Protective effect of dietary tomato against endothelial dysfunction in hypercholesterolemic mice.
The effects of dietary ingestion of tomato were studied in mice that had been made hypercholesterolemic by feeding atherogenic diets. Mice which had been fed on the atherogenic diet without tomato for 4 months had significantly increased plasma lipid peroxide, and the vaso-relaxing activity in the aorta induced by acetylcholine (ACh) was harmed when compared with mice fed on a common commercial diet. On the other hand, mice which had been fed on the atherogenic diet containing 20% (w/w) lyophilized powder of tomato showed less increase in the plasma lipid peroxide level, and ACh-induced vaso-relaxation was maintained at the same level as that in normal mice. These results indicate that tomato has a preventive effect on atherosclerosis by protecting plasma lipids from oxidation. (+info)
Protective effects of transient HO-1 overexpression on susceptibility to oxygen toxicity in lung cells.
Rat fetal lung cells (RFL-6) were transiently transfected with a full-length rat heme oxygenase (HO)-1 cDNA construct and then exposed to hyperoxia (95% O2-5% CO2) for 48 h. Total HO activity and HO-1 protein were measured as well as cell viability, lactate dehydrogenase (LDH) release, protein oxidation, lipid peroxidation, and total glutathione to measure oxidative injury. HO-1 overexpression resulted in increased total HO activity (2-fold), increased HO-1 protein (1.5-fold), and increased cell proliferation. Immunohistochemistry revealed perinuclear HO-1 localization, followed by migration to the nucleus by day 3. Decreased cell death, protein oxidation, and lipid peroxidation but increased LDH release and glutathione depletion were seen with HO-1 overexpression. Reactive iron content could not explain the apparent loss of cell membrane integrity. With the addition of tin mesoporphyrin, total HO activity was decreased and all changes in injury parameters were normalized to control values. We conclude that moderate overexpression of HO-1 is protective against oxidative injury, but we speculate that there is a beneficial threshold of HO-1 expression. (+info)
Protective effect of vitamin E on ischaemia-reperfusion injury in ovarian grafts.
Ovarian cortical tissue cryopreservation with subsequent autografting is a potential strategy for the preservation of fertility in patients undergoing systemic chemotherapy and pelvic radiotherapy. Non-vascular implants are first subjected to a period of ischaemia before revascularization and are, therefore, vulnerable to ischaemia-reperfusion injury from reactive oxygen species. Ischaemia-reperfusion injury was investigated during the first week after surgery in murine ovarian grafts and human ovarian xenografts in mice with severe combined immune deficiency (SCID) by measuring total lipid peroxides and malondialdehyde concentrations with a colorometric assay. The effects of administering an antioxidant, vitamin E, on these concentrations were also tested. Products of lipid peroxidation were higher in non-supplemented murine autografts compared with control ovaries (P < 0.05), and were significantly reduced on day 3 by vitamin E administration (P < 0.05). Similarly, in human xenografts, there was a significant reduction in lipid peroxidation with vitamin E administration. These results correspond to a significantly greater total follicle survival in the murine grafts of the supplemented group (45 versus 72%; P < 0.05). They suggest that antioxidant treatment improves the survival of follicles in ovarian grafts by reducing ischaemia-reperfusion injury. (+info)
A mechanistic study of self-inactivation of the peroxidase activity in prostaglandin H synthase-1.
Prostaglandin H synthase (PGHS) is a self-activating and self-inactivating enzyme. Both the peroxidase and cyclooxygenase activities have a limited number of catalytic turnovers. Sequential stopped-flow measurements were used to analyze the kinetics of PGHS-1 peroxidase self-inactivation during reaction with several different hydroperoxides. The inactivation followed single exponential kinetics, with a first-order rate constant of 0.2-0.5 s-1 at 24 degrees C. This rate was independent of the peroxide species and concentration used, strongly suggesting that the self-inactivation process originates after formation of Compound I and probably with Intermediate II, which contains an oxyferryl heme and a tyrosyl radical. Kinetic scan and rapid scan experiments were used to monitor the heme changes during the inactivation process. The results from both experiments converged to a simple, linear, two-step mechanism in which Intermediate II is first converted in a faster step (0.5-2 s-1) to a new compound, Intermediate III, which undergoes a subsequent slower (0.01-0.05 s-1) transition to a terminal species. Rapid-quench and high pressure liquid chromatography analysis indicated that Intermediate III likely retains an intact heme group that is not covalently linked with the PGHS-1 protein. (+info)
Activation of NADPH oxidase by docosahexaenoic acid hydroperoxide and its inhibition by a novel retinal pigment epithelial protein.
