The objective of this study was to examine the effect of reactive oxygen species (ROS) and cryopreservation on DNA fragmentation of equine spermatozoa. In experiment 1, equine spermatozoa were incubated (1 hour, <em>3</em>8 degrees C) according to the following treatments: 1) sperm alone; 2) sperm + xanthine (X, 0.<em>3</em> mM)-xanthine oxidase (XO, 0.025 U/mL); <em>3</em>) sperm + X (0.6 mM)-XO (0.05 U/mL); and 4) sperm + X (1 mM)-XO (0.1 U/mL). In experiment 2, spermatozoa were incubated (1 hour, <em>3</em>8 degrees C) with X (1 mM)-XO (0.1 U/mL) and either catalase (200 U/mL), superoxide dismutase (<em>SOD</em>, 200 U/mL), or reduced glutathione (GSH, 10 mM). Following incubation, DNA fragmentation was determined by the single cell gel electrophoresis (comet) assay. In experiment <em>3</em>, equine spermatozoa were cryopreserved, and DNA fragmentation was determined in fresh, processed, and postthaw sperm samples. In experiment 1, incubation of equine spermatozoa in the presence of ROS, generated by the X-XO system, increased DNA fragmentation (P <.005). In Experiment 2, the increase in DNA fragmentation associated with X-XO treatment was counteracted by the addition of catalase and GSH but not by <em>SOD</em>, suggesting that hydrogen peroxide and not superoxide appears to be the ROS responsible for such damage. In experiment <em>3</em>, cryopreservation of equine spermatozoa was associated with an increase (P <.01) in DNA fragmentation when compared with fresh or processed samples. This study indicates that ROS and cryopreservation promote DNA fragmentation in equine spermatozoa; the involvement of ROS in cryopreservation-induced DNA damage remains to be determined.

Atherosclerosis is a chronic inflammatory process with increased oxidative stress in vascular endothelium. Ginkgo biloba extract (GbE), extracted from Ginkgo biloba leaves, has commonly been used as a therapeutic agent for cardiovascular and neurological disorders. The aim of this study was to investigate how GbE protects vascular endothelial cells against the proatherosclerotic stressor oxidized low-density lipoprotein (oxLDL) in vitro. Human umbilical vein endothelial cells (HUVECs) were incubated with GbE (12.5-100 microg/ml) for 2 h and then incubated with oxLDL (150 microg/ml) for an additional 24 h. Subsequently, reactive oxygen species (ROS) generation, antioxidant enzyme activities, adhesion to monocytes, cell morphology, viability, and several apoptotic indexes were assessed. Our data show that ROS generation is an upstream signal in oxLDL-treated HUVECs. Cu,Zn-<em>SOD</em>, but not Mn-<em>SOD</em>, was inactivated by oxLDL. In addition, oxLDL diminished expression of endothelial NO synthase and enhanced expression of adhesion molecules (ICAM, VCAM, and E-selectin) and the adherence of monocytic THP-1 cells to HUVECs. Furthermore, oxLDL increased intracellular calcium, disturbed the balance of Bcl-2 family proteins, destabilized mitochondrial membrane potential, and triggered subsequent cytochrome c release into the cytosol and activation of caspase-<em>3</em>. These detrimental effects were ameliorated dose dependently by GbE (P < 0.05). Results from this study may provide insight into a possible molecular mechanism underlying GbE suppression of the oxLDL-mediated vascular endothelial dysfunction.

Interleukin (IL)-10, an anti-inflammatory cytokine, preserves endothelial function during acute inflammation. We tested the hypotheses that IL-10 plays a protective role in blood vessels during diabetes by suppressing impairment of endothelium-dependent relaxation and that protection by IL-10 is mediated by effects on superoxide (O(2-)). Streptozotocin (150 mg/kg i.p.) or citrate buffer was injected into IL-10-deficient (IL-10(-/-)) mice and wild-type controls (IL-10(+/+)). In IL-10(+/+) and IL-10(-/-) mice, blood glucose levels were approximately 120 mg/dl after citrate administration and approximately 400 mg/dl after streptozotocin administration. Vasorelaxation was examined in arteries in vitro 12-16 weeks later. Maximum relaxation to acetylcholine (<em>3</em>0 micromol/l) was 88 +/- <em>3</em>% (means +/- SE) in nondiabetic mice and 84 +/- <em>3</em>% in diabetic IL-10(+ /+) mice (P>> 0.05). Thus, at this time point, diabetes did not impair endothelium-dependent relaxation in vessels in wild-type mice. In contrast, maximum relaxation in vessels from diabetic IL-10(-/-) mice was significantly decreased (74 +/- 5%) compared with nondiabetic IL-10(-/-) mice (9<em>3</em> +/- 2%, P < 0.05). Superoxide dismutase with polyethylene glycol (PEG-<em>SOD</em>) restored impaired responses to acetylcholine to levels seen in controls. Responses to acetylcholine also were improved by allopurinol (an inhibitor of xanthine oxidase) in vessels from diabetic IL-10(- /-) mice. Thus, diabetes produces greater impairment of relaxation to acetylcholine in IL-10(-/-) mice than in IL-10(+/ +) mice. These findings provide direct evidence that IL-10 impedes mechanisms of endothelial dysfunction during diabetes. Restoration of vasorelaxation with PEG-<em>SOD</em> or allopurinol suggests that the mechanism(s) by which IL-10 preserves endothelium-dependent vasorelaxation involves O(2-), perhaps by reducing production of O(2-) by xanthine oxidase.

OBJECTIVE

To establish the antioxidant status of the aqueous humour in glaucoma associated with exfoliation syndrome (XFG) and to compare it to primary open-angle glaucoma (POAG) and cataract patients.

