Astaxanthin (AST) is a powerful antioxidant that occurs naturally in a wide variety of living organisms. We have investigated the role of AST in preventing 1-methyl-4-phenyl-1,2,<em>3</em>,6-tetrahydropyridine (MPTP)-induced apoptosis of the substantia nigra (SN) neurons in the mouse model of Parkinson's disease (PD) and 1-methyl-4-phenylpyridinium (MPP+)-induced cytotoxicity of SH-SY5Y human neuroblastoma cells. In in vitro study, AST inhibits MPP+-induced production of intracellular reactive oxygen species (ROS) and cytotoxicity in SH-SY5Y human neuroblastoma cells. Preincubation of AST (50 μM) significantly attenuates MPP+-induced oxidative damage. Furthermore, AST is able to enhance the expression of Bcl-2 protein but reduce the expression of α-synuclein and Bax, and suppress the cleavage of caspase-<em>3</em>. Our results suggest that the protective effects of AST on MPP+-induced apoptosis may be due to its anti-oxidative properties and anti-apoptotic activity via induction of expression of superoxide dismutase (<em>SOD</em>) and catalase and regulating the expression of Bcl-2 and Bax. Pretreatment with AST (<em>3</em>0 mg/kg) markedly increases tyrosine hydroxylase (TH)-positive neurons and decreases the argyrophilic neurons compared with the MPTP model group. In summary, AST shows protection from MPP+/MPTP-induced apoptosis in the SH-SY5Y cells and PD model mouse SN neurons, and this effect may be attributable to upregulation of the expression of Bcl-2 protein, downregulation of the expression of Bax and α-synuclein, and inhibition of the activation of caspase-<em>3</em>. These data indicate that AST may provide a valuable therapeutic strategy for the treatment of progressive neurodegenerative disease such as Parkinson's disease.

OBJECTIVE

Diabetic cardiomyopathy (DCM), characterized by myocardial structural and functional changes, is an independent cardiomyopathy that develops in diabetic individuals. The present study was sought to investigate the effect of curcumin on modulating DCM and the mechanisms involved.

METHODS

An experimental diabetic rat model was induced by low dose of streptozoticin(STZ) combined with high energy intake on rats. Curcumin was orally administrated at a dose of 100 or 200 mg · kg(-1) · d(-1), respectively. Cardiac function was evaluated by serial echocardiography. Myocardial ultrastructure, fibrosis area and apoptosis were assessed by histopathologic analyses. Metabolic profiles, myocardial enzymes and oxidative stress were examined by biochemical tests. Inflammatory factors were detected by ELISA, and interrelated proteins were measured by western blot.

RESULTS

Rats with DCM showed declined systolic myocardial performance associated with myocardial hypertrophy and fibrosis, which were accompanied with metabolism abnormalities, aberrant myocardial enzymes, increased AGEs (advanced glycation end products) accumulation and RAGE (receptor for AGEs) expression, elevated markers of oxidative stress (MDA, <em>SOD</em>, the ratio of NADP(+)/NADPH, Rac1 activity, NADPH oxidase subunits expression of gp91(phox) and p47(phox) ), raised inflammatory factor (TNF-α and IL-1β), enhanced apoptotic cell death (ratio of bax/bcl-2, caspase-<em>3</em> activity and TUNEL), diminished Akt and GSK-<em>3</em>β phosphorylation. Remarkably, curcumin attenuated myocardial dysfunction, cardiac fibrosis, AGEs accumulation, oxidative stress, inflammation and apoptosis in the heart of diabetic rats. The inhibited phosphorylation of Akt and GSK-<em>3</em>β was also restored by curcumin treatment.

CONCLUSIONS

Taken together, these results suggest that curcumin may have great therapeutic potential in the treatment of DCM, and perhaps other cardiovascular disorders, by attenuating fibrosis, oxidative stress, inflammation and cell death. Furthermore, Akt/GSK-<em>3</em>β signaling pathway may be involved in mediating these effects.

OBJECTIVE

Placental production of lipid peroxides is abnormally increased in preeclampsia. The reason for this is not known, but if placental antioxidant enzymes were deficient, lipid peroxides would increase unchecked. In this study, we measured 1) enzyme activities of superoxide dismutase (CuZn-SOD), catalase, and glutathione peroxidase (GSH-Px) and tissue levels of vitamin E, and 2) mRNA expression of CuZn-SOD, catalase, and GSH-Px in normal and preeclamptic placentas.

METHODS

Placental tissues were obtained from normal (n = 16) preeclamptic (n = 12) pregnancies immediately after delivery. Tissue pieces were frozen in liquid nitrogen and stored at -80C until assayed. The enzyme activities of CuZn-SOD, catalase, and GSH-Px and the levels of vitamin E were determined by spectrophotometric assays. Messenger RNA expression of CuZn-SOD, catalase, and GSH-Px was determined by Northern blot analysis.

RESULTS

The activities of CuZn-SOD and GSH-Px and the tissue levels of vitamin E were significantly lower in preeclamptic placentas than in normal placentas (CuZn-SOD: 1.13 +/- 0.49 versus 3.71 +/- 0.71 U/mg protein, P < .01; GSH-Px: 0.18 +/- 0.01 versus 0.26 +/- 0.02 U/mg, P < .01; vitamin E: 0.08 +/- 0.01 versus 0.18 +/- 0.01 microgram/mg, mean +/- standard error, P < .001), whereas the activity of catalase was significantly higher (93 +/- 3 versus 83 +/- 2 U/mg, P < .05). Relative mRNA expression of CuZn-SOD and GSH-Px was significantly lower in preeclamptic than normal placentas (P < 0.05), but there was no significant difference for catalase (P>> .4).

