Nimesulide, an anti-inflammatory and analgesic drug, is reported to cause severe hepatotoxicity. In this study, molecular mechanisms involved in deranged oxidant-antioxidant homeostasis and mitochondrial dysfunction during nimesulide-induced hepatotoxicity and its attenuation by plant derived terpenes, camphene and geraniol has been explored in male Sprague-Dawley rats. Hepatotoxicity due to nimesulide (80 mg/kg BW) was evident from elevated SGPT, SGOT, bilirubin and histo-pathological changes. Antioxidants and key redox enzymes (iNOS, mtNOS, Cu/Zn-<em>SOD</em>, Mn-<em>SOD</em>, GPx and GR) were altered significantly as assessed by their mRNA expression, Immunoblot analysis and enzyme activities. Redox imbalance along with oxidative stress was evident from decreased NAD(P)H and GSH (56% and 74% respectively; P<0.001), increased superoxide and secondary ROS/RNS generation along with oxidative damage to cellular macromolecules. Nimesulide reduced mitochondrial activity, depolarized mitochondria and caused membrane permeability transition (MPT) followed by release of apoptotic proteins (AIF; apoptosis inducing factor, EndoG; endonuclease G, and Cyto c; cytochrome c). It also significantly activated caspase-9 and caspase-<em>3</em> and increased oxidative DNA damage (level of 8-Oxoguanine glycosylase; P<0.05). A combination of camphene and geraniol (CG; 1:1), when pre-administered in rats (10 mg/kg BW), accorded protection against nimesulide hepatotoxicity in vivo, as evident from normalized serum biomarkers and histopathology. mRNA expression and activity of key antioxidant and redox enzymes along with oxidative stress were also normalized due to CG pre-treatment. Downstream effects like decreased mitochondrial swelling, inhibition in release of apoptotic proteins, prevention of mitochondrial depolarization along with reduction in oxidized NAD(P)H and increased mitochondrial electron flow further supported protective action of selected terpenes against nimesulide toxicity. Therefore CG, a combination of natural terpenes prevented nimesulide induced cellular damage and ensuing hepatotoxicity.

Phyllanthus amarus Schum. et Thonn. (Bhuia amla; Euphorbiacae) is a herb common to central and southern India. It is an ayurvedic herb and has a wide range of traditional uses in different diseases. The aim of this work was to evaluate the hepatoprotective effect of ethanolic extract of Phyllanthus amarus (Phyllanthus amarus) on aflatoxin B(1)-induced liver damage in mice using different biochemical parameters and histopathological studies. Aflatoxin was administered orally (66.6 microg kg(-1)BW 0.2 ml(-1)day(-1)) to the mice of each group except control to which normal saline and ascorbic acid (0.1g kg(-1)BW 0.2 ml(-1)day(-1)) were given, respectively. Ethanolic extract of Phyllanthus amarus (0.<em>3</em>g kg(-1)BW 0.2 ml(-1)day(-1)) was given to all groups except control groups (gp. I and gp. V) after <em>3</em>0 min of aflatoxin administration. The entire study was carried out for <em>3</em> months and animals were sacrificed after an interval of <em>3</em>0 days till the completion of study. Phyllanthus amarus extract was found to show hepatoprotective effect by lowering down the content of thiobarbituric acid reactive substances (TBARS) and enhancing the reduced glutathione level and the activities of antioxidant enzymes, glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (<em>SOD</em>) and catalase (CAT). Histopathological analyses of liver samples also confirmed the hepatoprotective value and antioxidant activity of the ethanolic extract of the herb, which was comparable to the standard antioxidant, ascorbic acid. The overall data indicated that Phyllanthus amarus possesses a potent protective effect against aflatoxin B(1)-induced hepatic damage, and the main mechanism involved in the protection could be associated with its strong capability to reduce the intracellular level of reactive oxygen species by enhancing the level of both enzymatic and non-enzymatic antioxidants.

BACKGROUND

This study investigates the levels of superoxide dismutase (SOD) activity in serum and saliva of patients with chronic periodontitis (CP). In addition, the outcome of scaling and root planing (SRP) with and without vitamin E supplementation is evaluated in terms of changes in periodontal parameters and SOD activity in patients with CP.

METHODS

Serum and salivary SOD activity in 38 patients with CP were compared with those of 22 systemically and periodontally healthy individuals (control group). At periodontal examination, serum and saliva samples were obtained. Patients with CP were randomly divided into treatment groups 1 (TG-1) and 2 (TG-2). SRP was performed for both groups, and TG-2 also received 200 mg (300 IU) vitamin E every other day. Periodontal parameters and SOD activity were evaluated after 3 months. SOD activity was determined using an SOD assay and enzyme-linked immunosorbent assay reader at 450 nm.

