METHODS

To evaluate curcumin loaded solid lipid nanoparticles (C-SLNs) in the experimental paradigm of cerebral ischemia (BCCAO model) in rats.

RESULTS

Oral administration of free curcumin and C-SLNs (25 and 50 mg/kg) was started 5 days prior and continued for <em>3</em> days after BCCAO. Alleviation in behavioral, oxidative and nitrosative stress, acetylcholinesterase, mitochondrial enzyme complexes, and physiological parameters were assessed. Confirmation of effective brain delivery of C-SLNs (p.o) was done using biodistribution studies in mice and confocal microscopy of rat brain section. There was an improvement of 90% in cognition and 52% inhibition of acetylcholinesterase versus cerebral ischemic group (I/R). Neurological scoring improved by 79%. Levels of superoxide dismutase, catalase, glutathione, and mitochondrial complex enzyme activities were significantly increased, while lipid peroxidation, nitrite, and acetylcholinesterase levels decreased (p<0.05) after C-SLNs administration. It is noteworthy to report the restoration of <em>SOD</em>, GSH, catalase, and mitochondrial complex enzyme levels equivalent to sham control values. Gamma-scintigraphic studies show 16.4 and <em>3</em>0 times improvement in brain bioavailability (AUC) upon oral and i.v administration of C-SLNs versus solubilized curcumin (C-S).

CONCLUSIONS

Study indicates protective role of curcumin against cerebral ischemic insult; provided it is packaged suitably for improved brain delivery.

Rice (Oryza sativa L.) seedlings, when kept at 42 degrees C for 24 h before being kept at 5 degrees C for 7 d, did not develop chilling injury. Chilling resistance was enhanced in parallel with the period of heat-treatment. The level of APX activity was higher in heated seedlings whereas CAT activity was decreased by heat stress. There was no significant difference in <em>SOD</em> activity between heated and unheated seedlings. The elevated activity of APX was sustained after 7 d of chilling. The cytosolic APX gene expression in response to high and low temperature was analysed with an APXa gene probe. APXa mRNA levels increased within 1 h after seedlings were exposed to 42 degrees C. Elevated APXa mRNA levels could also be detected after 24 h of heating. The APXa mRNA level in preheated seedlings was still higher than unheated seedlings under cold stress. The promoter of the APXa gene was cloned from rice genomic DNA by TAIL-PCR, and characterized by DNA sequencing. The promoter had a minimal heat shock factor-binding motif, 5'-nGAAnnTTCn-<em>3</em>', located in the 81 bp upstream of the TATA box. Heat shock induction of the APXa gene could be a possible cause of reduced chilling injury in rice seedlings.

OBJECTIVE

Oxidative stress and inflammation are leading risk factors for age-associated functional declines. We assessed aspirin effects on endogenous oxidative-stress levels, lifespan, and age-related functional declines, in the nematode Caenorhabditis elegans.

RESULTS

Both aspirin and its salicylate moiety, at nontoxic concentrations (0.5-1 mM), attenuated endogenous levels of reactive oxygen species (p<0.001), and upregulated antioxidant genes encoding superoxide dismutases (especially <em>sod</em>-<em>3</em>, p<0.001), catalases (especially ctl-2, p<0.0001), and two glutathione-S-transferases (gst-4 and gst-10; each p<0.005). Aspirin, and to a lesser degree salicylate, improved survival of hydrogen peroxide, and in the absence of exogenous stress aspirin extended lifespan by 21%-2<em>3</em>% (each p<10(-9)), while salicylate added 14% (p<10(-6)). Aspirin and salicylate delayed age-dependent declines in motility and pharyngeal pumping (each p<0.005), and decreased intracellular protein aggregation (p<0.0001)-all established markers of physiological aging-consistent with slowing of the aging process. Aspirin fails to improve stress resistance or lifespan in nematodes lacking DAF-16, implying that it acts through this FOXO transcription factor.

METHODS

Studies in mice and humans suggest that aspirin may protect against multiple age-associated diseases by reducing all-cause mortality. We now demonstrate that aspirin markedly slows many measures of aging in the nematode.

CONCLUSIONS

Aspirin treatment is associated with diminished endogenous oxidant stress and enhanced resistance to exogenous peroxide, both likely mediated by activation of antioxidant defenses. Our evidence indicates that aspirin attenuates insulin-like signaling, thus protecting against oxidative stress, postponing age-associated functional declines and extending C. elegans lifespan under benign conditions.

Ionizing radiation promotes formation of reactive oxygen species, including the superoxide anion (O2-). To evaluate whether O2- or O2--mediated perturbations may contribute to the known atherogenic effects of radiation, we examined aortic lesion formation in irradiated C57BL/6 mice and evaluated the effects of CuZn-superoxide dismutase (CuZn-<em>SOD</em>) overexpression. Ten-week-old mice were exposed to a 2-, 4-, or 8-Gy dose of 250-keV x-rays to the upper thorax and then placed on a high-fat diet for 18 weeks. Based on quantitative lipid staining of serial sections of the proximal aorta, mean lesion area was increased with increasing radiation dose and was <em>3</em>-fold greater in 8-Gy-irradiated than sham-irradiated mice (7800+/-2140 versus 26<em>3</em>5+/-709 micrometer(2), P<0.05). These effects were absolutely dependent on a high-fat diet, which had to be introduced within 1 to 2 weeks of the radiation exposure, suggesting the early involvement of atherogenic lipoproteins that were elevated in response to the diet. The importance of radiation-induced oxidative stress was supported by the observation of a 2-fold lower mean lesion area in irradiated CuZn-<em>SOD</em> transgenic mice than in their irradiated, nontransgenic littermates (<em>3</em>026+/-1590 versus 6102+/-18<em>3</em>4 micrometer(2), P<0.05). Lucigenin-enhanced chemiluminescence, used as an index of aortic O2- concentrations, was significantly elevated in the postradiation period, and this response was reduced in CuZn-<em>SOD</em> transgenics. On the basis of these results, we propose that radiation may be a useful tool for initiating oxidative or redox-regulated events that promote atherogenesis and for testing the antiatherogenic properties of antioxidants.