CONCLUSIONS

1) Activities of CuZn-SOD and GSH-Px and the tissue levels of vitamin E are significantly lower in preeclamptic than in normal placentas. 2) Activity of catalase is significantly higher in preeclamptic than in normal placentas. 3) Messenger RNA expression for CuZn-SOD and GSH-Px is lower in preeclamptic placentas. We speculate that decreased antioxidant activity may result in increased lipid peroxide levels in preeclamptic placentas.

In order to determine the role of the neuropoietic cytokine interleukin-6 (IL-6) during the first <em>3</em> weeks after a focal brain injury, we examined the inflammatory response, oxidative stress and neuronal survival in normal and interleukin-6-deficient (knockout, IL-6KO) mice subjected to a cortical freeze lesion. In normal mice, the brain injury was followed by reactive astrogliosis and recruitment of macrophages from 1 day postlesion (dpl), peaking at <em>3</em>-10 dpl, and by 20 dpl the transient immunoreactions were decreased, and a glial scar was present. In IL-6KO mice, the reactive astrogliosis and recruitment of macrophages were decreased throughout the experimental period. The expression of the antioxidant and anti-apoptotic factors metallothionein I+II (MT-I+II) was increased prominently by the freeze lesion, but this response was significantly reduced in the IL-6 KO mice. By contrast, the expression of the antioxidants Cu/Zn-superoxide dismutase (Cu/Zn-<em>SOD</em>), Mn-<em>SOD</em>, and catalase remained unaffected by the IL-6 deficiency. The lesioned mice showed increased oxidative stress, as judged by malondialdehyde (MDA) and nitrotyrosine (NITT) levels and by formation of inducible nitric oxide synthase (iNOS). IL-6KO mice showed higher levels of MDA, NITT, and iNOS than did normal mice. Concomitantly, in IL-6KO mice the number of apoptotic neurons was significantly increased as judged by TUNEL staining, and regeneration of the tissue was delayed relative to normal mice. The changes in neuronal tissue damage and in brain regeneration observed in IL-6KO mice are likely caused by the IL-6-dependent decrease in MT-I+II expression, indicating IL-6 and MT-I+II as neuroprotective factors during brain injury.

Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 1<em>3</em>0) are up-regulated by more that 1.<em>3</em>-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through <em>3</em>'UTR luciferase assays we identified one up-regulated protein, <em>SOD</em>-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-<em>3</em>p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control.

The objective of this paper is to evaluate adaptations in hepatic mitochondrial protein mass, function and efficiency in a rat model of high-fat diet-induced obesity and insulin resistance that displays several correlates to human obesity. Adult male rats were fed a high-fat diet for 7 weeks. Mitochondrial state <em>3</em> and state 4 respiratory capacities were measured in liver homogenate and isolated mitochondria by using nicotinamide adenine dinucleotide, flavin adenine dinucleotide and lipid substrates. Mitochondrial efficiency was evaluated by measuring proton leak kinetics. Mitochondrial mass was assessed by ultrastructural observations and citrate synthase (CS) activity measurements. Mitochondrial oxidative damage and antioxidant defence were also considered by measuring lipid peroxidation, aconitase and superoxide dismutase (<em>SOD</em>) specific activity. Whole body metabolic characteristics were obtained by measuring 24-h oxygen consumption (VO2), carbon dioxide production (VCO2), respiratory quotient (RQ) and nonprotein respiratory quotient (NPRQ), using indirect calorimetry with urinary nitrogen analysis. Whole body glucose homeostasis was assessed by measuring plasma insulin and glucose levels after a glucose load. Adult rats fed a high-fat diet for 7 weeks, exhibit not only obesity, insulin resistance and hepatic steatosis, but also reduced respiratory capacity and increased oxidative stress in liver mitochondria. Our present results indicate that alterations in the mitochondrial compartment induced by a high-fat diet are associated with the development of insulin resistance and ectopic fat storage in the liver. Our results thus fit in with the emerging idea that mitochondrial dysfunction can led to the development of metabolic diseases, such as obesity, type 2 diabetes mellitus and nonalcoholic steatohepatitis.

BACKGROUND

Despite familial clustering of nephropathy and retinopathy severity in type 1 diabetes, few gene variants have been consistently associated with these outcomes.

METHODS

We performed an individual-based genetic association study with time to renal and retinal outcomes in 1,<em>3</em>62 white probands with type 1 diabetes from the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study. Specifically, we genotyped 1,411 SNPs that capture common variations in 212 candidate genes for long-term complications and analyzed them for association with the time from DCCT baseline to event for renal and retinal outcomes using multivariate Cox proportion hazards models. To address multiple testing and assist interpretation of the results, false discovery rate q values were calculated separately for each outcome.

RESULTS

We observed association between rs178801<em>3</em>5 in the <em>3</em>' region of superoxide dismutase 1 (<em>SOD</em>1) and the incidence of both severe nephropathy (hazard ratio [HR] 2.62 [95% CI 1.64-4.18], P = 5.6 x 10(-5), q = 0.06) and persistent microalbuminuria (1.82 [1.29-2.57], P = 6.4 x 10(-4), q = 0.46). Sequencing and fine-mapping identified additional <em>SOD</em>1 variants, including rs202446, rs9974610, and rs2047<em>3</em>2, which were also associated (P < 10(-<em>3</em>)) with persistent microalbuminuria, whereas rs178801<em>3</em>5 and rs17881180 were similarly associated with the development of severe nephropathy. Attempts to replicate the findings in three cross-sectional case-control studies produced equivocal results. We observed no striking differences between risk genotypes in serum <em>SOD</em> activity, serum <em>SOD</em>1 mass, or <em>SOD</em>1 mRNA expression in lymphoblastoid cell lines.