RESULTS

SOD activity in both serum (P <0.05) and saliva (P <0.001) was lower in patients with CP compared with controls. After 3 months of follow-up, SOD activity improved in both treatment groups; however, the improvement in TG-2 was higher than in TG-1, along with more improvement in periodontal parameters. Serum SOD levels in TG-2 increased even above the level of the control group.

CONCLUSIONS

Systemic and local SOD levels are lowered in CP. Adjunctive vitamin E supplementation improves periodontal healing as well as antioxidant defense.

In many liver disorders, oxidative stress-related inflammation and apoptosis are important pathogenic components, finally resulting in acute liver failure. Erythropoietin and its analogues are well known to influence the interaction between apoptosis and inflammation in brain and kidney. The study is to clarify the effect of curcumin, a natural plant phenolic food additive, on lipopolysaccharides (LPS)-induced acute liver injury of mice with endotoxemia and associated molecular mechanism from inflammation, apoptosis and oxidative stress levels. And curcumin, lowered serum cytokines, including Interleukin 1beta (IL-1β), Interleukin 6 (IL-6) and tumor necrosis factor (TNF-α), and improved liver apoptosis through suppressing phosphatidylinositol <em>3</em>-kinase/protein kinase B (PI<em>3</em>K/AKT) signaling pathway and inhibiting Cyclic AMP-responsive element-binding protein (CREB)/Caspase expression, and decreased oxidative stress-associated protein expression, mainly involving 2E1 isoform of cytochrome P450/nuclear factor E2-related factor 2/reactive oxygen species (CYP2E/Nrf2/ROS) signaling pathway, as well as liver nitric oxide (NO) production in LPS-induced mice. Moreover, curcumin regulated serum alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP), accelerated liver antioxidant enzymes, such as superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione (GSH) and glutathione peroxidase (GSH-px) levels, and inhibited activation of the mitogen-activated protein kinases/c-Jun NH2-terminal kinase (P<em>3</em>8/JNK) cascade in the livers of LPS-induced rats. Thus, curcumin treatment attenuates LPS-induced PI<em>3</em>K/AKT and CYP2E/Nrf2/ROS signaling and liver injury. Strategies to inhibit inflammation and apoptosis signaling may provide alternatives to the current clinical approaches to improve oxidative responses of endotoxemia.

To examine the influence of oxidative stress on oxidative protein damage, we studied 51 young Type 1 diabetic patients clinically free of complications and 48 healthy normolipidaemic age-matched controls. We determined: (1) plasma carbonyl (PCO), plasma total thiol (T-SH), and nitrotyrosine (NT) levels as markers of oxidative protein damage; (2) plasma lipid hydroperoxide (LHP), and nitric oxide (NO) levels as markers of oxidative stress; (<em>3</em>) plasma total antioxidant capacity (TAO), ceruloplasmin (Cp), transferrin (TRF), unsaturated iron binding capacity (UIBC), erythrocyte glutathione (GSH), and erythrocyte superoxide dismutase (<em>SOD</em>) as markers of free radical scavengers. There were no significant differences in the levels of these markers between prepubertal diabetic patients and the controls. The levels of both of PCO and LHP were increased in adolescent and young adult Type 1 diabetic patients with respect to their controls. In the adolescent group, patient versus control values for PCO were 1.04+/-0.067 versus 0.67+/-0.0274 nmol/mg and for LHP they were 2. 10+/-1.09 versus 1.00+/-0.4 nmol/mg. In the young adult group, patient versus control values for PCO were 0.99+/-0.054 versus 0. 66+/-0.02 nmol/mg and for LHP they were 1.96+/-0.78 versus 1.15+/-0. 4 nmol/mg. TAO levels were significantly decreased in adolescent diabetic patients compared to their controls (0.92+/-0.27 vs. 1. 86+/-0.<em>3</em>7) and in young adult diabetic patients compared to their controls (0.80+/-0.27 vs. 2.11+/-0.54 nmol/mg). T-SH was not different between diabetic patients and the controls. Serum NT, NO, and erythrocyte <em>SOD</em> levels were not different either between three groups of diabetic patients or between the patients and their controls. We attribute this lack of difference to limited disease duration. Changes in markers of oxidative stress other than NT, NO, and <em>SOD</em> observed in adolescent and young adult early stage Type 1 diabetic patients contribute to the imbalance in the redox status of the plasma. We attribute this imbalance to metal-catalyzed protein oxidation in both groups of Type 1 diabetic patients clinically free from complications.