Doxorubicin (DOX) is one of the most effective chemotherapeutic drugs; however, its incidence of cardiotoxicity compromises its therapeutic index. Chrysin, a natural flavone, possesses multiple biological activities, such as antioxidant, anti-inflammatory and anti-cancer. The present study was designed to investigate whether chrysin could protect against DOX-induced acute cardiotoxicity; and if so, unravel the molecular mechanisms of this protective effect. Chrysin was administered to male albino rats once daily for 12 consecutive days at doses of 25 and 50mg/kg orally. DOX (15 mg/kg; i.p.) was administered on day 12. Chrysin pretreatment significantly protected against DOX-induced myocardial damage which was characterized by conduction abnormalities, increased serum creatine kinase isoenzyme-MB (CK-MB), and lactate dehydrogenase (LDH) and myofibrillar disarrangement. As indicators of oxidative stress, DOX caused significant glutathione depletion, lipid peroxidation and reduction in activities of antioxidant enzymes; catalase (CAT) and superoxide dismutase (<em>SOD</em>). Chrysin pretreatment significantly attenuated DOX-induced oxidative injury. Additionally, DOX provoked inflammatory responses by increasing the expressions of nuclear factor kappa-B (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and the levels of tumor necrosis factor-alpha (TNF-α) and nitric oxide while chrysin pretreatment significantly inhibited these inflammatory responses. Furthermore, DOX induced apoptotic tissue damage by increasing Bax and cytochrome c expressions and caspase-<em>3</em> activity while decreasing the expression of Bcl-2. Chrysin pretreatment significantly ameliorated these apoptotic actions of DOX. Collectively, these findings indicate that chrysin possesses a potent protective effect against DOX-induced acute cardiotoxicity via suppressing oxidative stress, inflammation and apoptotic tissue damage.

DL-<em>3</em>-n-butylphthalide (NBP) has been used for stroke treatment in China for years. Recently, we found that NBP can reduce the incidence of stroke and have protective action on cerebral microvessels, suggesting a direct action of NBP on endothelial cells. However, it is difficult to evaluate the direct action of NBP on endothelial cells in vivo because of the interactions of endothelial cells with other types of neuronal cells. Therefore, we investigated whether NBP protects against oxygen glucose deprivation (OGD)-induced cell injury in an immortalized human umbilical vein endothelial cells (HUVEC) in vitro. Cells were exposed to OGD, leading to endothelial damage. Endothelial injury was assessed by measuring MTT and the changes in chromatin morphology. Mitochondrial superoxide, mitochondrial membrane potential and mitochondrial morphology were assessed using MitoSOX Red. Rhodamine 12<em>3</em> and MitoTracker, respectively. Nitrosative stress was assessed by measuring the production of peroxynitrite. The activity of superoxide dismutase (<em>SOD</em>) is evaluated using <em>SOD</em> assay kit-WST. The expression of hypoxia inducible factor-1 alpha (HIF-1alpha) was assessed at the protein level by immunofluorescence and Western blotting. NBP at doses between 0.01 and 100 micromol/L dose-dependently protected against OGD-induced cell death. In addition, NBP attenuated OGD-induced mitochondria superoxide, cellular formation of peroxynitrite, and decrease in <em>SOD</em> activity, mitochondria fragmentation and loss of mitochondrial membrane potential. In parallel, NBP enhanced OGD-induced HIF-1alpha expression. This study demonstrates that NBP can protect HUVEC against OGD-induced oxidative/nitrosative stress, mitochondrial damage and subsequent cell death. This protective effect is, at least in part, associated with its enhancement on OGD-induced HIF-1alpha expression.

Exposure of newborn calves to chronic hypoxia causes pulmonary artery (PA) hypertension and remodeling. Previous studies showed that the redox-sensitive transcription factor, early growth response-1 (Egr-1), is upregulated in the PA of chronically hypoxic calves and regulates cell proliferation. Furthermore, we established in mice a correlation between hypoxic induction of Egr-1 and reduced activity of extracellular superoxide dismutase (EC-<em>SOD</em>), an antioxidant that scavenges extracellular superoxide. We now hypothesize that loss of EC-<em>SOD</em> in chronically hypoxic calves leads to extracellular superoxide-mediated upregulation of Egr-1. To validate our hypothesis and identify the signaling pathways involved, we utilized PA tissue from normoxic and chronically hypoxic calves and cultured calf and human PA smooth muscle cells (PASMC). Total <em>SOD</em> activity was low in the PA tissue, and only the extracellular <em>SOD</em> component decreased with hypoxia. PA tissue of hypoxic calves showed increased oxidative stress and increased Egr-1 mRNA. To mimic the in vivo hypoxia-induced extracellular oxidant imbalance, cultured calf PASMC were treated with xanthine oxidase (XO), which generates extracellular superoxide and hydrogen peroxide. We found that 1) XO increased Egr-1 mRNA and protein, 2) XO induced the phosphorylation of ERK1/2 and, <em>3</em>) pretreatment with an ERK1/2 inhibitor prevented induction of Egr-1 by XO. siRNA knock-down of EC-<em>SOD</em> in human PASMC also upregulated Egr-1 mRNA and protein, activated ERK1/2, and enhanced SMC proliferation and reduced apoptosis. We conclude that an oxidant/antioxidant imbalance arising from loss of EC-<em>SOD</em> in the PA with chronic hypoxia induces Egr-1 via activation of ERK1/2 and contributes to pulmonary vascular remodeling.