METHODS

Patients were diagnosed with POAG, XFG, or cataract (n=25 for each group). Total reactive antioxidant potential (TRAP) was measured by chemiluminescence. Ascorbic acid levels and the activities of catalase, glutathione peroxidase (GPx), and superoxide dismutase (<em>SOD</em>) were measured spectrophotometrically.ResultsTRAP value was lower in XFG (28+/-2 microM Trolox) than in POAG (55+/-8 microM Trolox; P<0.001). TRAP values in both glaucomas were lower than the cataract value (124+/-5 microM Trolox; P<0.001). A decrease in ascorbic acid was measured in XFG (2<em>3</em>0+/-20 microM) compared with POAG (415+/-17 microM; P<0.001). Ascorbic acid in both glaucomas was lower than in cataract (720+/-<em>3</em>0 microM; P<0.001). A significant increase in GPx was found in XFG (<em>3</em>0+/-2 U/ml) compared with POAG (16+/-<em>3</em> U/ml). GPx activity in both glaucomas was increased when compared with cataracts (6+/-2 U/ml; P<0.001). A significant increase of 67% in <em>SOD</em> activity was observed in the glaucoma group vscataract group (27+/-<em>3</em> U/ml; P<0.001), but no changes were found between both glaucomas.

CONCLUSIONS

The antioxidant status of the aqueous humour may play a role in the pathophysiology of both glaucomas.

The hyperglycemia triggers several chronic diabetic complications mediated by increased oxidative stress that eventually causes diabetic nephropathy. The aim of this study was to examine if the sodium-glucose cotransporter (SGLT2) inhibition prevents the oxidative stress in the kidney of diabetic rats.

METHODS

The diabetic rat model was established by intraperitoneal injection of streptozotocin (50 mg/kg). The inhibition of SGLT2 was induced by daily subcutaneous administration of phlorizin (0.4 g/kg). Oxidative stress was assessed by catalase (CAT), glutathione peroxidase (GPx), and superoxide dismutase (<em>SOD</em>) activities and by immunohistochemical analysis of <em>3</em>-nitrotyrosine (<em>3</em>-NT).

RESULTS

Streptozotocin-induced diabetes caused hyperglycemia and lower body weight. The CAT activity decreased in cortex and medulla from diabetic rats; in contrast, the GPx activity increased. Furthermore the <em>3</em>-NT staining of kidney from diabetic rats increased compared to control rats. The inhibition of SGLT2 decreased hyperglycemia. However, significant diuresis and glucosuria remain in diabetic rats. The phlorizin treatment restores the CAT and GPX activities and decreases <em>3</em>-NT staining.

CONCLUSIONS

The inhibition of SGLT2 by phlorizin prevents the hyperglycemia and oxidative stress in kidney of diabetic rats, suggesting a prooxidative mechanism related to SGLT2 activity.

Diclofenac (DCLF) is a nonsteroidal anti-inflammatory drug that is widely used for the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, and acute muscle pain conditions. Toxic doses of DCLF can cause nephrotoxicity in humans and experimental animals. However, whether this DCLF-induced nephrotoxicity involves apoptotic cell death in addition to necrosis is unknown. The goals of this investigation were to determine whether DCLF-induced nephrotoxicity involves oxidative stress and apoptotic type genomic DNA fragmentation, and if so, whether DCLF-induced oxidative stress and DNA fragmentation cause apoptotic cell death in mouse kidneys. Male ICR mice (CD-1; 25-45 g), fed ad libitum, were administered nephrotoxic doses of DCLF (100, 200, <em>3</em>00 mg/Kg, po) and sacrificed 24 h later. Blood was collected to evaluate renal injury (BUN), lipid peroxidation (MDA: malondialdehyde levels), and superoxide dismutase (<em>SOD</em>) activity (a marker of oxidative stress). Kidney tissues were analyzed both quantitatively and qualitatively to determine the degree and type of DNA damage, and evaluated histopathologically for the presence of apoptotic characteristics in the nucleus of diverse types of kidney cells. Results show that diclofenac is a powerful nephrotoxicant (at 100, 200, and <em>3</em>00 mg/kg: 4.7-, 4.9-, and 5.0-fold increases in BUN compared to the control, respectively) and a strong inducer of oxidative stress (significant increase in MDA levels). Oxidative stress induced by DCLF was also coupled with massive kidney DNA fragmentation (100, 200, and <em>3</em>00 mg/kg: <em>3</em>-, 8-, and 10-fold increases compared to control, respectively). A dose-dependent increase in MDA levels and <em>SOD</em> activity was also observed, which indicated a link between oxidative stress and nephrotoxicity. Qualitative analysis of DNA fragmentation by gel electrophoresis showed a DNA ladder indicative of Ca2+-Mg2+-endonuclease activation. Histopathological examination of kidney sections revealed numerous apoptotic nuclei across proximal and distal tubular cell linings. Collectively, these data for the first time suggest that DCLF-induced nephrotoxicity may involve production of reactive oxygen species leading to oxidative stress and massive genomic DNA fragmentation, and these two free radical mediated events may ultimately translate into apoptotic cell death of kidney cells in vivo, and reveal a DNA-active role for DCLF.