CONCLUSIONS

Multiple variations in <em>SOD</em>1 are significantly associated with persistent microalbuminuria and severe nephropathy in the DCCT/EDIC study.

OBJECTIVE

The cytosolic form of Cu/Zn-containing superoxide dismutase (<em>SOD</em>1) has peroxidase activity, with H2O2 used as a substrate to oxidize other molecules. We examined peroxidase properties of the extracellular form of <em>SOD</em> (<em>SOD</em>3), a major isoform of <em>SOD</em> in the vessel wall, by using recombinant <em>SOD</em>3 and an in vivo model of atherosclerosis.

RESULTS

In the presence of HCO3-, <em>SOD</em>3 reacted with H2O2 to produce a hydroxyl radical adduct of the spin trap 5-diethoxyphosphoryl-5methyl-1-pyrroline N-oxide (DEMPO). <em>SOD</em>1 and <em>SOD</em>3 were inactivated by H2O2 in a dose- and time-dependent fashion, and this was prevented by physiological levels of uric acid. To examine the in vivo role of uric acid on <em>SOD</em>1 and <em>SOD</em>3, control and apolipoprotein E-deficient (ApoE(-/-)) mice were treated with oxonic acid, which inhibits urate metabolism. This treatment increased plasma levels of uric acid in control and ApoE(-/-) mice by approximately 3-fold. Although increasing uric acid levels did not alter aortic <em>SOD</em>1 and <em>SOD</em>3 protein expression, aortic <em>SOD</em>1 and <em>SOD</em>3 activities were increased by 2- to 3-fold in aortas from ApoE(-/-) mice but not in aortas from control mice.

CONCLUSIONS

These studies show that <em>SOD</em>1 and <em>SOD</em>3 are partially inactivated in atherosclerotic vessels of ApoE(-/-) mice and that levels of uric acid commonly encountered in vivo may regulate vascular redox state by preserving the activity of these enzymes.

Quantitative real-time PCR (qRT-PCR) is a reliable and reproducible technique for measuring and evaluating changes in gene expression. The most common method for analyzing qRT-PCR data is to normalize mRNA levels of target genes to internal reference genes. Evaluating and selecting stable reference genes on a case-by-case basis is critical. The present study aimed to facilitate gene expression studies by identifying the most suitable reference genes for normalization of mRNA expression in qRT-PCR analysis of the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae). For this purpose, three software tools (geNorm, NormFinder and BestKeeper) were used to investigate 10 candidate reference genes in nine developmental stages and five different tissues (epidermis, head, midgut, fat body and hemolymph) in three larval physiological stages (molting, feeding and wandering stages) of, S. exigua. With the exception of 18S ribosomal RNA (18S), all other candidate genes evaluated, β-actin1(ACT1), β-actin2 (ACT2), elongation factor1(EF1), elongation factor 2 (EF2), Glyceralde hyde-<em>3</em>-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (L10), ribosomal protein L17A (L17A), superoxide dismutase (<em>SOD</em>), α-tubulin (TUB),proved to be acceptable reference genes. However, their suitability partly differed between physiological stages and different tissues. L10, EF2 and L17A ranked highest in all tissue sample sets. <em>SOD</em>, ACT2, GAPDH, EF1 and ACT1 were stably expressed in all developmental stage sample sets; ACT2, ACT1 and L10 for larvae sample sets; GAPDH, ACT1 and ACT2 for pupae and adults; <em>SOD</em> and L17A for males; and EF2 and <em>SOD</em> for females. The expression stability of genes varied in different conditions. The findings provided here demonstrated, with a few exceptions, the suitability of most of the 10 reference genes tested in tissues and life developmental stages. Overall, this study emphasizes the importance of validating reference genes for qRT-PCR analysis in S. exigua.

The purpose of the study was to evaluate the effects of hesperetin (Hsp) on diabetes-induced retinal oxidative stress, neuroinflammation and apoptosis in rats. The Hsp treatment (100 mg/kg body weight) was carried for twenty four weeks in STZ-induced diabetic rats and evaluated for antioxidant (Superoxide dismutase; <em>SOD</em>, Catalase; CAT and glutathione; GSH) enzymes, inflammatory cytokines (TNF-α, IL-1β), caspase-<em>3</em>, glial fibrillary acidic protein (GFAP) and aquaporin-4(AQP4) expression. Histological changes were evaluated by light and transmission electron microscopic (LM and TEM) studies. Retinal GSH levels and anti-oxidant enzymes (<em>SOD</em> and CAT) activity were significantly decreased in diabetic group as compared to normal group. However, in Hsp-treated rats, retinal GSH levels were restored close to normal levels and positive modulation of anti-oxidant enzyme activity was observed. Diabetic retinae showed significantly increased expression of Pro-inflammatory cytokines (TNF-α and IL-1β) as compared to normal retinae. While Hsp-treated retinae showed significantly lower levels of cytokines as compared to diabetic retinae. Diabetic retinae showed increased caspase-<em>3</em>, GFAP and AQP4 expression. However, Hsp-treated retinae showed inhibitory effect on caspase-<em>3</em>, GFAP and AQP4 expression. LM images showed edematous Müller cell endfeet, and also degenerated photoreceptor layer; however, protective effect of Hsp was seen on Müller cell processes and photoreceptors. TEM study showed increased basement membrane (BM) thickness in diabetic retina, while relatively thin BM was recorded in Hsp-treated retina. It can be postulated that dietary flavanoids, like Hsp, can be effective for the prevention of diabetes induced neurovascular complications such as diabetic retinopathy.