Oxidative stress has been increasingly postulated as a major contributor to endothelial dysfunction in preeclampsia (PE), although evidence supporting this hypothesis remains inconsistent. This study aimed to analyze in depth the potential role of oxidative stress as a mechanism underlying endothelial damage in PE and the pregnant woman's susceptibility to the disease. To this end, indicative markers of lipoperoxidation and protein oxidation and changes in antioxidant defense systems were measured in blood samples from 5<em>3</em> women with PE and <em>3</em>0 healthy pregnant controls. Results, analyzed in relation to disease severity, showed PE women, compared with women with normal pregnancy, to have: (1) significantly enhanced antioxidant enzyme <em>SOD</em> and GPx activities in erythrocytes; (2) similar plasma alpha-tocopherol levels and significantly increased alpha-tocopherol/lipids in both mild and severe PE; (<em>3</em>) significantly decreased plasma vitamin C and protein thiol levels; (4) similar erythrocyte glutathione content, total plasma antioxidant capacity, and whole plasma oxidizability values; (5) significantly elevated plasma total lipid hydroperoxides, the major initial reaction products of lipid peroxidation, in severe PE; (6) no intracellular or extracellular increases in any of the secondary end-products of lipid peroxidation, malondialdehyde or lipoperoxides; (7) similar oxidative damage to proteins quantified by plasma carbonyl levels, immunoblot analysis, and advanced oxidation protein products assessment; and (8) significantly elevated and severity-related soluble vascular cell adhesion molecule-1 serum levels reflecting endothelial dysfunction. No correlations were found among plasma levels of circulating adhesion molecules with regard to lipid and protein oxidation markers. Globally, these data reflect mild oxidative stress in blood of preeclamptic women, as oxidative processes seem to be counteracted by the physiologic activation of antioxidant enzymes and by the high plasma vitamin E levels that would prevent further oxidative damage. These results do not permit us to conclude that oxidative stress might be a pathogenetically relevant process causally contributing to the disease.

Type 2 diabetes (T2D) is a leading risk factor for a variety of cardiovascular diseases including coronary heart disease and atherosclerosis. Exercise training (ET) has a beneficial effect on these disorders, but the basis for this effect is not fully understood. This study was designed to investigate whether the ET abates endothelial dysfunction in the aorta in T2D. Heterozygous controls (m Lepr(db)) and type 2 diabetic mice (db/db; Lepr(db)) were either exercise entrained by forced treadmill exercise or remained sedentary for 10 wk. Ex vivo functional assessment of aortic rings showed that ET restored acetylcholine-induced endothelial-dependent vasodilation of diabetic mice. Although the protein expression of endothelial nitric oxide synthase did not increase, ET reduced both IFN-γ and superoxide production by inhibiting gp91(phox) protein levels. In addition, ET increased the expression of adiponectin (APN) and the antioxidant enzyme, <em>SOD</em>-1. To investigate whether these beneficial effects of ET are APN dependent, we used adiponectin knockout (APNKO) mice. Indeed, impaired endothelial-dependent vasodilation occurred in APNKO mice, suggesting that APN plays a central role in prevention of endothelial dysfunction. APNKO mice also showed increased protein expression of IFN-γ, gp91(phox), and nitrotyrosine but protein expression of <em>SOD</em>-1 and -<em>3</em> were comparable between wild-type and APNKO. These findings in the aorta imply that APN suppresses inflammation and oxidative stress in the aorta, but not <em>SOD</em>-1 and -<em>3</em>. Thus ET improves endothelial function in the aorta in T2D via both APN-dependent and independent pathways. This improvement is due to the effects of ET in inhibiting inflammation and oxidative stress (APN-dependent) as well as in improving antioxidant enzyme (APN-independent) performance in T2D.

Nephrotoxicity is the dose-limiting factor for colistin, but the exact mechanism is unknown. This study aimed to investigate the roles of the mitochondrial, death receptor, and endoplasmic reticulum pathways in colistin-induced nephrotoxicity. Mice were intravenously administered 7.5 or 15 mg of colistin/kg of body weight/day (via a <em>3</em>-min infusion and divided into two doses) for 7 days. Renal function, oxidative stress, and apoptosis were measured. Representative biomarkers involved in the mitochondrial, death receptor, and endoplasmic reticulum pathways were investigated, and the key markers involved in apoptosis and autophagy were examined. After 7-day colistin treatment, significant increase was observed with blood urea nitrogen, serum creatinine, and malondialdehyde, while activities of superoxide dismutase (<em>SOD</em>) and catalase decreased in the kidneys. Acute tubular necrosis and mitochondrial dysfunction were detected, and colistin-induced apoptosis was characterized by DNA fragmentation, cleavage of poly(ADP-ribose) polymerase (PARP-1), increase of 8-hydroxydeoxyguanosine (8-OHdG), and activation of caspases (caspase-8, -9, and -<em>3</em>). It was evident that colistin-induced apoptosis involved the mitochondrial pathway (downregulation of Bcl-2 and upregulation of cytochrome C [cytC] and Bax), death receptor pathway (upregulation of Fas, FasL, and Fas-associated death domain [FADD]), and endoplasmic reticulum pathway (upregulation of Grp78/Bip, ATF6, GADD15<em>3</em>/CHOP, and caspase-12). In the 15-mg/kg/day colistin group, expression of the cyclin-dependent kinase 2 (CDK2) and phosphorylated JNK (p-JNK) significantly increased (P < 0.05), while in the 7.5-mg/kg/day colistin group, a large number of autophagolysosomes and classic autophagy were observed. Western blot results of Beclin-1 and LC<em>3</em>B indicated that autophagy may play a protective role in colistin-induced nephrotoxicity. In conclusion, this is the first study to demonstrate that all three major apoptosis pathways and autophagy are involved in colistin-induced nephrotoxicity.