Colon cancer is one of the serious health problems in most developed countries and its incidence rate is increasing in India. Hesperetin (HN) (<em>3</em>',5,7-trihydroxy-4'-methoxyflavonone) and hesperetin analogue (HA) were tested for their apoptosis inducing ability. Methyl thiazolyl tetrazolium assay revealed a dose as well as duration-dependent reduction of HT-29 (colon adenocarcinoma) cellular growth in response to HN and HA treatment. At 24 h 70 μM of HN and <em>3</em>2 μM of HA showed 50% reduction of HT-29 cellular growth. Acridine orange/ethidium bromide staining showed apoptotic features of cell death induced by HN and HA. Rhodamine 12<em>3</em> staining showed significant reduction in mitochondrial membrane potential induced by HN and HA. HN and HA induced DNA damage was confirmed by comet tail formation. Lipid peroxidation markers (TBARS) and protein oxidation marker (PCC) were significantly elevated in HN and HA treated groups. Enzymic antioxidants such as superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GPx) were slightly decreased in their activities compared to control (untreated HT-29 cells). Results of Western blot analysis of apoptosis associated genes revealed an increase in cytochrome C, Bax, cleaved caspase-<em>3</em> expression and a decrease in Bcl-2 expression. These findings indicate that HN and HA induce apoptosis on HT-29 via Bax dependent mitochondrial pathway involving oxidant/antioxidant imbalance.

Flavonoids are considered therapeutic agents in neurodegenerative disease because of their neuroprotective activity. This study investigated the neuroprotective effects of hesperetin in the brains of mice administered hesperetin at 10 or 50 mg/kg body weight (BW) for five weeks. Hesperetin inhibited biomarkers of oxidative stress, such as the level of thiobarbituric acid-reactive substance (TBARS) and carbonyl content, although there was a significant reduction at the higher dose of hesperetin. Moreover, at the higher dose, hesperetin significantly activated the catalase and total superoxide dismutase (<em>SOD</em>) activities. The same patterns were observed in the protein expression, and the expression of CuZn-<em>SOD</em> was more pronounced than that of Mn-<em>SOD</em>. The reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio was increased significantly in a dose-dependent manner, as well as the glutathione peroxidase (GSH-px) and glutathione reductase (GR) activities. Moreover, hesperetin did not induce apoptosis, even at the higher dose, as evidenced by caspase-<em>3</em> expression and its activity. Based on these results, hesperetin may have a neuroprotective effect via the inhibition of oxidative damage, together with activation of the antioxidant enzyme system.

Quercetin, a flavonoid, effectively improved the lead-induced histology changes including structure damage and leukocyte infiltration in rat liver. The present study was designed to explore the protective mechanism of quercetin against lead-induced hepatic injury. We found that quercetin markedly decreased the MDA and H(2)O(2) levels and lowered the GSH/GSSG ratio in the liver of lead-treated rat. Moreover, quercetin markedly restored Cu/Zn-<em>SOD</em>, Mn-<em>SOD</em>, CAT and GPx activities and upregulated mRNA expression levels of these proteins in the liver of lead-treated rat. Western blot analysis showed that quercetin significantly inhibited apoptosis by modulating the ratio of Bax to Bcl-2 expression and suppressing the expression of phosphorylated JNK1/2 and cleaved caspase-<em>3</em> in the liver of lead-treated rat. In conclusion, these data suggest that quercetin protects the rat liver from lead-induced injury by attenuating lipid peroxidation, renewing the activities of antioxidant enzymes and inhibiting apoptosis.

Experimental studies have demonstrated significant secondary damage (including cell apoptosis, blood-brain barrier disruption, inflammatory responses, excitotoxic damage, and free radical production) after traumatic brain injury (TBI). Quercetin is a natural flavonoid found in high quantities in fruits and vegetables, and may be a potential antioxidant and free radical scavenger. The purpose of this study was to determine the effects of quercetin on TBI-induced upregulation of oxidative stress, inflammation, and apoptosis in adult Sprague-Dawley rats. Animals were subjected to Feeney's weight-drop injury, thus inducing the parietal contusion brain injury model. Quercetin was administered (<em>3</em>0 mg/kg intraperitoneal injection) 0, 24, 48, and 72 h after TBI. Quercetin reduced cognitive deficits, the number of TUNEL- and ED-1-positive cells, the protein expressions of Bax and cleaved-caspase-<em>3</em> proteins, and the levels of TBARS and proinflammatory cytokines, and increased the activity of antioxidant enzymes (GSH-Px, <em>SOD</em>, and CAT) at 1 week after TBI. Our results suggest that in TBI rats, quercetin improves cognitive function owing to its neuroprotective action via the inhibition of oxidative stress, leading to a reduced inflammatory response, thereby reducing neuronal death.