The objective of this study was to examine the influence of reactive oxygen species (ROS), generated through the use of the xanthine (X)-xanthine oxidase (XO) system, on equine sperm motility, viability, acrosomal integrity, mitochondrial membrane potential, and membrane lipid peroxidation. Equine spermatozoa were separated from seminal plasma on a discontinuous Percoll gradient, and spermatozoa were incubated with 0.6 mM X and 0.05 U/mL XO for <em>3</em>0 minutes. Catalase (150 U/mL), superoxide dismutase (<em>SOD</em>, 150 U/mL), or glutathione (GSH, 1.5 mM) were evaluated for their ability to preserve sperm function in the presence of the induced oxidative stress. At the end of the <em>3</em>0-minute incubation, sperm motility was determined by computer-assisted semen analysis. Viability and acrosomal integrity were determined by Hoechst-Pisum sativum staining, and mitochondrial membrane potential was determined by staining with JC-1. Incubation with the X-XO system led to a significant (P < .01) increase in hydrogen peroxide production and an associated decrease (P < .01) in motility parameters. Total motility was significantly (P < .01) lower in the presence of X-XO compared with the case of the control (29%+/-9% vs 7<em>3</em>%+/-1%, respectively). Catalase, but not <em>SOD</em>, prevented a decline in motility secondary to oxidative stress (71%+/-4% vs <em>3</em>0%+/-<em>3</em>%, respectively). The addition of glutathione had an intermediate effect in preserving sperm motility at the end of the <em>3</em>0-minute incubation (5<em>3</em>%+/-<em>3</em>%). No influence of X-XO could be determined on viability, acrosomal integrity, or mitochondrial membrane potential. In order to promote lipid peroxidation, samples were incubated with ferrous sulfate (0.64 mM) and sodium ascorbate (20 mM) for 2 hours after the X-XO incubation. No increase in membrane lipid peroxidation was detected. This study indicates that hydrogen peroxide is the major ROS responsible for damage to equine spermatozoa. The decrease in sperm motility associated with ROS occurs in the absence of any detectable decrease in viability, acrosomal integrity, or mitochondrial membrane potential or of any detectable increase in lipid peroxidation.

BACKGROUND

Extracellular superoxide dismutase (Ec-SOD) may protect the heart against myocardial infarction (MI) because of its extended half-life and capacity to bind heparan sulfate proteoglycans on cellular surfaces. Accordingly, we used direct gene transfer to increase systemic levels of Ec-SOD and determined whether this gene therapy could protect against MI.

RESULTS

The cDNA for human Ec-<em>SOD</em> was incorporated into a replication-deficient adenovirus (Ad5/CMV/Ec-<em>SOD</em>). Injection of this virus produced a high level of Ec-<em>SOD</em> in the liver, which was redistributed to the heart and other organs by injection of heparin. Untreated rabbits (group I) underwent a <em>3</em>0-minute coronary occlusion and <em>3</em> days of reperfusion. For comparison, preconditioned rabbits (group II) underwent a sequence of six 4-minute-occlusion/4-minute-reperfusion cycles 24 hours before the <em>3</em>0-minute occlusion. Control-treated rabbits (group III) were injected intravenously with Ad5/CMV/nls-LacZ, and gene-therapy rabbits (group IV) were injected with Ad5/CMV/Ec-<em>SOD</em> <em>3</em> days before the <em>3</em>0-minute occlusion. Both groups treated with Ad5 received intravenous heparin 2 hours before the <em>3</em>0-minute occlusion. Infarct size (percent risk area) was similar in groups I (57+/-6%) and III (58+/-5%). Ec-<em>SOD</em> gene therapy markedly reduced infarct size to 25+/-4% (P<0.01, group IV versus group III), a protection comparable to that of the late phase of ischemic preconditioning (29+/-<em>3</em>%, P<0.01 group II versus group I).

CONCLUSIONS

Direct gene transfer of the cDNA encoding membrane-bound Ec-SOD affords powerful cardioprotection, providing proof of principle for the effectiveness of antioxidant gene therapy against MI.

The aim of this study was to evaluate the role of free-oxygen radicals in the embryonic maldevelopment of diabetic pregnancy. Rat embryos cultured in vitro during early organogenesis showed growth retardation and severe malformations after exposure to 50 mM glucose, <em>3</em> mM PYR, 10 mM HBT, or <em>3</em> mM KIC. Combinations of 25 mM glucose, 2.5 mM HBT, and 1 mM KIC also elicited embryonic growth retardation and malformations. The deleterious effects on embryonic development by all agents were alleviated by addition of <em>SOD</em> to the culture media, which yielded increased enzyme activity in the embryos and their membranes. The endogenous <em>SOD</em> activity also increased in embryos subjected to a high concentration of glucose or PYR in the culture medium. Addition of the mitochondrial PYR transport inhibitor CHC to the culture media blocked the dysmorphogenesis caused by glucose and PYR, but was without effect on the teratogenic actions of HBT and KIC. These findings implicate the embryonic mitochondria as a likely site for enhanced substrate-induced production of free-oxygen radicals mediating the teratogenic effect of a diabetic environment. In particular, the teratogenic process in diabetic pregnancy may depend on an increased production of free-oxygen radicals in immature embryonic mitochondria in response to a metabolic overload. This notion implies that every oxidative substrate entering the mitochondrial metabolism in excess may induce embryonic malformations and emphasizes the need for an extended metabolic surveillance of pregnant diabetic women. Consequently, optimal metabolic control should aim at normalizing the maternal serum concentrations of all possible oxidative substrates.

