III had significantly lower body weights and significantly higher blood glucose levels than the rats of group I (P<0.001 and P<0.001, respectively). There were no significant differences in body weight or blood glucose levels between groups II and III. MDA levels in untreated diabetic rats were higher than those in control group rats and in diabetic rats treated with melatonin (P<0.01 and P<0.05, respectively). However, MDA levels in diabetic rats treated with melatonin were not different from those of the control group. The GSH, GSH-Px and <em>SOD</em> levels of untreated diabetic rats were significantly lower than those of the control group (P<0.02, P<0.002 and P<0.05, respectively). In group III, however, melatonin prevented decreases in the thiol antioxidant and the associated enzymes, and so these levels were not significantly different from those in the control group. These results confirm the presence of oxidative stress in STZ-induced experimental diabetes and indicate the beneficial free radical-scavenging and antioxidant properties of melatonin.