The mechanism by which hyperuricemia induced-endothelial dysfunction contributes to cardiovascular diseases (CVDs) is not yet fully understood. In the present study, we used uric acid (UA) to trigger endothelial dysfunction in cultured endothelial cells, and investigated the effects of induced reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress induction, and the protein kinase C (PKC)-dependent endothelial nitric oxide synthase (eNOS) signaling pathway. Human umbilical vein endothelial cells (HUVECs) were incubated with 6, 9 or 12 mg/dl UA, ROS scavenger polyethylene glycol-superoxide dismutase (PEG‑<em>SOD</em>), ER stress inhibitor 4-phenylbutyric acid (4-PBA), and PKC inhibitor polymyxin B for 6-48 h. Nitric oxide (NO) production, eNOS activity, intracellular ROS, ER stress levels, and the interaction between eNOS and calmodulin (CaM) and cytosolic calcium levels were assessed using fluorescence microscopy and western blot analysis. Apoptosis was assessed by annexin V staining. UA increased HUVEC apoptosis and reduced eNOS activity and NO production in a dose- and time-dependent manner. Intracellular ROS was elevated after <em>3</em> h, while ER stress level increased after 6 h. UA did not alter intracellular Ca2+, CaM, or eNOS concentration, or eNOS Ser1177 phosphorylation. However, PKC-dependent eNOS phosphorylation at Thr495 was greatly enhanced, and consequently interaction between eNOS and CaM was reduced. Cellular ROS depletion, ER stress inhibition and PKC activity reduction inhibited the effect of UA on eNOS activity, NO release and apoptosis in HUVECs. Thus, we concluded that UA induced HUVEC apoptosis and endothelial dysfunction by triggering oxidative and ER stress through PKC/eNOS-mediated eNOS activity and NO production.

Although diabetic hepatopathy is potentially less common, it may be appropriate for addition to the list of target organ conditions related to diabetes. This study was designed to evaluate the hepatoprotective properties of green tea extract (GTE) in STZ-induced diabetes in rats. Wistar rats were made diabetic through single injection of STZ (75 mg/kg i.p.). The rats were randomly divided into four groups of 10 animals each: Group 1, healthy control; Group 2, nondiabetics treated with GTE administered orally (1.5%, w/v); Group <em>3</em>, diabetics; Group 4, diabetics treated with GTE (1.5%, w/v) for 8 weeks. Serum biomarkers were assessed to determine hepatic injury. Malondialdehyde (MDA) and reduced glutathione (GSH) contents were measured to assess free radical activity in the liver tissue. Hepatic antioxidant activities of glutathione peroxidase (GSH-Px), superoxide dismutase (<em>SOD</em>), and catalase (CAT) were also determined. The biochemical findings were matched with histopathological verifications. Liver MDA content and serum levels of ALT, AST, ALP, and bilirubin in Group <em>3</em> significantly increased compared to Group 1 (P < 0.05) and significantly decreased in Group 4 compared to Group <em>3</em> (P < 0.05). Serum albumin level and GSH, <em>SOD</em>, CAT, and GSH-Px contents of the liver in Group <em>3</em> were significantly decreased compared to Group 1 (P < 0.05) and were significantly increased in Group 4 compared to Group <em>3</em> (P < 0.05). Histopathologically, the changes were in the same direction with biochemical findings. This study proved the hepatoprotective activity of GTE in experimentally induced diabetic rats.

Acinetobacter baumannii and Pseudomonas aeruginosa are nosocomial pathogens with overlapping sites of infection. This work reports that the two can coexist stably in mixed-culture biofilms. In a study intended to improve our understanding of the mechanism of their coexistence, it was found that pyocyanin, produced by P. aeruginosa that generally eliminates competition from other pathogens, led to the generation of reactive oxygen species (ROS) in A. baumannii cells, which in response showed a significant (P ≤ 0.05) increase in production of enzymes, specifically, catalase and superoxide dismutase (<em>SOD</em>). This work shows for the first time that the expression of catalase and <em>SOD</em> is under the control of a quorum-sensing system in A. baumannii. In support of this observation, a quorum-sensing mutant of A. baumannii (abaI::Km) was found to be sensitive to pyocyanin compared to its wild type and showed significantly (P ≤ 0.001) lower levels of the antioxidant enzymes, which increased on addition of 5 μM N-(<em>3</em>-hydroxydodecanoyl)-l-homoserine lactone. Likewise, in wild-type A. baumannii, there was a significant (P < 0.01) decrease in the level of anti-oxidant enzymes in the presence of salicylic acid, a known quencher of quorum sensing. In the presence of amikacin and carbenicillin, A. baumannii formed 0.07 and 0.02% persister cells, which increased 4- and <em>3</em>-fold, respectively, in the presence of pyocyanin. These findings show that pyocyanin induces a protective mechanism in A. baumannii against oxidative stress and also increases its persistence against antibiotics which could be of clinical significance in the case of coinfections with A. baumannii and P. aeruginosa.