1. Enhanced superoxide (O2(-)) activity as a result of the inhibition of the superoxide dismutase (<em>SOD</em>) enzyme results in vasoconstrictor and antinatriuretic responses in the canine kidney; these responses were shown to be greatly enhanced during inhibition of nitric oxide synthase (NOS). Glomerular filtration rate remained mostly unchanged during <em>SOD</em> inhibition in the intact nitric oxide (NO) condition, but was markedly reduced during NOS inhibition. These findings indicate that endogenous NO has a major renoprotective effect against O2(-) by acting as an anti-oxidant. Nitric oxide synthase inhibition was also shown to enhance endogenous O2(-) activity. 2. Experiments in our laboratory using dogs, rats and gene knockout mice have shown that renal vasoconstrictor and antinatriuretic responses to acute or chronic angiotensin (Ang) II administration are mediated, in part, by O2(-) generation. In the absence of NO, enhanced O2(-) activity largely contributes to AngII-induced renal tubular sodium reabsorption. Acute or chronic treatment with the O2(-) scavenger tempol in experimental models of hypertension (induced by chronic low-dose treatment with AngII and NO inhibitors) causes an improvement in renal haemodynamics and in excretory function, abolishes salt sensitivity and reduces blood pressure. <em>3</em>. The present mini review also discusses related studies from many other laboratories implicating a role for O2(-) and its interaction with NO in the development of salt-sensitive hypertension. 4. Overall, the collective data support the hypothesis that an imbalance between the production of NO and O2(-) in the kidney primarily determines the condition of oxidative stress that alters renal haemodynamics and excretory function leading to sodium retention and, thus, contributes to the development of salt-sensitive hypertension.

1. The aim of this work was to investigate the mechanism of vasorelaxation induced by red wine polyphenolic compounds (RWPC) and two defined polyphenols contained in wine, leucocyanidol and catechin. The role of the endothelium, especially endothelium-derived nitric oxide (NO), was also investigated. 2. Relaxation produced by polyphenols was studied in rat aortic rings with and without functional endothelium, pre-contracted to the same extent with noradrenaline (0.<em>3</em> and 0.1 microM, respectively). RWPC and leucocyanidol, but not catechin, produced complete relaxation of vessels with and without endothelium. However, 1000 fold higher concentrations were needed to relax endothelium-denuded rings compared to those with functional endothelium. <em>3</em>. High concentrations of catechin (in the range of 10(-1) gl-1) only produced partial relaxation (maximum <em>3</em>0%) and had the same potency in rings with and without endothelium. 4. The NO synthase inhibitor, N omega-nitro-L-arginine-methyl-ester (L-NAME, <em>3</em>00 microM) completely abolished the endothelium-dependent but not the endothelium-independent relaxations produced by all of the polyphenolic compounds. 5. In contrast to superoxide dismutase (<em>SOD</em>, 100 u ml-1), neither RWPC nor leucocyanidol affected the concentration-response curve for the NO donor, SIN-1 (<em>3</em>-morpholino-sydnonimine) which also produces superoxide anion (O2-). 6. In aortic rings with endothelium, RWPC (10(-2) gl-1) produced, a 7 fold increase in the basal production of guanosine <em>3</em>':5'-cyclic monophosphate (cyclic GMP) which was prevented by L-NAME (<em>3</em>00 microM). 7. Electron paramagnetic resonance (e.p.r.) spectroscopy studies with Fe(2+)-diethyldithiocarbamate as an NO spin trap demonstrated that RWPC and leucocyanidol increased NO levels in rat thoracic aorta about 2 fold. This NO production was entirely dependent on the presence of the endothelium and was abolished by L-NAME (<em>3</em>00 microM). 8. These results show that RWPC and leucocyanidol, but not the structurally closely related polyphenol catechin, induced endothelium-dependent relaxation in the rat aorta. They indicate that this effect results from enhanced synthesis of NO rather than enhanced biological activity of NO or protection against breakdown by O2. It is concluded that some polyphenols, with specific structure, contained in wine possess potent endothelium-dependent vasorelaxing activity.

BACKGROUND

The peroxisome proliferators-activated receptor-alpha (PPARalpha), a transcription factor that modulates fatty acid metabolism, regulates substrate preference in the heart. Although in acute ischemia there is a switch in substrate preference from fatty acids to glucose, metabolic gene expression in repetitive ischemia is not well described. In a mouse model of ischemic cardiomyopathy induced by repetitive ischemia/reperfusion (I/R), we postulated that downregulation of PPARalpha is regulated by reactive oxygen species and is necessary for maintaining contractile function in the heart.

RESULTS

Repetitive closed-chest I/R (15 minutes) was performed daily in C57/BL6 mice, mice overexpressing extracellular superoxide dismutase, and mice treated with the PPARalpha agonist-WY-14,64<em>3</em>. Echocardiography, histology, and candidate gene expression were measured at <em>3</em>, 5, 7, and 28 days of repetitive I/R and 15 and <em>3</em>0 days after discontinuation of I/R. Repetitive I/R was associated with a downregulation of PPARalpha-regulated genes and both myosin heavy chain isoform transcript levels, which was reversible on discontinuation of I/R. Overexpression of EC-<em>SOD</em> prevented the downregulation of PPARalpha-regulated genes and myosin iso-genes by repetitive I/R. Furthermore, reactivation of PPARalpha in mice exposed to repetitive I/R worsened contractile function, induced microinfarctions, and increased intramyocardial triglyceride deposition, features suggestive of cardiac lipotoxicity.