The present study has been carried out to investigate the role of taurine (2-aminoethanesulfonic acid), a conditionally essential amino acid, in ameliorating cadmium-induced renal dysfunctions in mice. Cadmium chloride (CdCl(2)) has been selected as the source of cadmium. Intraperitoneal administration of CdCl(2 )(at a dose of 4 mg/kg body weight for <em>3</em> days) caused significant accumulation of cadmium in renal tissues and lessened kidney weight to body weight ratio. Cadmium administration reduced intracellular ferric reducing/antioxidant power (FRAP) of renal tissues. Levels of serum marker enzymes related to renal damage, creatinine and urea nitrogen (UN) have been elevated due to cadmium toxicity. Cadmium exposure diminished the activities of enzymatic antioxidants, superoxide dismutase (<em>SOD</em>), catalase (CAT), glutathione-S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx) and glucose-6-phosphate dehydrogenase (G6PD) as well as non-enzymatic antioxidant, reduced glutathione (GSH) and total thiols. On the other hand, the levels of oxidized glutathione (GSSG), lipid peroxidation, protein carbonylation, DNA fragmentation, concentration of superoxide radicals and activities of cytochrome P450 enzymes (CYP P450s) have been found to increase due to cadmium intoxication. Treatment with taurine (at a dose of 100 mg/kg body weight for 5 days) before cadmium intoxication prevented the toxin-induced oxidative impairments in renal tissues. The beneficial role of taurine against cadmium-induced renal damage was supported from histological examination of renal segments. Vitamin C, a well-established antioxidant was used as the positive control in the study. Experimental evidence suggests that both taurine and vitamin C provide antioxidant defense against cadmium-induced renal oxidative injury. Combining all, results suggest that taurine protects murine kidneys against cadmium-induced oxidative impairments, probably via its antioxidative property.

To investigate the time-dependent effects of acrylamide (ACR) on the antioxidative status in rat nerve tissues, adult male Wistar rats were given ACR (40 mg/kg, i.p., <em>3</em> times/week) for 2, 4, 6 and 10 weeks, respectively. The time-dependent changes of the lipid peroxidation (malondialdehyde, MDA) and antioxidative status (glutathione, GSH; glutathione peroxidase, GSH-Px; glutathione reductase, GR; superoxide dismutase, <em>SOD</em> and anti-reactive oxygen species, anti-ROS) in nerve tissues were investigated. The electrophysiology indices (nerve conduction velocity, NCV; compound action potential duration, CAPD; compound action potential amplitude, CAPA; compound action potential latency, CAPL) in the sciatic nerve were determined using BL-420E Biologic Function Determining System. The results showed that MDA levels increased significantly (P < 0.05) in nerve tissues, while GSH levels markedly decreased (P < 0.05) in a time-dependent manner. <em>SOD</em> activity (in the spinal cord and sciatic nerve) and GR activity (in the sciatic nerve) increased significantly after 4 weeks ACR treatment (P < 0.01), but then decreased (P < 0.05). The anti-ROS activity in the sciatic nerve was markedly decreased at the end of week 6 and 10 (P < 0.01). The above indices changed most in the sciatic nerve. The levels of GSH, MDA and anti-ROS in rat sciatic nerve were in high correlation (P < 0.05, |r|>> 0.80) with the electrophysiology indices according to the exposure time. Thus, ACR-induced neurotoxicity may be associated with the enhancement of lipid peroxidation and reduction of the antioxidative capacity. Depletion of neural GSH level might be one of the primary events in ACR-induced neuropathy.

We propose a novel combination of high-throughput luminescent bacterial tests for the evaluation of the reactive oxygen species (ROS)-generating potential of engineered nanoparticles (eNPs) and the role of solubilised metal ions in this process. The set of tests consists of differently engineered recombinant Escherichia coli strains: (1) a new sensor strain, which bioluminescence is induced by superoxide anions; (2) six recombinant E. coli strains (superoxide dismutase (<em>sod</em>) single, double and triple mutants and a respective wild-type strain), transformed with luxCDABE genes responding to toxic compounds by decreasing their luminescence; and (<em>3</em>) three strains in which bioluminescence is specifically induced by bioavailable metals (Cu, Zn and Ag). The applicability of this battery of tests in profiling oxidative potential of eNPs was evaluated on nTiO(2), nCuO, nZnO and nAg (25, <em>3</em>0, 70 and <100 nm, respectively) NPs and fullerenes. As controls for the size or solubility, the bulk formulations (bTiO(2), bCuO and bZnO) and soluble salts (ZnSO(4), CuSO(4) and AgNO(<em>3</em>)) were also analysed. Bacterial toxicity tests showed that nCuO was four-fold more toxic, and nAg was 15-fold more toxic to triple <em>sod</em> mutant than to wild type (2-h EC(50) values were 8.1 and 2.0 mg Cu l(-1), respectively, and 46 and <em>3</em>.1 mg Ag l(-1), respectively). Formation of ROS by nCuO and nAg was proved by superoxide anion-inducible strain. The metal sensor bacteria showed that the ROS formation by CuO NPs was caused by solubilised Cu ions, but in case of nAg, particles also had an effect. nZnO was remarkably more toxic to <em>sod</em> triple mutant than to wild type strain (2-h EC(50) were 4.5 and 54 mg Zn l(-1), respectively). Fullerenes inhibited the bioluminescence of <em>sod</em> triple mutant at <em>3</em>,882 mg l(-1) but had no effect on the wild-type strain even at 20,800 mg l(-1). Nano and bTiO(2) showed some effect on viability of bacteria only at high concentrations (>4,000 mg l(-1)) although nTiO(2) (but not bTiO(2)) induced the bioluminescence of the superoxide anion sensing bacteria starting from 100 mg l(-1). Thus, our innovative combined approach is expected to provide more consistent and informative data concerning the general toxicity, ROS-production potential and also solubilisation of metals in the case of metallic NPs.