Transfection of V79 Chinese hamster cells produced clones in which CuZn-superoxide dismutase (CuZn-<em>SOD</em>) activities were 2.2- to <em>3</em>. 5-fold higher than in the parental cells. An overall reduction of antioxidant enzyme activities and both total and oxidized glutathione levels had been found in these clones. Aconitase activities in these cells were determined to indirectly measure the O2- steady-state levels. As expected, in cells overexpressing CuZn-<em>SOD</em>, both total and cytosolic aconitase activities have increased. Because these clones showed reduced oxidized glutathione contents, it is unlikely that they present higher H2O2 steady-state levels as a consequence of the higher <em>SOD</em> levels. This was confirmed by measuring H2O2 steady-state levels in cells by flow cytometric analysis of 2',7'-dichlorofluorescein diacetate-treated cells. Despite the decreased antioxidant defenses, three of the clones overexpressing CuZn-<em>SOD</em> showed reduced H2O2 steady-state levels. These reduced H2O2 steady-state levels were found even when the cells were treated with the O2- generator 2,<em>3</em>-dimethoxy-1, 4-naphthoquinone. These data provide in vivo support for the hypothesis proposed by Liochev and Fridovich [Liochev, S. I. & Fridovich, I. (1994) Free Radical Biol. Med. 16, 29-<em>3</em><em>3</em>] that O2- dismutation prevents the formation of higher H2O2 levels by other reactions.

UNASSIGNED

Dihydromyricetin (DMY) is the most abundant ingredient in vine tea. Here, we investigated the cytoprotective effects and possible mechanisms of DMY on hydrogen peroxide (H2O2)-induced oxidative stress damage in human umbilical vein endothelial cells (HUVECs).

METHODS

The percentage of cell viability was evaluated using the <em>3</em>-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We determined the antioxidant properties of DMY by measuring the activity of superoxide dismutase (<em>SOD</em>) and malondialdehyde (MDA). Flow cytometry was used to measure apoptosis in HUVECs that were double stained with Hoechst <em>3</em><em>3</em><em>3</em>42 and propidium iodide (PI). The generation of intracellular reactive oxygen species (ROS) was measured in 2',7'-dichlorofluorescin diacetate (DCFH-DA)-loaded HUVECs using a fluorescent microscope. Moreover, the expression of apoptosis-related proteins was determined by Western blotting. In addition, the release of nitric oxide (NO) was analyzed using a commercial kit.

RESULTS

HUVECs treated with H2O2 had a notable decrease in cell viability that was attenuated when cells were pretreated with DMY (<em>3</em>7.5-<em>3</em>00μM). DMY pretreatment significantly attenuated H2O2-induced apoptosis in HUVECs and inhibited intracellular ROS overproduction. Finally, pretreatment of cells with DMY prior to H2O2 exposure resulted in the inhibition of p5<em>3</em> activation, followed by the regulation of the expression of Bcl-2 and Bax, the release of cytochrome c, the cleavage (activation) of caspase-9 and caspase-<em>3</em>, and then the suppression of PARP cleavage in H2O2-induced HUVECs.

CONCLUSIONS

Our study is the first to report that DMY can protect HUVECs from oxidative stress damage, an effect that is mediated by the mitochondrial apoptotic pathways.

Oxidative damage has been suggested to be a contributory factor in the development and complications of diabetes. The antioxidant effect of an aqueous extract of Scoparia dulcis, an indigenous plant used in Ayurvedic medicine in India was studied in rats with streptozotocin-induced diabetes. Oral administration of Scoparia dulcis plant extract (SPEt) (200 mg/kg body weight) for <em>3</em> weeks resulted in a significant reduction in blood glucose and an increase in plasma insulin. The aqueous extract also resulted in decreased free radical formation in tissues (liver and kidney) studied. The decrease in thiobarbituric acid reactive substances (TBARS) and hydroperoxides (HPX) and increase in the activities of superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH) and glutathione-S-transferase (GST) clearly show the antioxidant properties of SPEt in addition to its antidiabetic effect. The effect of SPEt at 200 mg/kg body weight was better than glibenclamide, a reference drug.

The effect of n-<em>3</em> and n-6 fatty acids (FAs) on the growth of human cervical carcinoma (HeLa) cells was studied. Of all the FAs tested, docosahexaenoic acid (DHA, 22:6 n-<em>3</em>) and eicosapentaenoic acid (EPA, 20:5 n-<em>3</em>) were found to be the most potent in their cytotoxic action on HeLa cells and the potency of various fatty acids with regard to their cytotoxic action was as follows: DHA>> EPA>> dihomo-gamma-linolenic acid (DGLA) = gamma-linolenic acid (GLA)>> linoleic acid (LA)>> arachidonic acid (AA)>> alpha-linolenic acid (ALA). The cycloxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguaretic acid (NDGA), the antioxidants vitamin E, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT), the superoxide anion quencher superoxide dismutase (<em>SOD</em>), the hydroxyl and hydrogen peroxide quenchers mannitol and catalase, respectively, and the calmodulin antagonists trifluoperazine (TFP) and chlorpromazine (CPZ) could all block the cytotoxic action of GLA, which was used as a representative cytotoxic FA, on HeLa cells. On the other hand, copper and iron salts and buthionine sulfoxamine, a glutathione (GSH) depletor, potentiated the cytotoxic action of suboptimal doses of GLA. GLA-induced radical generation and lipid peroxidation in HeLa cells could be blocked by indomethacin, NDGA and calmodulin antagonists. The cytotoxic action of cis-unsaturated fatty acids (c-UFAs) is not dependent on the alteration in the protein kinase C levels since no alteration in the diacylglycerol levels was observed. Hydroxy and hydroperoxy products of GLA were found to be toxic to HeLa cells, whereas prostaglandin (PG)E1, PGF2 alpha, and prostacyclin stimulated cell growth. From these results, it is evident that radicals are the modulators of the cytotoxic action of c-UFAs, that their formation is a calmodulin-dependent process, and that lipoxygenase products may mediate the tumoricidal action of FAs.

Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (<em>SOD</em>). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,Zn<em>SODs</em>, suggests that periplasmic <em>SODs</em> protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,Zn<em>SOD</em> in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, <em>3</em>-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,Zn<em>SOD</em>. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,Zn<em>SOD</em> that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.

OBJECTIVE

Neutrophil accumulation contributes to the pathogenesis of rheumatoid arthritis. This study was undertaken to examine the ability of H2O2 to influence neutrophilic inflammation in a model of antigen-induced arthritis (AIA) in mice.

METHODS

AIA was induced by administration of antigen into the knee joints of previously immunized mice. Neutrophil accumulation was measured by counting neutrophils in the synovial cavity and assaying myeloperoxidase activity in the tissue surrounding the mouse knee joint. Apoptosis was determined by morphologic and molecular techniques. The role of H2O2 was studied using mice that do not produce reactive oxygen species (gp91phox-/- mice) and drugs that enhance the generation or enhance the degradation of H2O2.

RESULTS

Antigen challenge of immunized mice induced neutrophil accumulation that peaked at 12-24 hours after challenge. H2O2 production peaked at 24 hours, after which time, the inflammation resolved. Neutrophil recruitment was similar in wild-type and gp91phox-/- mice, but there was delayed resolution in gp91phox-/- mice or after administration of catalase. In contrast, administration of H2O2 or superoxide dismutase (<em>SOD</em>) resolved neutrophilic inflammation. The resolution of inflammation induced by <em>SOD</em> or H2O2 was accompanied by an increase in the number of apoptotic neutrophils. Apoptosis was associated with an increase in Bax and caspase <em>3</em> cleavage and was secondary to phosphatidylinositol <em>3</em>-kinase (PI<em>3</em>K)/Akt activation.

CONCLUSIONS

Our findings indicate that levels of H2O2 increase during neutrophil influx and are necessary for the natural resolution of neutrophilic inflammation. Mechanistically, enhanced levels of H2O2 (endogenous or exogenous) inhibit p-Akt/NF-κB and induce apoptosis of migrated neutrophils. Modulation of H2O2 production may represent a novel strategy for controlling neutrophilic inflammation in the joints.

OBJECTIVE

Rheumatoid arthritis (RA) is a systemic autoimmune inflammatory disease involving the breakdown of cartilage and juxta-articular bone, which is often accompanied by decreased bone mineral density (BMD) and increased risk of fracture. Anti-inflammatory omega-<em>3</em> fatty acids may prevent arthritis and bone loss in MRL/lpr mice model of arthritis and in humans.

METHODS

In this study, the effect of long term feeding of 10% dietary n-<em>3</em> (fish oil (FO)) and n-6 (corn oil (CO)) fatty acids begun at 6 weeks of age on bone mineral density (BMD) in different bone regions in an MRL/lpr female mouse model of RA was measured at 6, 9, and 12 months of age by dual energy x-ray absorptiometry (DEXA). After sacrificing the mice at 12 months of age, antioxidant enzyme activities were measured in spleen, mRNA for receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG) was measured by RT-PCR in lymph nodes, and synovitis was measured in leg joints.

RESULTS

At 6, 9 and 12 months of age, BMD was significantly higher (p < 0.05) in distal femur, proximal tibia, and lumbar spine of FO fed mice than those of CO fed mice. Spleen catalase (CAT) and superoxide dismutase (SOD) activities were also significantly higher (p < 0.01) in FO fed mice than in CO fed mice. Histology of knee joints revealed mild synovitis in CO fed mice, which was not present in FO fed mice. RT-PCR analysis of lymph nodes revealed decreased RANKL mRNA (p < 0.001) expression and enhanced OPG mRNA expression (p < 0.01) in FO fed mice compared to CO fed mice.

CONCLUSIONS

These results suggest beneficial effects of long-term FO feeding in maintaining higher BMD and lower synovitis in this mouse model. These beneficial effects may be due, in part, to increased activity of antioxidant enzymes, decreased expression of RANKL, and increased expression of OPG in FO fed mice thereby altering the RANKL/OPG ratio. These significant beneficial effects on BMD suggest that FO may serve as an effective dietary supplement to prevent BMD loss in patients with RA.