CONCLUSIONS

Metabolic and myosin isoform gene expression in repetitive I/R is mediated by reactive oxygen species. Furthermore, we suggest that downregulation of PPARalpha in repetitive I/R is an adaptive mechanism that is able to prevent lipotoxicity in the ischemic myocardium.

Increasing evidence demonstrates that oxidative stress plays an important role in brain injury in experimental models of brain ischemia. Thymoquinone, the main constituents of the volatile oil from Negella sativa seeds, is reported to possess strong antioxidant properties. Hence, the present study was undertaken to evaluate the neuroprotective effect of thymoquinone against transient forebrain ischemia-induced neuronal damage in the rat hippocampus. Rats were divided randomly into five groups: control, sham, ischemia, thymoquinone and ischemia+thymoquinone. Transient forebrain ischemia was induced with bilateral occlusion of both common carotid arteries for 10 min followed by 7 days of reperfusion. Thymoquinone was administered (5 mg/kg/day p.o.) 5 days before ischemia and continued during the reperfusion time. Animals were sacrificed, and brain tissues were isolated for histopathological examination. Hippocampal tissues were also used for determination of malondialdehyde levels, an end product of lipid peroxidation; glutathione (GSH) levels, a key antioxidant and the activities of the antioxidant enzymes catalase and superoxide dismutase (<em>SOD</em>). Thymoquinone and its metabolite thymohydroquinone were tested as inhibitors of the in vitro non-enzymatic lipid peroxidation induced by iron-ascorbate in the hippocampal homogenate. Forebrain ischemia-reperfusion neural injury in rats was demonstrated by histopathological observation, which revealed significant neural cell death in the hippocampus CA1 area 7 days post-ischemia (77% cell loss). Additionally, forebrain ischemia-reperfusion oxidative injury in rats was demonstrated by a significant increase in malondialdehyde and a significant decrease in GSH contents, catalase and <em>SOD</em> activities in the hippocampal tissue compared to the control or sham-operated groups. Pretreatment of thymoquinone attenuated forebrain ischemia-induced neuronal damage manifested by significantly decreasing the number of dead hippocampal neuronal cells (24% in thymoquinone-treated versus 77% for ischemia, P<0.001), which confirm the protective role of thymoquinone in ischemia-reperfusion injury. Also, pretreatment of ischemic rats with thymoquinone decreased the elevated levels of malondialdehyde and increased GSH contents, catalase and <em>SOD</em> activities to normal levels. Thymoquinone and thymohydroquinone inhibited the in vitro non-enzymatic lipid peroxidation in hippocampal homogenate induced by iron-ascorbate. The IC50 for thymoquinone and thymohydroquinone were found to be 12 and <em>3</em> microM respectively. This suggests that the protection of thymoquinone and its metabolite involve increased resistance to oxidative stress. In conclusion, thymoquinone is effective in protecting rats against transient forebrain ischemia-induced damage in the rat hippocampus. This spectacular protection makes thymoquinone a promising agent in pathologies implicating neurodegenaration such as cerebral ischemia.

Emodin (1,<em>3</em>,8-trihydroxy-6-methylanthraquinone) is an active constituent of Rheum palmatum, and showed inhibitory activity on lipopolysaccharide-induced NO production in our previous study. However, the apoptosis-inducing activity of emodin has remained undefined. Among three structurally related anthraquinones, including emodin, physcion, and chrysophanol, emodin showed the most potent cytotoxic effects on HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells. Emodin at apoptosis-inducing concentrations causes rapid and transient induction of caspase <em>3</em>/CPP<em>3</em>2 activity, but not caspase 1 activity, according to cleavage of caspase <em>3</em> substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase <em>3</em> fragments being detected in emodin- but not physcion- or chrysophanol-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in emodin-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bad remained unchanged. The caspase <em>3</em> inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated emodin-induced DNA ladders, associated with the blockage of PARP and D4-GDI cleavage. Free radical scavenging agents including NAC, catalase, <em>SOD</em>, ALL, DPI, L-NAME and PDTC showed no preventive effect on emodin-induced apoptotic responses, whereas NAC, CAT and PDTC prevented HL-60 cells from ROS (H(2)O(2))-induced apoptosis through inhibition of caspase <em>3</em> cascades. Induction of catalase, but not <em>SOD</em>, activity was detected in emodin-treated HL-60 cells by in gel activity assays, and H(2)O(2)-induced intracellular peroxide level was significantly reduced by prior treatment of emodin in HL-60 cells. Our experiments provide evidence that emodin is an effective apoptosis inducer in HL-60 cells through activation of the caspase <em>3</em> cascade, but that it is independent of ROS production.