BACKGROUND

Nitric oxide inactivation by superoxide impairs endothelium-dependent vasodilation and plays a role in various forms of hypertension. Almost no data exist regarding hypertension secondary to chronic renal failure. Previous studies have shown that endothelium-related relaxations, secondary to decreased nitric oxide bioactivity, are impaired in resistance vessels from rats <em>3</em> to 10 days after renal mass reduction (RMR).

METHODS

The membrane-permeable superoxide dismutase (SOD)-mimetic (tempol) was administered IP (1.5 mmol/kg/day for 10 days) to RMR rats and sham-operated controls (SN). Systolic blood pressure (SBP) was measured by tail cuff manometry at days 0, <em>3</em>, 6 and 10. The increase of flow induced by acetylcholine (10-6 mol/L) was measured in isolated perfused mesenteric arteries from RMR and SN rats pre-contracted with noradrenaline (1 to 5 micromol/L), with or without exogenous SOD. Plasma levels of advanced oxidative protein products (AOPPs; chloramine-T equivalents) were measured in SN and RMR rats.

RESULTS

Tempol prevented the increase of SBP: 118 +/- 2.2 mm Hg at baseline and 122 +/- 1.6 mm Hg at 10 days in tempol-treated vs 118.14 +/- 1.65 mm Hg at baseline and 145 +/- 7.69 mm Hg at 10 days in untreated RMR rats (P < 0.01). Responsiveness to acetylcholine was reduced in RMR rats (peak flow increase: 1<em>3</em>9 +/- 7.8% vs. 176 +/- 11% in SN, P=0.028 at <em>3</em> days and 140 +/- 6.4% vs. 187 +/- 16.9% in SN at 10 days, P=0.007). In arteries pre-incubated with SOD (200 U/mL) the peak flows were 175 +/- 9.4% at <em>3</em> days and 157 +/- 5.8% at 10 days (P=0.007 and P=0.051, respectively, vs. control RMR vessels). AOPP values were significantly increased in plasma from RMR rats <em>3</em> days after 5/6 nephrectomy (747 +/- 107 vs. 481 +/- 77 micromol/L, P < 0.05) but returned to normal by day 10. AOPP levels were not significantly reduced by tempol.

CONCLUSIONS

Increased vascular superoxide production plays a central role in the development of vascular endothelial dysfunction and hypertension early after 5/6 nephrectomy.

Understanding the factors that determine the ability of Mn porphyrins to scavenge reactive species is essential for tuning their in vivo efficacy. We present herein the revised structure-activity relationships accounting for the critical importance of electrostatics in the Mn porphyrin-based redox modulation systems and show that the design of effective <em>SOD</em> mimics (per se) based on anionic porphyrins is greatly hindered by inappropriate electrostatics. A new strategy for the beta-octabromination of the prototypical anionic Mn porphyrins Mn(III) meso-tetrakis(p-carboxylatophenyl)porphyrin ([Mn(III)TCPP](<em>3</em>-) or MnTBAP(<em>3</em>-)) and Mn(III) meso-tetrakis(p-sulfonatophenyl)porphyrin ([Mn(III)TSPP](<em>3</em>-)), to yield the corresponding anionic analogues [Mn(III)Br(8)TCPP](<em>3</em>-) and [Mn(III)Br(8)TSPP](<em>3</em>-), respectively, is described along with characterization data, stability studies, and their ability to substitute for <em>SOD</em> in <em>SOD</em>-deficient Escherichia coli. Despite the Mn(III)/Mn(II) reduction potential of [Mn(III)Br(8)TCPP](<em>3</em>-) and [Mn(III)Br(8)TSPP](<em>3</em>-) being close to the <em>SOD</em>-enzyme optimum and nearly identical to that of the cationic Mn(III) meso-tetrakis(N-methylpyridinium-2-yl)porphyrin (Mn(III)TM-2-PyP(5+)), the <em>SOD</em> activity of both anionic brominated porphyrins ([Mn(III)Br(8)TCPP](<em>3</em>-), E(1/2)=+21<em>3</em> mV vs NHE, log k(cat)=5.07; [Mn(III)Br(8)TSPP](<em>3</em>-), E(1/2)=+209 mV, log k(cat)=5.56) is considerably lower than that of Mn(III)TM-2-PyP(5+) (E(1/2)=+220 mV, log k(cat)=7.79). This illustrates the impact of electrostatic guidance of O(2)(-) toward the metal center of the mimic. With low k(cat), the [Mn(III)TCPP](<em>3</em>-), [Mn(III)TSPP](<em>3</em>-), and [Mn(III)Br(8)TCPP](<em>3</em>-) did not rescue <em>SOD</em>-deficient E. coli. The striking ability of [Mn(III)Br(8)TSPP](<em>3</em>-) to substitute for the <em>SOD</em> enzymes in the E. coli model does not correlate with its log k(cat). In fact, the protectiveness of [Mn(III)Br(8)TSPP](<em>3</em>-) is comparable to or better than that of the potent <em>SOD</em> mimic Mn(III)TM-2-PyP(5+), even though the dismutation rate constant of the anionic complex is 170-fold smaller. Analyses of the medium and E. coli cell extract revealed that the major species in the [Mn(III)Br(8)TSPP](<em>3</em>-) system is not the Mn complex, but the free-base porphyrin [H(2)Br(8)TSPP](4-) instead. Control experiments with extracellular MnCl(2) showed the lack of E. coli protection, indicating that "free" Mn(2+) cannot enter the cell to a significant extent. We proposed herein the alternative mechanism where a labile Mn porphyrin [Mn(III)Br(8)TSPP](<em>3</em>-) is not an <em>SOD</em> mimic per se but carries Mn into the E. coli cell.