Reactive oxygen species (ROS) are crucial in long-term diabetes complications, including peripheral artery disease (PAD). In this study, we have investigated the potential clinical impact of unacylated ghrelin (UnAG) in a glucose intolerance and PAD mouse model. We demonstrate that UnAG is able to protect skeletal muscle and endothelial cells (ECs) from ROS imbalance in hind limb ischemia-subjected ob/ob mice. This effect translates into reductions in hind limb functional impairment. We show that UnAG rescues sirtuin 1 (SIRT1) activity and superoxide dismutase-2 (<em>SOD</em>-2) expression in ECs. This leads to SIRT1-mediated p5<em>3</em> and histone <em>3</em> lysate 56 deacetylation and results in reduced EC senescence in vivo. We demonstrate, using small interfering RNA technology, that SIRT1 is also crucial for <em>SOD</em>-2 expression. UnAG also renews micro-RNA (miR)-126 expression, resulting in the posttranscriptional regulation of vascular cell adhesion molecule 1 expression and a reduced number of infiltrating inflammatory cells in vivo. Loss-of-function experiments that target miR-126 demonstrate that miR-126 also controls SIRT1 and <em>SOD</em>-2 expression, thus confirming its role in driving UnAG-mediated EC protection against ROS imbalance. These results indicate that UnAG protects vessels from ROS imbalance in ob/ob mice by rescuing miR-126 expression, thus emphasizing its potential clinical impact in avoiding limb loss in PAD.

The second member of the transient receptor potential-melastatin channel family (TRPM2) is expressed in the heart and vasculature. TRPM2 channels were expressed in the sarcolemma and transverse tubules of adult left ventricular (LV) myocytes. Cardiac TRPM2 channels were functional since activation with H2O2 resulted in Ca(2+) influx that was dependent on extracellular Ca(2+), was significantly higher in wild-type (WT) myocytes compared with TRPM2 knockout (KO) myocytes, and inhibited by clotrimazole in WT myocytes. At rest, there were no differences in LV mass, heart rate, fractional shortening, and +dP/dt between WT and KO hearts. At 2-<em>3</em> days after ischemia-reperfusion (I/R), despite similar areas at risk and infarct sizes, KO hearts had lower fractional shortening and +dP/dt compared with WT hearts. Compared with WT I/R myocytes, expression of the Na(+)/Ca(2+) exchanger (NCX1) and NCX1 current were increased, expression of the α1-subunit of Na(+)-K(+)-ATPase and Na(+) pump current were decreased, and action potential duration was prolonged in KO I/R myocytes. Post-I/R, intracellular Ca(2+) concentration transients and contraction amplitudes were equally depressed in WT and KO myocytes. After 2 h of hypoxia followed by <em>3</em>0 min of reoxygenation, levels of ROS were significantly higher in KO compared with WT LV myocytes. Compared with WT I/R hearts, oxygen radical scavenging enzymes (<em>SODs</em>) and their upstream regulators (forkhead box transcription factors and hypoxia-inducible factor) were lower, whereas NADPH oxidase was higher, in KO I/R hearts. We conclude that TRPM2 channels protected hearts from I/R injury by decreasing generation and enhancing scavenging of ROS, thereby reducing I/R-induced oxidative stress.

1-Methylnicotinamide (MNA) is one of the major derivatives of nicotinamide, which was recently shown to exhibit antithrombotic and antiinflammatory actions. However, it is not yet known whether MNA affects gastric mucosal defense. The effects of exogenous MNA were studied on gastric secretion and gastric lesions induced in rats by <em>3</em>.5 h of water immersion and water restraint stress (WRS) or in rats administered 75% ethanol. MNA [6.25-100 mg/kg intragastrically (i.g.)] led to a dose-dependent rise in the plasma MNA level, inhibited gastric acid secretion, and attenuated these gastric lesions induced by WRS or ethanol. The gastroprotective effect of MNA was accompanied by an increase in the gastric mucosal blood flow and plasma calcitonin gene-related peptide (CGRP) levels, the preservation of prostacyclin (PGI(2)) generation (measured as 6-keto-PGF1alpha), and an overexpression of mRNAs for cyclooxygenase (COX)-2 and CGRP in the gastric mucosa. R-<em>3</em>-(4-Fluoro-phenyl)-2-[5-(4-fluoro-phenyl)-benzofuran-2-ylmethoxycarbonylamino]-propionic acid (RO <em>3</em>24479), which is the selective antagonist of IP/PGI(2) receptors, reversed the effects of MNA on gastric lesions and GBF. MNA-induced gastroprotection was attenuated by suppression of COX-1 [5-(4-chlorophenyl)-1-(4-methoxyphenyl)-<em>3</em>-(trifluoromethyl)-1H-pyrazole; SC-560] and COX-2 [4-(4-methylsulfonylphenyl)-<em>3</em>-phenyl-5H-furan-2-one; rofecoxib] activity, capsaicin denervation, and by the pretreatment with CGRP(8-<em>3</em>7) or capsazepine. Addition of exogenous PGI(2) or CGRP restored the MNA-induced gastroprotection in rats treated with COX-1 and COX-2 inhibitors or in those with capsaicin denervation. WRS enhanced MDA content while decreasing superoxide dismutase (<em>SOD</em>) activity in the gastric mucosa, but pretreatment with MNA reversed these changes. MNA exerts potent gastroprotection against WRS damage via mechanisms involving cooperative action of PGI(2) and CGRP in preservation of microvascular flow, antioxidizing enzyme <em>SOD</em> activity, and reduction in lipid peroxidation.