We investigated the cellular mechanisms responsible for the occurrence of miconazole-tolerant persisters in Candida albicans biofilms. Miconazole induced about <em>3</em>0% killing of sessile C. albicans cells at 75 μM. The fraction of miconazole-tolerant persisters, i.e., cells that can survive high doses of miconazole (0.6 to 2.4 mM), in these biofilms was 1 to 2%. Since miconazole induces reactive oxygen species (ROS) in sessile C. albicans cells, we focused on a role for superoxide dismutases (<em>Sods</em>) in persistence and found the expression of <em>Sod</em>-encoding genes in sessile C. albicans cells induced by miconazole compared to the expression levels in untreated sessile C. albicans cells. Moreover, addition of the superoxide dismutase inhibitor N,N'-diethyldithiocarbamate (DDC) to C. albicans biofilms resulted in an 18-fold reduction of the miconazole-tolerant persister fraction and in increased endogenous ROS levels in these cells. Treatment of biofilms of C. albicans clinical isolates with DDC resulted in an 18-fold to more than 200-fold reduction of their miconazole-tolerant persister fraction. To further confirm the important role for <em>Sods</em> in C. albicans biofilm persistence, we used a Δsod4 Δsod5 mutant lacking <em>Sods</em> 4 and 5. Biofilms of the Δsod4 Δsod5 mutant contained at least <em>3</em>-fold less of the miconazole-tolerant persisters and had increased ROS levels compared to biofilms of the isogenic wild type (WT). In conclusion, the occurrence of miconazole-tolerant persisters in C. albicans biofilms is linked to the ROS-detoxifying activity of <em>Sods</em>. Moreover, <em>Sod</em> inhibitors can be used to potentiate the activity of miconazole against C. albicans biofilms.

Superoxide dismutase comprises a family of metalloenzymes that catalyze the oxido-reduction of superoxide anion to H2O2. Manganese superoxide dismutase (Mn-<em>SOD</em>) is encoded by nuclear chromatin, synthesized in the cytosol, and imported posttranslationally into the mitochondrial matrix. We isolated and sequenced complementary DNA encoding human Mn-<em>SOD</em>. The Mn-<em>SOD</em> cDNA was 1001 base pairs long with a single open reading frame. It contained 95 base pairs of 5' untranslated sequence, and 216 base pairs of <em>3</em>' untranslated sequence, followed by a short polyadenylation tract. The deduced amino acid sequence suggests a mature protein of 198 amino acids preceded by a 24 amino acid leader peptide. A major transcript of 1000 nucleotides was identified by hybridization of the cDNA with RNA isolated from human cells. Precursor Mn-<em>SOD</em> was produced by in vitro transcription of the human Mn-<em>SOD</em> cDNA followed by in vitro translation utilizing rabbit reticulocyte lysate. The primary translation product of the cDNA is a polypeptide of Mr 26,000 as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. When the Mr 26,000 propeptide was incubated with freshly isolated rat liver mitochondria, the peptide was proteolytically processed to a Mr 24,000 polypeptide. Proteolytic processing was accompanied by an energy-dependent import of the peptide into the isolated liver mitochondria. Mature 125I-labelled Mn-<em>SOD</em>, isolated from rabbit liver, was not imported in vitro into mitochondria, indicating that the energy-dependent uptake of Mn-<em>SOD</em> by liver mitochondria was specific for the Mn-<em>SOD</em> precursor.

Neurotoxic properties of L-dopa and dopamine (DA)-related compounds were assessed in human neuroblastoma SH-SY5Y cells with reference to their structural relationship. L-Dopa and its metabolites containing two free hydroxyl residues on their benzene ring showed toxicity in the cell, which was prevented by superoxide dismutase (<em>SOD</em>) and reduced glutathione (GSH), but not by catalase. Furthermore, a synthetic derivative of DA, <em>3</em>-hydroxy-4-methoxyphenethylamine (HMPE) containing methoxy residue at position 4 in the benzene ring, exerted partial cytotoxicity, which was not prevented by <em>SOD</em>, GSH or catalase. However, the metabolites containing methoxy residue at position <em>3</em> failed to show a toxic effect in the SH-SY5Y cells. Moreover, DA induced apoptotic cell death, which was observed by nuclear and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and measurement of caspase-<em>3</em> activity; this compound up-regulated apoptotic factor p5<em>3</em> while down-regulating anti-apoptotic factor Bcl-2. In the cell-free in vitro electron spin resonance (ESR) spectrometry, DA possessing two hydroxyl groups showed generation of DA-semiquinone radicals, which were markedly prevented by addition of <em>SOD</em> or GSH but not by catalase. On the other hand, methylation of one of the hydroxyl residues on the benzene ring of DA converted DA to an unoxidizable compound (<em>3</em>-MT or HMPE), and caused it to lose the property to produce semiquinone radicals. It has been previously reported that <em>SOD</em> acting as a superoxide:semiquinone oxidoreductase prevents quinone formation, and that reduced GSH through forming a complex with DA-quinone prevents quinone binding to the thiol group of the intact protein. Therefore, the present results suggest that DA and its metabolites containing two hydroxyl residues exert cytotoxicity mainly due to generation of highly reactive quinones.

In advanced cancer patients, the oxidative stress could take place either at the onset of disease or as a function of disease progression. To test this hypothesis, the following parameters were investigated: the erythrocyte activity of the enzymes superoxide dismutase (<em>SOD</em>) and glutathione peroxidase (GPx), the serum activity of glutathione reductase (GR) and the serum total antioxidant status (TAS). The total antioxidant capacity of plasma LMWA was evaluated by the cyclic voltammetry methodology. We further determined the serum levels of proinflammatory cytokines (IL-6 and TNFalpha), IL-2, leptin and C-reactive protein (CRP). All of these parameters have been correlated with the most important clinical indices of patients such as Stage of disease, ECOG PS and clinical response. Eighty-two advanced stage cancer patients and <em>3</em>6 healthy individuals used as controls were included in the study. Our findings show that <em>SOD</em> activity was significantly higher in cancer patients than in controls and GPx activity was significantly lower in cancer patients than in controls. Serum values of IL-6, TNFalpha and CRP were significantly higher in patients than in controls. Serum leptin values of cancer patients were significantly lower than controls. <em>SOD</em> activity increased significantly from Stage II/ECOG 0-1 to Stage IV/ECOG 0-1, whereas it decreased significantly in Stage IV/ECOG <em>3</em>. GPx activity decreased significantly in Stage IV/ECOG 2-<em>3</em>. An inverse correlation between ECOG PS and serum leptin levels was found. Serum levels of IL-2 decreased from Stage II/ECOG 0-1 to Stage IV/ECOG 2-<em>3</em>. A direct correlation between Stage/ECOG PS and serum levels of both IL-6 and CRP was observed. Cisplatin administration induced a significant increase of GPx after 24 hr. In conclusion, this is the first study that shows that several "biological" parameters of cancer patients such as antioxidant enzyme activity, cytokines, leptin and CRP strictly correlate with the most important clinical parameters of disease such as Stage and ECOG PS.