The vascular endothelium is a significant site for tissue injury following exposure to reactive oxygen species derived from a number of sources. In order to develop a better understanding of the mechanism(s) of oxidative damage, monolayer cultures of endothelial cells obtained from bovine pulmonary arteries were exposed to reactive oxygen species generated from the oxidation of dihydroxyfumarate (DHF) to diketosuccinate. Exposure to oxidizing DHF caused a loss of cell membrane integrity that was delayed in onset; that is, it did not begin until 2 h after the addition of DHF although reactive oxygen species are produced immediately by DHF in solution. Endothelial cell lysis by DHF was prevented by the simultaneous addition of superoxide dismutase (<em>SOD</em>), catalase (CAT), or deferoximine (DFX). This oxidant-induced lysis was unaffected by N,N,-diphenyl-p-phenylenediamine (DPPD), a potent inhibitor of lipid peroxidation. However, simultaneous addition of <em>3</em>-aminobenzamide (<em>3</em>AB) and nicotinamide (NA), inhibitors of poly(ADP-ribose) polymerase, prevented cell lysis. Oxidant-induced loss of membrane integrity was preceded by the early appearance of DNA strand breaks, by increased levels of poly(ADP-ribose), the product of polymerase activity, and by depletion of NAD+ and ATP, followed by a decline in the energy charge ratio of the cells. None of these intracellular changes occurred when either <em>SOD</em>, CAT, or DFX were added at the same time as DHF, suggesting that O2-., H2O2, and HO. mediated these changes. The O2-. appears to be important in the autoxidation reaction of DHF. The latter two reactive oxygen species may be part of cellular-catalyzed Fenton chemistry. The increase in poly(ADP-ribose), depletion of NAD+, and the decline in ATP were also prevented by the addition of <em>3</em>AB. The oxidant-induced DNA strand breakage was, however, unaffected by either <em>3</em>AB or NA. Addition of <em>3</em>AB immediately prior to the onset of cell lysis (2 h after the addition of DHF), prevented cell lysis, i.e., "rescued" the cells when neither <em>SOD</em>, CAT, nor DFX addition were effective. Concurrent with the "rescue" from lysis by <em>3</em>AB, there was an increase in NAD+ content and a return of the energy charge ratio to control levels. The data presented in this study suggests that in endothelial cells, DNA is a very sensitive target for reactive oxygen species and HO. is the likely proximal damaging species.(ABSTRACT TRUNCATED AT 400 WORDS)

Peroxisome proliferator-activated receptor (PPAR)-α, a lipid-sensing transcriptional factor, serves an important role in lipotoxicity. We evaluated whether fenofibrate has a renoprotective effect by ameliorating lipotoxicity in the kidney. Eight-week-old male C57BLKS/J db/m control and db/db mice, divided into four groups, received fenofibrate for 12 weeks. In db/db mice, fenofibrate ameliorated albuminuria, mesangial area expansion and inflammatory cell infiltration. Fenofibrate inhibited accumulation of intra-renal free fatty acids and triglycerides related to increases in PPARα expression, phosphorylation of AMP-activated protein kinase (AMPK), and activation of Peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α)-estrogen-related receptor (ERR)-1α-phosphorylated acetyl-CoA carboxylase (pACC), and suppression of sterol regulatory element-binding protein (SREBP)-1 and carbohydrate regulatory element-binding protein (ChREBP)-1, key downstream effectors of lipid metabolism. Fenofibrate decreased the activity of phosphatidylinositol-<em>3</em> kinase (PI<em>3</em>K)-Akt phosphorylation and FoxO<em>3</em>a phosphorylation in kidneys, increasing the B cell leukaemia/lymphoma 2 (BCL-2)/BCL-2-associated X protein (BAX) ratio and superoxide dismutase (<em>SOD</em>) 1 levels. Consequently, fenofibrate recovered from renal apoptosis and oxidative stress, as reflected by 24 hr urinary 8-isoprostane. In cultured mesangial cells, fenofibrate prevented high glucose-induced apoptosis and oxidative stress through phosphorylation of AMPK, activation of PGC-1α-ERR-1α, and suppression of SREBP-1 and ChREBP-1. Our results suggest that fenofibrate improves lipotoxicity via activation of AMPK-PGC-1α-ERR-1α-FoxO<em>3</em>a signaling, showing its potential as a therapeutic modality for diabetic nephropathy.