1. The aim of the present study was to investigate the role of oxidative stress in renal injury and to determine whether erythropoietin (EPO) acts as an anti-oxidant in vancomycin (VCM)-induced renal impairment. 2. Twenty-four rats were divided into three groups as follows: (i) control (Group 1); (ii) VCM treated (Group 2); and (iii) VCM + EPO treated (Group <em>3</em>). Vancomycin (200 mg/kg, i.p.) was administered to Groups 2 and <em>3</em> for 7 days. Erythropoietin (150 IU/kg, i.p.) treatment was started 24 h before VCM and lasted for 7 days. On Day 8, renal tissues were excised and blood samples were collected. Serum creatinine and blood urea nitrogen were measured, along with renal malondialdehyde (MDA) levels, superoxide dismutase (<em>SOD</em>) and catalase (CAT) activity and tissue VCM levels. The kidneys were examined for any histopathological changes. <em>3</em>. Renal MDA levels were found to be increased, whereas <em>SOD</em> and CAT activity was decreased, in the VCM-treated group compared with the control group. There was a marked decrease in MDA levels and an increase in <em>SOD</em> activity, but not CAT activity, after VCM + EPO treatment. Marked histopathological alterations, including interstitial oedema, tubular dilatation, tubular epithelial cell desquamation and vacuolization, were observed in VCM-treated rats. Histopathological changes were significantly improved after EPO administration. 4. In conclusion, the present data suggest that oxidative stress plays an important role in VCM-induced nephrotoxicity. Erythropoietin seems to act as an anti-oxidant, diminishing the toxic oxidative effects of VCM on renal tissues.

Superoxide dismutase (<em>SOD</em>) removes excess superoxide radicals via dismutation to oxygen and hydrogen peroxide. In this work, we have characterized TcFe<em>SOD</em>-A gene from 25 Trypanosoma cruzi populations and clones susceptible, naturally resistant or with in vitro-induced (17 LER) or in vivo-selected resistance to benznidazole (BZR). In the 17 LER T. cruzi population, the levels of TcFe<em>SOD</em>-A mRNA were at least <em>3</em>-fold higher than its drug-susceptible counterpart 17 WTS. The levels of TcFe<em>SOD</em>-A mRNA were similar among the other T. cruzi populations and clones regardless of the drug-resistance phenotype. We determined whether the increase in mRNA levels was due to gene amplification using Southern blot analysis of the T. cruzi populations and clones. We found that the number of TcFe<em>SOD</em>-A gene copies was similar for all samples tested, except for 17 LER that presented twice as many copies. The chromosomal location of the TcFe<em>SOD</em>-A gene and polymorphisms detected in nucleotide and amino acid sequences of TcFe<em>SOD</em>-A were associated with the zymodeme of the T. cruzi strain but not with drug-resistance phenotype. We observed a 2<em>3</em> kDa TcFe<em>SOD</em>-A polypeptide in all analysed T. cruzi strains. The level of this polypeptide was increased only in the 17 LER population. Specific enzyme activity analysis of TcFe<em>SOD</em> in the T. cruzi samples revealed a correlation between expression and activity. Our findings show an increased expression of the TcFe<em>SOD</em>-A enzyme in the T. cruzi population with in vitro-induced resistance to benznidazole.

OBJECTIVE

Luteolin is a falconoid compound that has an antioxidant effect, but its contribution to ROS-activated MAPK pathways in ischemia/reperfusion injury is seldom reported. Here, we have confirmed that it exhibits an antioxidant effect in myocardial ischemia/reperfusion injury (MIRI) by inhibiting ROS-activated MAPK pathways.

METHODS

We exposed rat hearts into the left anterior descending coronary artery (LAD) ligation for <em>3</em>0min followed by 1h of reperfusion. Observations were carried out using electrocardiography; detection of hemodynamic parameters; and testing levels of lactate dehydrogenase (LDH), creatine kinase (CK), total superoxide dismutase (T-<em>SOD</em>), and malondialdehyde (MDA). Mitogen-activated protein kinase (MAPK) pathway was measured by western blot and transmission electron microscopy was applied to observe the myocardial ultrastructure. Rat H9c2 cell in 95% N2 and 5% CO2 stimulated the MIRI. Oxidation system mRNA levels were measured by real-time PCR; mitochondrial membrane potential and apoptosis were measured by confocal microscopy and flow cytometry; western blot analysis was used to assay caspase-<em>3</em>, -8, and -9 and MAPK pathway protein expression; the MAPK pathway was inhibited using SB20<em>3</em>580 (p<em>3</em>8 MAPK inhibitor) and SP600125 (c-Jun NH2-terminal kinase inhibitor) before H9c2 cells were exposed to hypoxia/reoxygenation injury to show the modulation of the changes in ROS generation, cell viability and apoptosis.

RESULTS

In vivo, luteolin can ameliorate the impaired mitochondrial morphology, regulating the MAPK pathway to protect MIRI. In vitro, luteolin can affect the oxidation system, mitochondrial membrane potential and MAPK pathway to anti-apoptosis.

CONCLUSIONS

These results reveal a ROS-MAPK mediated mechanism and mitochondrial pathway through which luteolin can protect myocardial ischemia/reperfusion injury.

The goal of the present study was to investigate the antifibrotic role of inducible nitric oxide synthase (iNOS) in Peyronie's disease (PD) by determining whether a plasmid expressing iNOS (piNOS) injected into a PD-like plaque can induce regression of the plaque. A PD-like plaque was induced with fibrin in the penile tunica albuginea of mice and then injected with a luciferase-expressing plasmid (pLuc), either alone or with piNOS, following luciferase expression in vivo by bioluminescence imaging. Rats were treated with either piNOS, an empty control plasmid (pC), or saline. Other groups were treated with pC or piNOS, in the absence of fibrin. Tissue sections were stained for collagen, transforming growth factor (TGF) beta1, and plasminogen-activator inhibitor (PAI-1) as profibrotic factors; copper-zinc superoxide dismutase (CuZn <em>SOD</em>) as scavenger of reactive oxygen species (ROS); and nitrotyrosine to detect nitric oxide reaction with ROS. Quantitative image analysis was applied. Both iNOS and xanthine oxido-reductase (XOR; oxidative stress) were estimated by Western blot analysis. Luciferase reporter expression was restricted to the penis, peaked at <em>3</em> days after injection, but continued for at least <em>3</em> wk. In rats receiving piNOS, iNOS expression also peaked at <em>3</em> days, but expression decreased at the end of treatment, when a considerable reduction of plaque size occurred. Protein nitrotyrosine, XOR, and CuZn <em>SOD</em> increased, and TGFbeta1 and PAI-1 decreased. The piNOS gene transfer regressed the PD plaque and expression of profibrotic factors, supporting the view that endogenous iNOS induction in PD is defense mechanism by the tissue against fibrosis.