Drought is a world-spread problem seriously influencing grain production and quality, the loss of which is the total for other natural disasters, with increasing global climate change making the situation more serious. Wheat is the staple food for more than <em>3</em>5% of world population, so wheat anti-drought physiology study is of importance to wheat production and biological breeding for the sake of coping with abiotic and biotic conditions. Much research is involved in this hot topic, but the pace of progress is not so large because of drought resistance being a multiple-gene-control quantitative character and wheat genome being larger (16,000Mb). On the other hand, stress adaptive mechanisms are quite different, with stress degree, time course, materials, soil quality status and experimental plots, thus increasing the complexity of the issue in question. Additionally, a little study is related to the whole life circle of wheat, which cannot provide a comprehensive understanding of its anti-drought machinery. We selected 10 kinds of wheat genotypes as materials, which have potential to be applied in practice, and measured change of relative physiological indices through wheat whole growing-developmental circle (i.e. seedling, tillering and maturing). Here, we reported the anti-oxidative results of maturation stage (the results of seedling and tillering stage have been published) in terms of activities of POD, <em>SOD</em>, CAT and MDA content as follows: (1) 10 wheat genotypes can be grouped into three kinds (A-C, respectively) according to their changing trend of the measured indices; (2) A group performed better resistance drought under the condition of treatment level 1 (appropriate level), whose activities of anti-oxidative enzymes (POD, <em>SOD</em>, CAT) were higher and MDA lower; (<em>3</em>) B group exhibited stronger anti-drought under treatment level 2 (light-stress level), whose activities of anti-oxidative enzymes were higher and MDA lower; (4) C group expressed anti-drought to some extent under treatment level <em>3</em> (serious-stress level), whose activities of anti-oxidative enzymes were stronger, MDA lower; (5) these results demonstrated that different wheat genotypes have different physiological mechanisms to adapt themselves to changing drought stress, whose molecular basis is discrete gene expression profiling (transcriptom); (6) our results also showed that the concept and method accepted and adopted by most researchers [T.C. Hsiao, Plant response to water stress, Ann. Rev. Plant Physiol. 24 (197<em>3</em>) 519-570], that 75% FC is a proper supply for higher plants, was doubted, because this level could not reflect the true suitable level of different wheat genotypes. The study in this respect is the key to wheat anti-drought and biological-saving water agriculture; (7) our research can provide insights into physiological mechanisms of crop anti-drought and direct practical materials for wheat anti-drought breeding; (8) the physiological study of wheat is more urgent up-to-date and molecular aspects are needed, but cannot substitute this important part. The combination of both is an important strategy and a key and (9) POD, <em>SOD</em> and CAT activities and MDA content of different wheat genotypes had quite different changing trend at different stages and under different soil water stress conditions, which was linked with their origin of cultivation and individual soil water threshold.

Glutathione reductase (GR; EC 1.6.4.2) and superoxide dismutase (<em>SOD</em>; EC 1.15.1.1) are two well-known enzymes involved in the scavenging of reactive oxygen intermediates. However, little is known about the regulation of Gor and <em>Sod</em> genes in plant cells. To obtain information about hypothetical redox regulatory mechanisms controlling Gor and <em>Sod</em> gene expression we artificially enhanced the levels of reduced and oxidized forms of glutathione (GSH and GSSG) in Pinus sylvestris L. needles. Scots pine shoots were placed for 12 h in beakers containing 5 mM GSH, 5 mM GSSG or water. Increased levels of both GSSG and GSH were observed in the GSSG-treated needles after <em>3</em> h. In contrast, only the GSH level was increased by the GSH treatment. Thus, the GSH/GSSG ratio increased up to 15-fold during the GSH treatment and decreased approximately two-fold during the GSSG treatment. The GR activity was significantly higher (60%) when GSSG was applied, without any apparent change in the amount and isoform population of GR or accumulation of Gor gene transcripts. This indicates that the GR activity increased per se in the GSSG treatment. The level of cytosolic CuZn-<em>Sod</em> transcripts was decreased significantly by the GSH treatment without any change in enzyme activity. The chloroplastic CuZn-<em>Sod</em> gene generally showed a more stable transcript level in the different treatments. However, a similarity between the cytosolic and chloroplastic levels of CuZn-<em>Sod</em> transcripts could be observed in different treatments. This suggests that the redox state of glutathione plays an important role in the in vivo regulation of CuZn-<em>Sod</em> gene expression in plants.