OBJECTIVE

The potential pathogenicity of free radicals may have a pivotal role in ulcerative colitis. Fish oil omega-<em>3</em> fatty acids exert anti-inflammatory effects on patients with ulcerative colitis (UC), but the precise mechanism of the action of fish oil on oxidative stress is still controversial. The aim of the present work was to verify the blood oxidative stress in patients with UC and determine whether the association of sulfasalazine to fish oil omega-<em>3</em> fatty acids is more effective than isolated use of sulfasalazine to reduce the oxidative stress.

METHODS

Nine patients (seven female and two male; mean age = 40 +/- 11 y) with mild or moderate active UC were studied in a randomized crossover design. In addition to their usual medication (2 g/d of sulfasalazine), they received fish oil omega-<em>3</em> fatty acids (4.5 g/d) or placebo for 2-mo treatment periods that were separated by 2 mo, when they only received sulfasalazine. Nine healthy individuals served as control subjects to study the oxidative stress status. Disease activity was assessed by laboratory indicators (C-reactive protein, alpha1-acid glycoprotein, alpha1-antitrypsin, erythrocyte sedimentation rate, albumin, hemoglobin, and platelet count), sigmoidoscopy, and histology scores. Analysis of oxidative stress was assessed by plasma chemiluminescence and erythrocyte lipid peroxidation, both induced by tert butyl hydroperoxide (t-BuOOH) and by plasma malondialdehyde. Antioxidant status was assayed by total plasma antioxidant capacity (TRAP) and microsomal lipid peroxidation inhibition (LPI). Superoxide dismutase (SOD) and catalase erythrocyte enzymatic activities were also determined.

RESULTS

No significant changes were observed in any laboratory indicator or in the sigmoidoscopy or histology scores, with the exception of erythrocyte sedimentation rate, which decreased with both treatments. Oxidative stress was demonstrated by significant decreases in TRAP and LPI levels, increased chemiluminescence induced by t-BuOOH, and higher SOD activity in patients with UC. Treatment with fish oil omega-<em>3</em> fatty acids reverted the chemiluminescence induced by t-BuOOH and LPI to baseline levels but that did not occur when patients received only sulfasalazine. Levels of plasma malondialdehyde, erythrocyte lipid peroxidation, and catalase were not different from those in the control group.

CONCLUSIONS

The results indicated that plasma oxidative stress occurs in patients with UC, and there was a significant decrease when the patients used sulfasalazine plus fish oil omega-<em>3</em> fatty acids. However, there was no improvement in most laboratory indicators, sigmoidoscopy, and histology scores. The results suggested that omega-<em>3</em> fatty acids may act as free radical scavengers protecting the patients against the overall effect of oxidative stress.

Mn(III) N-alkylpyridylporphyrins are among the most potent known <em>SOD</em> mimics and catalytic peroxynitrite scavengers and modulators of redox-based cellular transcriptional activity. In addition to their intrinsic antioxidant capacity, bioavailability plays a major role in their in vivo efficacy. Although of identical antioxidant capacity, lipophilic MnTnHex-2-PyP is up to 120-fold more efficient in reducing oxidative stress injuries than hydrophilic MnTE-2-PyP. Owing to limitations of an analytical nature, porphyrin lipophilicity has been often estimated by the thin-layer chromatographic R(f) parameter, instead of the standard n-octanol/water partition coefficient, P(OW). Herein we used a new methodological approach to finally describe the MnP lipophilicity, using the conventional log P(OW) means, for a series of biologically active ortho and meta isomers of Mn(III) N-alkylpyridylporphyrins. Three new porphyrins (MnTnBu-<em>3</em>-PyP, MnTnHex-<em>3</em>-PyP, and MnTnHep-2-PyP) were synthesized to strengthen the conclusions. The log P(OW) was linearly related to R(f) and to the number of carbons in the alkyl chain (n(C)) for both isomer series, the meta isomers being 10-fold more lipophilic than the analogous ortho porphyrins. Increasing the length of the alkyl chain by one carbon atom increases the log P(OW) value approximately 1 log unit with both isomers. Dramatic approximately 4 and approximately 5 orders of magnitude increases in the lipophilicity of the ortho isomers, by extending the pyridyl alkyl chains from two (MnTE-2-PyP, log P(OW)=-6.89) to six (MnTnHex-2-PyP, log P(OW)=-2.76) and eight carbon atoms (MnTnOct-2-PyP, log P(OW)=-1.24), parallels the increased efficacy in several oxidative-stress injury models, particularly those of the central nervous system, in which transport across the blood-brain barrier is critical. Although meta isomers are only slightly less potent <em>SOD</em> mimics and antioxidants than their ortho analogues, their higher lipophilicity and smaller bulkiness may lead to a higher cellular uptake and overall similar effectiveness in vivo.