Patients with uncontrolled essential hypertension have elevated concentrations of superoxide anion (O(2)(-*)), hydrogen peroxide (H(2)O(2)), lipid peroxides, endothelin, and transforming growth factor-beta (TGF-beta) with a simultaneous decrease in endothelial nitric oxide (eNO), superoxide dismutase (<em>SOD</em>), vitamin E, and long-chain polyunsaturated fatty acids (LCPUFAs). Physiological concentrations of angiotensin II activate NAD(P)H oxidase and trigger free radical generation (especially that of O(2)(-*)). Normally, angiotensin II-induced oxidative stress is abrogated by adequate production and release of eNO, which quenches O(2)(-*) to restore normotension. Angiotensin II also stimulates the production of endothelin and TGF-beta. TGF-beta enhances NO generation, which in turn suppresses TGF-beta production. Thus, NO has a regulatory role on TGF-beta production and is also a physiological antagonist of endothelin. Antihypertensive drugs suppress the production of O(2)(-*) and TGF-beta and enhance eNO synthesis to bring about their beneficial actions. LCPUFAs suppress angiotensin-converting enzyme (ACE) activity, reduce angiotensin II formation, enhance eNO generation, and suppress TGF-beta expression. Perinatal supplementation of LCPUFAs decreases insulin resistance and prevents the development of hypertension in adult life, whereas deficiency of LCPUFAs in the perinatal period results in raised blood pressure later in life. Patients with essential hypertension have low concentrations of various LCPUFAs in their plasma phospholipid fraction. Based on this, it is proposed that LCPUFAs serve as endogenous regulators of ACE activity, O(2)(-*), eNO generation, and TGF-beta expression. Further, LCPUFAs have actions similar to statins, inhibit (especially omega-<em>3</em> fatty acids) cyclooxygenase activity and suppress the synthesis of proinflammatory cytokines, and activate the parasympathetic nervous system, all actions that reduce the risk of major vascular events. Hence, it is proposed that availability of adequate amounts of LCPUFAs during the critical periods of growth prevents the development of hypertension in adulthood.

FGF21 is recently discovered with pleiotropic effects on glucose and lipid metabolism. However, the potential protective effect of FGF21 against D-gal-induced injury in the liver has not been demonstrated. The aim of this study is to investigate the pathophysiological role of FGF21 on hepatic oxidative injury and apoptosis in mice induced by D-gal. The <em>3</em>-month-old Kunming mice were subcutaneously injected with D-gal (180 mg kg(-1) d(-1)) for 8 weeks and administered simultaneously with FGF21 (5 or 1 mg kg(-1) d(-1)). Our results showed that the administration of FGF21 significantly alleviated histological lesion including structure damage, degeneration, and necrosis of hepatocytes induced by D-gal, and attenuated the elevation of liver injury markers, serum AST, and ALP in a dose-dependent manner. FGF21 treatment also suppressed D-gal-induced profound elevation of ROS production and oxidative stress, as evidenced by an increase of the MDA level and depletion of the intracellular GSH level in the liver, and restored the activities of antioxidant enzymes <em>SOD</em>, CAT, GSH-Px, and T-AOC. Moreover, FGF21 treatment increased the nuclear abundance of Nrf2 and subsequent up regulation of several antioxidant genes. Furthermore, a TUNEL assay showed that D-gal-induced apoptosis in the mouse liver was significantly inhibited by FGF21. The expression of caspase-<em>3</em> was markedly inhibited by the treatment of FGF21 in the liver of D-gal-treated mice. The levels of PI<em>3</em>K and PBK/Akt were also largely enhanced, which in turn inactivated pro-apoptotic signaling events, restoring the balance between pro- and anti-apoptotic Bcl-2 and Bax proteins in the liver of D-gal-treated mice. In conclusion, these results suggest that FGF21 protects the mouse liver against D-gal-induced hepatocyte oxidative stress via enhancing Nrf2-mediated antioxidant capacity and apoptosis via activating PI<em>3</em>K/Akt pathway.

BACKGROUND

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive multifactorial disease with limited therapeutic options. Glycosides based standardized fenugreek seed extract (SFSE-G) possesses potent anti-inflammatory and anti-oxidant property.

OBJECTIVE

To evaluate the efficacy of SFSE-G against bleomycin (BLM) induced pulmonary fibrosis by assessing behavioral, biochemical, molecular and ultrastructural changes in the laboratory rats.

METHODS

IPF was induced in male Sprague-Dawley rats by single intratracheal BLM (6IU/kg) injection followed by SFSE-G (5, 10, 20 and 40mg/kg, p.o.) or methylprednisolone (10mg/kg, p.o.) treatment for 28day. Various parameters were analyzed in lung and bronchoalveolar lavage fluid (BALF) after 14 and 28days of the drug treatment.