Evidence from clinical and experimental studies indicate that oxidative stress is involved in pathogenesis of Parkinson's disease. The present study was designed to investigate the neuroprotective potential of lycopene on oxidative stress and neurobehavioral abnormalities in rotenone induced PD. Rats were treated with rotenone (<em>3</em> mg/kg body weight, intraperitoneally) for <em>3</em>0 days. NADH dehydrogenase a marker of rotenone action was observed to be significantly inhibited (<em>3</em>5%) in striatum of treated animals. However, lycopene administration (10 mg/kg, orally) to the rotenone treated animals for <em>3</em>0 days increased the activity by <em>3</em>9% when compared to rotenone treated animals. Rotenone administration increased the MDA levels (75.15%) in striatum, whereas, lycopene administration to rotenone treated animals decreased the levels by 24.<em>3</em><em>3</em>%. Along with this, significant decrease in GSH levels (42.69%) was observed in rotenone treated animals. Lycopene supplementation on the other hand, increased the levels of GSH by 75.<em>3</em>5% when compared with rotenone treated group. The activity of <em>SOD</em> was inhibited by 69% in rotenone treated animals and on lycopene supplementation; the activity increased by 12% when compared to controls. This was accompanied by cognitive and motor deficits in rotenone administered animals, which were reversed on lycopene treatment. Lycopene treatment also prevented release of cytochrome c from mitochondria. Collectively, these observations suggest that lycopene supplementation along with rotenone for <em>3</em>0 days prevented rotenone-induced alterations in antioxidants along with the prevention of rotenone induced oxidative stress and neurobehavioral deficits. The results provide an evidence for beneficial effect of lycopene supplementation in rotenone-induced PD and suggest therapeutic potential in neurodegenerative diseases involving accentuated oxidative stress.

In Neurospora crassa, several biological phenomena such as the synthesis of carotenoids in the mycelia and polarity of perithecia are regulated by light. We found that a <em>sod</em>-1 mutant, with a defective Cu,Zn-type superoxide dismutase (<em>SOD</em>), showed accelerated light-dependent induction of carotenoid accumulation in the mycelia compared with the wild type. The initial rate of light-induced carotenoid accumulation in the <em>sod</em>-1 mutant was faster than that in the vvd mutant known to accumulate high concentrations. This acceleration was suppressed by treatment with antioxidant reagents. Light-induced transcription of genes involved in carotenoid synthesis, al-1, -2, and -<em>3</em>, was sustained in the <em>sod</em>-1 mutant, whereas it was transient in the wild type. Moreover <em>sod</em>-1 was defective in terms of light-induced polarity of perithecia. By genetic analysis, the enhancement in light-inducible carotenoid synthesis in <em>sod</em>-1 was dependent on the wild type alleles of wc-1 and wc-2. However, the <em>sod</em>-1;vvd double mutant showed additive effects on the carotenoid accumulation in the mycelia. These results suggested that intracellular reactive oxygen species regulated by <em>SOD</em>-1 could affect the light-signal transduction pathway via WC proteins.

BACKGROUND

Increased intrahepatic vascular tone in cirrhosis has been attributed to a decrease of hepatic nitric oxide (NO) secondary to disturbances in the post-translational regulation of the enzyme eNOS. NO scavenging by superoxide (O(2)(-)) further contributes to a reduction of NO bioavailability in cirrhotic livers.

OBJECTIVE

To investigate whether removing increased O(2)(-) levels could be a new therapeutic strategy to increase intrahepatic NO, improve endothelial dysfunction and reduce portal pressure in cirrhotic rats with portal hypertension.

METHODS

Adenoviral vectors expressing extracellular superoxide dismutase (<em>SOD</em>) (AdEC<em>SOD</em>) or beta-galactosidase (Adbetagal) were injected intravenously in control and CCl(4)-induced cirrhotic rats. After <em>3</em> days, liver O(2)(-) levels were determined by dihydroethidium staining, NO bioavailability by hepatic cGMP levels, nitrotyrosinated proteins by immunohistochemistry and western blot, and endothelial function by responses to acetylcholine in perfused rat livers. Mean arterial pressure (MAP) and portal pressure were evaluated in vivo.

RESULTS

Transfection of cirrhotic livers with AdEC<em>SOD</em> produced a significant reduction in O(2)(-) levels, a significant increase in hepatic cGMP, and a decrease in liver nitrotyrosinated proteins which were associated with a significant improvement in the endothelium-dependent vasodilatation to acetylcholine. In addition, in cirrhotic livers AdEC<em>SOD</em> transfection produced a significant reduction in portal pressure (17.<em>3</em> (SD 2) mm Hg vs 15 (SD 1.6) mm Hg; p<0.05) without significant changes in MAP. In control rats, AdEC<em>SOD</em> transfection prevents the increase in portal perfusion pressure promoted by an ROS-generating system.

CONCLUSIONS

In cirrhotic rats, reduction of O(2)(-) by AdECSOD increases NO bioavailability, improves intrahepatic endothelial function and reduces portal pressure. These findings suggest that scavenging of O(2)(-) might be a new therapeutic strategy in the management of portal hypertension.

A number of reports have described the effects of oxidative stress on tumor growth. Therefore, these experiments were designed to test the hypothesis that overexpression of extracellular superoxide dismutase (ec<em>SOD</em>) would inhibit the growth of tumors arising from s.c. implantation of syngenic B16-F1 melanoma cells. C57BL/6 mice were infected i.m. with adenovirus containing either beta-galactosidase (Ad.lacZ) as control or the secreted extracellular isoform of <em>SOD</em> (Ad.ec<em>SOD</em>) <em>3</em> days before s.c. implantation of B16-F1 tumor cells. Serum <em>SOD</em> activity was elevated nearly approximately 5-fold over control animals. Two weeks after implantation, B16-F1 tumor size was 65% smaller in mice infected with Ad.ec<em>SOD</em> in comparison with mice infected with Ad.lacZ. However, the presence of <em>SOD</em> did not affect growth rates of B16-F1 cells in vitro. Consistent with smaller tumor volume, tumors from Ad.ec<em>SOD</em>-infected mice also expressed less vascular endothelial growth factor (VEGF). Moreover, in vitro studies using B16-F1 cells confirm that <em>SOD</em> blunts oxidant-dependent VEGF expression. Importantly, CD<em>3</em>1 expression and vessel density were markedly reduced in tumors from Ad.ec<em>SOD</em>-infected mice compared with controls. These data suggest that tumor oxidative stress may facilitate tumor vascularization and thus promote tumor growth.