RESULTS

SFSE-G (20 and 40mg/kg, p.o.) administration significantly prevented the BLM induced alteration in body weight, lung index, lung function test and hematology. The altered total and differential cell count in BALF and blood was significantly prevented by SFSE-G treatment. The decreased peripheral blood oxygen content after BLM instillation was significantly increased by SFSE-G treatment. SFSE-G significantly enhanced the BALF and lung antioxidant status, through modulating the <em>SOD</em>, GSH, T-AOC, MDA, NO level and Nrf2, HO-1 mRNA expression. There was a significant reduction in lung 5-HT level by SFSE-G treatment. The altered mRNA expression of biomarkers of lung inflammation (TNF-α, IL-1β, IL-6 and IL-8), fibrosis (TGF-β, collagen-1, ET-1, Muc5ac, NF-κB, VEGF, Smad-<em>3</em>) and apoptosis (Bax, Bcl-2 and Caspase-<em>3</em>) were significantly prevented by SFSE-G treatment. BLM induced histological inflammatory and fibrotic insult in the lung were reduced by SFSE-G treatment. It also ameliorated BLM induced lung ultrastructural changes as observed by transmission electron microscopic studies. However, administration of SFSE-G (5mg/kg, p.o.) failed to show any protective effect against BLM-induced PF whereas SFSE-G (10mg/kg, p.o.) showed significant amelioration in BLM-induced PF except lung function test, BALF and lung antioxidant level.

CONCLUSIONS

SFSE-G showed anti-fibrotic efficacy executed through induction of Nrf2, which in turn may modulate anti-inflammatory molecules, inhibit fibrogenic molecules and decreased apoptosis to ameliorate BLM induced pulmonary fibrosis.

Guanosine, a guanine-based purine, is an extracellular signaling molecule that is released from astrocytes and shows neuroprotective effects in several in vivo and in vitro studies. Our group recently showed that guanosine presents antioxidant properties in C6 astroglial cells. The heme oxygenase 1 signaling pathway is associated with protection against oxidative stress. Azide, an inhibitor of the respiratory chain, is frequently used in experimental models to induce oxidative and nitrosative stress. Thus, the goal of this study was to investigate the effect of guanosine on azide-induced oxidative damage in C6 astroglial cells. Azide treatment of these cells resulted in several detrimental effects, including induction of cytotoxicity and mitochondrial dysfunction, increased levels of reactive oxygen/nitrogen species, inducible nitric oxide synthase expression and NADPH oxidase, decreased glutamate uptake and EAAC1 glutamate transporter expression, decreased glutathione (GSH) levels, and decreased activities of glutamine synthetase (GS), superoxide dismutase and catalase (CAT). The treatment also increased nuclear factor-κB activation and the release of proinflammatory cytokines tumor necrosis factor α and IL-1β. Guanosine strongly prevented these effects, protecting glial cells against azide-induced cytotoxicity and modulating glial, oxidative and inflammatory responses through the activation of the heme oxygenase 1 pathway. These observations reinforce and support the role of guanosine as an antioxidant molecule against oxidative damage. Guanosine protects against azide-induced oxidative damage in C6 astroglial cells. Azide-induced mitochondrial dysfunction (1); increased reactive oxygen species/reactive nitrogen species levels (2); decreased glutamate uptake (<em>3</em>), GS activity (4), GSH levels (5), and <em>SOD</em> (6) and CAT (7) activities; increased glutathione peroxidase (GPx) (8) and NADPH oxidase (9) activities and cellular superoxide levels (10); increased NF-κB activation (11), TNF-α and IL-1β levels (12); and induced iNOS expression (1<em>3</em>). Guanosine prevented these effects through the HO1 signaling pathway, thus our findings support the antioxidant effects of guanosine.

OBJECTIVE

To examine the safety and pharmacokinetics of multiple intratracheal (IT) doses of recombinant human CuZn superoxide dismutase (rhSOD) in premature infants with respiratory distress syndrome who are at risk for developing bronchopulmonary dysplasia (BPD). Methods. Thirty-three infants (700 to 1300 g) were randomized and blindly received saline, 2.5 mg/kg or 5 mg/kg rhSOD IT within 2 hours of surfactant administration. Infants were treated every 48 hours (as long as endotracheal intubation was required) up to 7 doses. Serial blood and urine studies, chest radiographs, neurosonograms, SOD concentration and activity measurements, and tracheal aspirate (TA) inflammatory markers were assessed throughout the 28-day study.

RESULTS

SOD concentrations in serum (0.1 [0.05/0.15] microg/mL-geometric mean with lower/upper confidence intervals), tracheal aspirates (TA) (0.2 [0.1/0.3] microg/mL) and urine (0.3 [0.2/0.4] microg/mL) were similar at baseline in all 3 groups and did not change significantly in the placebo group. In the rhSOD treatment groups, SOD concentrations were increased on day 3 and did not change significantly thereafter over the 14-day dosing period (also measured on days 5, 7, and 13). SOD concentrations averaged 0.4 [0.3/0.5] microg/mL in serum, 0.8 [0.6/1.2] microg/mL in TA and 1.1 [1.0/1.3] microg/mL in urine for the low-dose group and 0.6 [0.5/0.7] microg/mL in serum, 1.1 [0.9/1.5] microg/mL in TA, and 2.2 [1.6/2.9] microg/mL in urine for the high-dose group over the 14-day dosing period. Enzyme activity directly correlated with SOD concentration and rhSOD was active even when excreted in urine. TA markers of acute lung injury (neutrophil chemotactic activity, albumin concentration) were lower in the rhSOD agroups compared with placebo. No significant differences in any clinical outcome variable were noted between groups.

CONCLUSIONS

These data indicate that multiple IT doses of rhSOD increase the concentration and activity of the enzyme in serum, TA and urine, reduce TA lung injury markers and are well-tolerated. Further clinical trials examining the efficacy of rhSOD in the prevention of BPD are warranted.