The pathogenesis of influenza virus infections of the lungs is in part mediated by oxidative stress. Such infections might therefore be expected to induce expression of stress-response genes and genes encoding antioxidant enzymes and to activate transcriptional regulatory proteins. Mice (C57B1/6 and C<em>3</em>H/HeJ) were infected intranasally with influenza virus A/PR/8/<em>3</em>4 (H1N1). Expression of the genes encoding the antioxidant enzymes manganese superoxide dismutase (Mn- <em>SOD</em>), indoleamine-2, <em>3</em>-dioxygenase (IDO), heme oxygenase-1, and glutathione peroxidase were increased in the lungs of virus-infected animals. Cu/Zn<em>SOD</em> and catalase mRNA were not induced by viral infection. Activation of the transcriptional regulatory proteins AP-1, C/EBP, and NF-kappa B (which are known to be affected by oxidant stress) was demonstrated by electrophoretic mobility shift assay after viral infection. In the case of Mn<em>SOD</em>, despite increased gene expression enzyme activity was not increased. In contrast, for heme oxygenase-1 both mRNA and activity were increased. C<em>3</em>H/ HeJ and C57B1/6 mice, which are known to have different responses to other types of oxidant stress, also differed in their responses to viral infection. Induction of heme oxygenase-1 expression was greater in C57B1/6 mice than in C<em>3</em>H/ HeJ mice, although inhibiting this enzyme did not alter virus-induced mortality. In contrast, IDO was more strongly induced in C<em>3</em>H/HeJ mice. Activation of NF-kappa B was much more marked in C57B1/6 mice than in C<em>3</em>H/HeJ mice. Although virus replication and inflammatory responses were equivalent in the two strains, lung injury (as measured by wet-to-dry wt ratios) and mortality were greater in C<em>3</em>H/HeJ mice than in C57B1/6 mice, a difference that may be related to differing oxidant stress responses. Thus influenza pneumonia causes an oxidant stress response in the lungs, the nature of which is determined in part by the genetic background of the host.

Xanthine oxidoreductase (XDH + XO, EC 1.2.<em>3</em>.2) is released into the circulation from organs rich in XO activity. Herein we report the specific high affinity binding of XO to glycosaminoglycans (GAGs) and the preferential association of XO with heparin, compared with heparan sulfate, chondroitin sulfate, and dematan sulfate. The binding of XO to Sepharose 6B-conjugated heparin (HS6B) occurs at physiological ionic strength and increased with pH, with Scatchard analysis revealing a nonlinear binding pattern at pH 7.4. The dissociation constant (Kd) for XO binding was 0.4 to 1.8 x 10(-7) M, similar to the heparin-reversible binding of lipoprotein lipase to vascular endothelium. The binding energy of 9-1<em>3</em> kcal/mol was concordant with noncovalent electrostatic interactions. Xanthine oxidase immobilization to HS6B rendered a catalytically active enzyme from that had kinetic characteristics distinct from XO in free solution. While the Km and Ki for xanthine in phosphate buffer at pH 7.4 were <em>3</em> microM and 1.6 mM, respectively, for free XO, they were 15 microM and 2.8 mM for immobilized XO. Inhibition constants for guanine and uric acid were also increased upon XO binding to HS6B. Changes in kinetic parameters were related to a real and not apparent decrease in binding affinity for substrate and inhibitors and were not due to diffusion-controlled processes within the gel matrix. Changes in Km and Ki for xanthine also had a significant influence on the relative quantities of O2.- and H2O2 generated by a given substrate concentration. Superoxide formed by HS6B-bound XO was partially consumed within the gel microenvironment which electrostatically excluded CuZn <em>SOD</em>. Immobilization of XO increased the half-life of enzyme activity in buffer and in the absence of substrate from 67 to 120 h at 4 degrees C. These data indicate that binding to cell surfaces will strongly influence the catalytic properties, oxidant producing capacity, and stability of XO.

OBJECTIVE

The importance of different antioxidative enzymes for the defence of insulin-producing cells against the toxicity of nitric oxide (NO) was characterised in bioengineered RINm5F cells.

METHODS

RINm5F insulin-producing cells stably overexpressing glutathione peroxidase (GPX), catalase (CAT) or Cu/Zn superoxide dismutase (<em>SOD</em>) were exposed to S-nitroso-N-acetyl-D,L-penicillamine (SNAP), sodium nitroprusside (SNP) and <em>3</em> morpholinosydnonimine (SIN-1), which generate both NO and reactive oxygen species, and to the polyamine/ NO, complex DETA/NO which generates NO alone. The viability of the cells was tested by the MTT assay.

RESULTS

Overexpression of antioxidant enzymes provided significant protection against the toxicity of SNAP, SNP and SIN-1, with an individual specificity related to their chemical characteristics, but was without effect upon the toxicity of DETA/NO. Cells overexpressing GPX were well protected against SNP and SNAP, while CAT was most effective against SIN-1. SOD overexpression provided less protection against the toxicity of SNAP and SNP than overexpression of GPX but was more effective in protecting against SIN-1. Co-incubation of cells with NO donors and hydrogen peroxide or hypoxanthine and xanthine oxidase showed an overadditive synergism of toxicity.

CONCLUSIONS

The results emphasise the importance of a synergism between NO and reactive oxygen species for pancreatic beta-cell death. Such a synergism has also been observed after exposure of beta cells to cytokines. The component of the toxicity that is mediated by oxygen radicals can be suppressed effectively through overexpression of CAT, GPX or SOD or both.