The mycotoxin aflatoxin is a secondary metabolite and potent human carcinogen. We investigated one mechanism that links stress response with coordinate activation of genes involved in aflatoxin biosynthesis in Aspergillus parasiticus. Electrophoretic mobility shift assays demonstrated that AtfB, a basic leucine zipper (bZIP) transcription factor, is a master co-regulator that binds promoters of early (fas-1), middle (ver-1), and late (omtA) aflatoxin biosynthetic genes as well as stress-response genes (mycelia-specific cat1 and mitochondria-specific Mn <em>sod</em>) at cAMP response element motifs. A novel conserved motif 5'-T/GNT/CAAG CCNNG/AA/GC/ANT/C-<em>3</em>' was identified in promoters of the aflatoxin biosynthetic and stress-response genes. A search for transcription factors identified SrrA as a transcription factor that could bind to the motif. Moreover, we also identified a STRE motif (5'-CCCCT-<em>3</em>') in promoters of aflatoxin biosynthetic and stress-response genes, and competition EMSA suggested that MsnA binds to this motif. Our study for the first time provides strong evidence to suggest that at least four transcription factors (AtfB, SrrA, AP-1, and MsnA) participate in a regulatory network that induces aflatoxin biosynthesis as part of the cellular response to oxidative stress in A. parasiticus.

Several single-nucleotide polymorphisms of genes related to oxidative stress have been evaluated because intracellular reactive oxygen species are associated with development of diabetes and its microvascular complications. We performed a case-control study to investigate whether V16A polymorphism of manganese superoxide dismutase (Mn-<em>SOD</em>) gene is related to pathogenesis of diabetes and whether the polymorphism is associated with stages of albuminuria in Korean type 2 diabetic patients. Genotype distributions were studied in 178 nondiabetic subjects and <em>3</em>71 type 2 diabetic patients of <em>3</em> groups with a normoalbuminuria group (Normo group, n = 244), a microalbuminuria group (Micro group, n = 86), and an overt albuminuria group (Macro group, n = 41). The albumin/creatinine ratio (ACR) was defined as a urinary albumin/creatinine ratio. V16A genotypes were determined with polymerase chain reaction-restriction fragment length polymorphism method. Between nondiabetic subjects and type 2 diabetic patients, Mn-<em>SOD</em> genotype distribution (VV/VA + AA, 146/<em>3</em>2 vs <em>3</em>14/57) and A allele frequency (0.121 vs 0.104) were not different. Patients with nephropathy, Micro and Macro groups, had significantly lower A allele frequency, longer diabetic duration, higher prevalence of hypertension, and greater ACR than those of patients without nephropathy (P < .05). A allele was significantly less frequent with progression of nephropathy (Normo group, 0.119; Micro group, 0.07<em>3</em>; Macro group, 0.0<em>3</em>; P < .05). In type 2 diabetic patients, A allele carriers had significantly lower prevalence of hypertension and lesser ACR than those of A allele noncarriers (P < .01). In multivariate analysis, hypertension, duration of diabetes, serum total cholesterol level, and A allele of Mn-<em>SOD</em> gene were independently associated with stages of albuminuria. These results suggest that V16A polymorphism of Mn-<em>SOD</em> gene is not related to pathogenesis of diabetes but is associated with stages of albuminuria in Korean type 2 diabetes.

The purpose of the present study was to investigate the role of fresh garlic homogenate (FGH) and its bioactive sulphur compound S-allyl cysteine sulphoxide (SACS) in potentiating antihypertensive and cardioprotective activities of captopril in rats. SACS was extracted from the fresh garlic using ion exchange resins with yield of 890 mg/kg garlic. The dose of SACS was calculated based on the amount of SACS extracted from 125 to 250 mg of FGH. Albino rats weighing 150-200 g were fed with 10% fructose in fluid for <em>3</em> weeks for induction of hypertension and subsequently administered FGH (125 and 250 mg/kg, p.o.) or SACS (0.111 and 0.222 mg/kg/day, p.o.) for the next <em>3</em> weeks in their respective groups. In CAP alone and interactive groups (GH+CAP; SACS+CAP), captopril <em>3</em>0 mg/kg was given during sixth week of 10% fructose in fluid. At the end of drug treatment, animals were given isoproterenol 175 mg/kg subcutaneously for two consecutive days. Additionally, varying concentrations of SACS (4, 8, 16, <em>3</em>2 and 64 ng), CAP (1, 2, 4, 8 and 16 ng) and their combination (4:1) were checked for fall in blood pressure in hypertensive rats (10% fructose in fluid without pretreatment) as well as angiotensin-converting enzyme (ACE) inhibiting activity using guinea pig ileum. An isobolographic analysis was used to characterise the interaction between SACS and CAP for fall in blood pressure and ACE inhibiting evaluations. Administration of captopril, low and high doses of FGH (125, 250 mg/kg), either alone or together showed fall in fluid intake and body weight. The combined therapy of FGH 250 mg/kg and CAP was more effective in reducing systolic blood pressure, cholesterol, triglycerides and glucose. The <em>SOD</em> and catalase activities in heart tissue were significantly elevated in groups treated with FGH, SACS, CAP, FGH+CAP and SACS+CAP. Further, combined therapy of FGH 250 mg/kg and CAP caused significant fall in LDH and CK-MB activities in serum and elevation in heart tissue homogenate. SACS in low dose was less effective than low dose of FGH; similarly, high dose of FGH was more efficacious than high dose of SACS. Corroborating with this, combined therapy of garlic (250 mg/kg) with CAP demonstrated higher synergistic action than combination of SACS (0.222 mg/kg) with CAP suggesting the role of additional bioactive constituents apart from SACS, responsible for therapeutic efficacy of garlic. Moreover, combination of SACS and CAP exerted super-additive (synergistic) interaction with respect to fall in blood pressure and ACE inhibition. This study may represent an advertence on concomitant use of garlic or its bioactive constituent, SACS, with captopril.