Remodeling by its very nature implied synthesis and degradation of extracellular matrix (ECM) proteins. Although oxidative stress, matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) have been implicated in vascular remodeling, the differential role of MMPs versus TIMPs and oxidative stress in vascular remodeling was unclear. TIMP-<em>3</em> induced vascular cell apoptosis, therefore, we hypothesized that during vascular injury TIMP-<em>3</em>, MMP-9 and -12 (elastin-degrading MMP) were increased, whereas MMP-2 (constitutive MMP) and TIMP-4 (cardioprotective TIMP) decreased. Because of the potent anti-oxidant, vasorelaxing, anti-hypertensive agent, hydrogen sulfide (H2S) was used to mitigate the vascular remodeling due to the differential expression of MMP and TIMP. Carotid artery injury was created by inserting a PE-10 catheter and rotating several times before pulling out. The insertion hole was sealed. Mice were grouped: wild type (WT), wild-type damaged artery (WTD), WT+NaHS (sodium hydrogen sulfide, precursor of H2S) treatment (<em>3</em>0 µmol/L in drinking water/6 weeks) and WTD+NaHS treatment. Carotid arteries were analyzed for oxidative stress and remodeling, by measuring super oxide dismutase-1 (<em>SOD</em>1), p47 (NADPH oxidase subunit), nitrotyrosine, MMPs and TIMPs by in situ immunolabeling and by Western blot analyses. The results suggested robust increase in p47, nitrotyrosine, MMP-9, MMP-12, TIMP-<em>3</em> and decrease in <em>SOD</em>1 and MMP-2 levels in the injured arteries. The treatment with H2S ameliorated these effects. We concluded that p47, TIMP-<em>3</em>, MMP-9 and -12 were increased where as <em>SOD</em>-1, MMP-2 and TIMP-4 were decreased in the injured arteries. The treatment with H2S mitigated the vascular remodeling by normalizing the levels of redox stress, MMPs and TIMPs.

The goal of this study was to examine the role of endogenous copper/zinc (CuZn)-superoxide dismutase (<em>SOD</em>) on superoxide levels and on responses of cerebral blood vessels to stimuli that are mediated by nitric oxide (acetylcholine) and cyclooxygenase-dependent mechanisms (bradykinin and arachidonic acid). Levels of superoxide in the rabbit basilar artery were measured using lucigenin-enhanced chemiluminescence (5 microM lucigenin). Diethyldithiocarbamate (DDC; 10 mM), an inhibitor of CuZn-<em>SOD</em>, increased superoxide levels by approximately 2.4-fold (P < 0.05) from a baseline value of 1.0 +/- 0.2 relative light units x min(-1) x mm(-2) (means +/- SE). The diameter of cerebral arterioles (baseline diameter, 99 +/- <em>3</em> microm) was also measured using a closed cranial window in anesthetized rabbits. Topical application of DDC attenuated responses to acetylcholine, bradykinin, and arachidonate, but not nitroprusside. For example, 10 microM arachidonic acid dilated cerebral arterioles by 40 +/- 5 and 2 +/- 2 microm under control conditions and after DDC, respectively (P < 0.05). These inhibitory effects of DDC were reversed by the superoxide scavenger 4,5-dihydroxy-1,<em>3</em>-benzenedisulfonic acid (10 mM). Arachidonate increased superoxide levels in the basilar artery moderately under normal conditions and this increase was greatly augmented in the presence of DDC. These findings suggest that endogenous CuZn-<em>SOD</em> limits superoxide levels under basal conditions and has a marked influence on increases in superoxide in vessels exposed to arachidonic acid. The results also suggest that nitric oxide- and cyclooxygenase-mediated responses in the cerebral microcirculation are dependent on normal activity of CuZn-<em>SOD</em>.

BACKGROUND

Many studies indicate that oxygen free-radical formation after reoxygenation of liver may initiate the cascade of hepatocellular injury. It has been demonstrated that controlled ozone administration may promote an oxidative preconditioning or adaptation to oxidative stress, preventing the damage induced by reactive oxygen species and protecting against liver ischaemia-reperfusion (I/R) injury.

OBJECTIVE

In the present study, the effects of ozone oxidative preconditioning (OzoneOP) on nitric oxide (NO) generation and the cellular redox balance have been studied.

METHODS

Six groups of rats were classified as follows: (1). sham-operated; (2). sham-operated+l-NAME (N(omega)-nitro-l-arginine methyl ester); (<em>3</em>). I/R (ischaemia 90 min-reperfusion 90 min); (4). OzoneOP+I/R; (5). OzoneOP+l-NAME+I/R; and (6). l-NAME+I/R. The following parameters were measured: plasma transaminases (aspartate aminotransferase, alanine aminotransferase) as an index of hepatocellular injury; in homogenates of hepatic tissue: nitrate/nitrite as an index of NO production; superoxide dismutase (<em>SOD</em>), catalase (CAT) and glutathione levels as markers of endogenous antioxidant system; and finally malondialdehyde+4-hydroxyalkenals (MDA+4-HDA) and total hydroperoxides (TH) as indicators of oxidative stress.

RESULTS

A correspondence between liver damage and the increase of NO, CAT, TH, glutathione and MDA+4-HDA concentrations were observed just as a decrease of SOD activity. OzoneOP prevented and attenuated hepatic damage in I/R and OzoneOP+l-NAME+I/R, respectively, in close relation with the above-mentioned parameters.

CONCLUSIONS

These results show that OzoneOP protected against liver I/R injury through mechanisms that promote a regulation of endogenous NO concentrations and maintenance of cellular redox balance. Ozone treatment may have important clinical implications, particularly in view of the increasing hepatic transplantation programs.