We characterized the changes in nitric oxide (NO) levels in the brain during global forebrain ischemia and reperfusion and tested the ability of the natural flavonoid, quercetin, and a synthetic flavonoid, FB277, to increase the amount of available NO by elimination of the superoxide radicals produced during reperfusion. In Sprague-Dawley rats, we used a four-vessel occlusion model of forebrain ischemia (15 min) and reperfusion (<em>3</em>0 min). Brain NO was measured on samples of cerebral cortex and cerebellum ex vivo by electron paramagnetic resonance (EPR) spectroscopy. The spin trap used was diethyldithiocarbamate sodium salt (DETC) associated with ferrous citrate. The complex Fe(DETC)2NO was detected at 77 K as a triplet signal at g = 2.0<em>3</em>5. Groups of animals were treated with quercetin or FB277 (<em>3</em>-morpholinomethyl-<em>3</em>',4',5,7tetramethoxyflavone) or polyethylene glycol-conjugated superoxide dismutase (PEG-<em>SOD</em>). In control (intact anesthetized animals), the signal was about <em>3</em> times greater in the cortex than in the cerebellum. During ischemia, the signal rose to 110% in cortex (NS) and 28<em>3</em>% in cerebellum (P < 0.05). In reperfusion, it fell again to 91% of control in cerebellum (NS) and <em>3</em>5% in cortex (P < 0.05). Treatment by quercetin (5 mg/kg i.v.) of intact and ischemia-reperfusion groups did not significantly change the signal amplitude in the cerebellum, but did double it in the cortex (to 76% of control) for the ischemia-reperfusion group (P < 0.05). In contrast, FB277 (<em>3</em>.75 mg/kg i.v.) did not increase the signal in the cortex during ischemia-reperfusion, but did do so in the cerebellum (to 152% of control, P < 0.05). The results obtained for PEG-<em>SOD</em> (10,000 U/kg i.v.) were similar to those for FB277. In separate in vitro measurements, we found that quercetin but not FB277 efficiently scavenged superoxide. We hypothesize that quercetin but not FB277 scavenged superoxide anions released in the cortex during reperfusion, thus diminishing the amount of NO removed by the formation of peroxynitrite. The lack of effect of PEG-<em>SOD</em> may be related to the need for chronic treatment to obtain protection.

BACKGROUND

Obesity is associated with low-grade systemic inflammation, in part because of secretion of proinflammatory cytokines, resulting into peripheral insulin resistance (IR). Increased oxidative stress is proposed to link adiposity and chronic inflammation. The effects of endurance exercise in modulating these outcomes in insulin-resistant obese adults remain unclear. We investigated the effect of endurance exercise on markers of oxidative damage (4-hydroxy-2-nonenal (4-HNE), protein carbonyls (PCs)) and antioxidant enzymes (superoxide dismutase (SOD), catalase) in skeletal muscle; urinary markers of oxidative stress (8-hydroxy-2-deoxyguanosine (8-OHdG), 8-isoprostane); and plasma cytokines (C-reactive protein (CRP), interleukin-6 (IL-6), leptin, adiponectin).

METHODS

Age- and fitness-matched sedentary obese and lean men (n=9 per group) underwent 3 months of moderate-intensity endurance cycling training with a vastus lateralis biopsy, 24-h urine sample and venous blood samples taken before and after the intervention.

RESULTS

Obese subjects had increased levels of oxidative damage: 4-HNE (+37%; P0.03) and PC (+63%; P0.02); evidence of increased adaptive response to oxidative stress because of elevated levels of copper/zinc SOD (Cu/ZnSOD) protein content (+84%; P0.01); increased markers of inflammation: CRP (+737%; P0.0001) and IL-6 (+85%; P0.03), and these correlated with increased markers of obesity; and increased leptin (+262%; P0.0001) with lower adiponectin (-27%; P0.01) levels vs lean controls. Training reduced 4-HNE (-10%; P0.04), PC (-21%; P0.05), 8-isoprostane (-26%; P0.02) and leptin levels (-33%; P0.01); had a tendency to decrease IL-6 levels (-21%; P=0.07) and IR (-17%; P=0.10); and increased manganese SOD (MnSOD) levels (+47%; P0.01).

CONCLUSIONS

Endurance exercise reduced skeletal muscle-specific and systemic oxidative damage while improving IR and cytokine profile associated with obesity, independent of weight loss. Hence, exercise is a useful therapeutic modality to reduce risk factors associated with the pathogenesis of IR in obesity.

It is unknown whether nutritional deficiencies affect the morphology and function of structural cells, such as epithelial cells, and modify the susceptibility to viral infections. We developed an in vitro system of differentiated human bronchial epithelial cells (BEC) grown either under selenium-adequate (Se+) or selenium-deficient (Se-) conditions, to determine whether selenium deficiency impairs host defense responses at the level of the epithelium. Se- BECs had normal <em>SOD</em> activity, but decreased activity of the selenium-dependent enzyme GPX1. Interestingly, catalase activity was also decreased in Se- BECs. Both Se- and Se+ BECs differentiated into a mucociliary epithelium; however, Se- BEC demonstrated increased mucus production and increased Muc5AC mRNA levels. This effect was also seen in Se+ BEC treated with <em>3</em>-aminotriazole, an inhibitor of catalase activity, suggesting an association between catalase activity and mucus production. Both Se- and Se+ were infected with influenza A/Bangkok/1/79 and examined 24 h postinfection. Influenza-induced IL-6 production was greater while influenza-induced IP-10 production was lower in Se- BECs. In addition, influenza-induced apoptosis was greater in Se- BEC as compared to the Se+ BECs. These data demonstrate that selenium deficiency has a significant impact on the morphology and influenza-induced host defense responses in human airway epithelial cells.