PURPOSE: In an earlier study, a novel retinal pigment epithelial protective protein (RPP) was described, which suppresses the superoxide generation of activated polymorphonuclear leukocytes (PMNs). In experimental autoimmune uveitis, docosahexaenoic acid hydroperoxide (22:6OOH) has been shown to be the major lipid peroxidation product in photoreceptors. This hydroperoxide was also found to be chemotactic to PMNs. This study was undertaken to evaluate the activation capability of 22:6OOH in resting PMNs and the possible inhibition of this activation by RPP. METHODS: The 22:6OOH was obtained from pure 22:6 and was purified by thin-layer and high-performance liquid chromatography. Intact rabbit peritoneal PMNs (7-8 X 10(5)) were coincubated with 0.5 microM formyl-methionyl-leucyl-phenylalanine (fMLP), 1.3 microM 22:6OOH, or 5.0 microM 22:6. These systems were coincubated with and without 0.25 microg/ml RPP. From PMN cell-free preparations, the reconstitutes each containing 21 microg plasma membranes and 276 microg cytosolic factors were coincubated with arachidonate, 22:6OOH, or 22:6, each at 100 microM. The inhibition of superoxide production was estimated by adding 0.20 microg/ml RPP. Superoxide generation was measured by superoxide dismutase-inhibitable cytochrome C reduction. RESULTS: In 30 minutes, 22:6OOH-activated PMNs produced 11.10 +/- 0.68 nanomoles superoxide, and production was suppressed 72% by RPP. Under the same conditions, fMLP induced production of 34.6 +/- 2.77 nanomoles superoxide, and RPP inhibited 60% of production. In the PMN cell-free systems, 100 microM 22:6OOH induced 74.7 nanomoles superoxide per milligram plasma membrane proteins per 5 minutes, and RPP suppressed 50% of production. These results were comparable with those generated by arachidonate, a known stimulator for this system. RPP was effective only when it was added before assembly of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. CONCLUSIONS: The inflammation-mediated retinal peroxidation product 22:6OOH significantly activates resting PMNs, either in intact cells or in cell-free preparations, to increase further the release of superoxide from PMNs, thus accelerating inflammation-mediated tissue damage. This profound amplification process seems to be effectively downregulated by an RPE-generated protein RPP. (+info)
Developmental damage, increased lipid peroxidation, diminished cyclooxygenase-2 gene expression, and lowered prostaglandin E2 levels in rat embryos exposed to a diabetic environment.
Previous experimental studies suggest that diabetic embryopathy is associated with an excess of radical oxygen species (ROS), as well as with a disturbance of prostaglandin (PG) metabolism. We aimed to investigate the relationship between these pathways and used hyperglycemia in vitro (embryo culture for 24-48 h) and maternal diabetes in vivo to affect embryonic development. Subsequently, we assessed lipid peroxidation and gene expression of cyclooxygenase (COX)-1 and -2 and measured the concentration of prostaglandin E2 (PGE2) in embryos and membranes. Both hyperglycemia in vitro and maternal diabetes in vivo caused embryonic dysmorphogenesis and increased embryonic levels of 8-epi-PGF2alpha, an indicator of lipid peroxidation. Addition of N-acetylcysteine (NAC) to the culture medium normalized the morphology and 8-epi-PGF2alpha concentration of the embryos exposed to high glucose. Neither hyperglycemia nor diabetes altered COX-1 expression, but embryonic COX-2 expression was diminished on gestational day 10. The PGE2 concentration of day 10 embryos and membranes was decreased after exposure to high glucose in vitro or diabetes in vivo. In vitro addition of NAC to high glucose cultures largely rectified morphology and restored PGE2 concentration, but without normalizing the COX-2 expression in embryos and membranes. Hyperglycemia/diabetes-induced downregulation of embryonic COX-2 gene expression may be a primary event in diabetic embryopathy, leading to lowered PGE2 levels and dysmorphogenesis. Antioxidant treatment does not prevent the decrease in COX-2 mRNA levels but restores PGE2 concentrations, suggesting that diabetes-induced oxidative stress aggravates the loss of COX-2 activity. This may explain in part the antiteratogenic effect of antioxidant treatment. (+info)
Protective effect of flavonoids on endothelial cells against linoleic acid hydroperoxide-induced toxicity.
The protective effect of flavonoids against linoleic acid hydroperoxide (LOOH)-induced cytotoxicity was examined by using cultured endothelial cells. When the cells were incubated with both LOOH and flavonoids, most flavonols protected the cells from injury by LOOH. Flavones bearing an ortho-dihydroxy structure also showed a protective effect against the cytotoxicity of LOOH. However, flavanones had no effect. The structure-activity relationship revealed the presence of either the ortho-di-hydroxy structure in the B ring of the flavonoids or 3-hydroxyl and 4-oxo groups in the C ring to be important for the protective activities. The interaction between flavonoids and a-tocopherol was also examined in this system. Flavonoids that were protective against LOOH-induced cytotoxicity had at least an additive effect on the action of alpha-tocopherol against LOOH-induced damage. (+info)
Age-independent oxidative stress in elderly patients with non-insulin-dependent diabetes mellitus.
Impaired antioxidant defence is implicated in the development of cardiovascular complications in non-insulin-dependent diabetes (NIDDM). However, as many of these patients are elderly, observed changes in antioxidant status may be due to the patient's age rather than their disease. We sampled blood from 47 elderly NIDDM patients (21 male and 26 female; mean age +/- SD, 75.62 +/- 7.97 years), 66 young (30 male and 36 female; 24.52 +/- 4.72 years) and 58 healthy elderly volunteers (17 male and 41 female; 70.74 +/- 4.85 years), and measured the antioxidant glutathione, the marker for free-radical-damage lipid hydroperoxide products (LHP), vitamin E and total antioxidant capacity (TAC). There was a significant increase in LHP in the healthy elderly group compared with the young volunteers (3.14 +/- 1.5 vs. 2.14 +/- 1.38 mumol/l, p < 0.01). The values were much higher in NIDDM patients (7.02 +/- 2.29 mumol/l, p < 0.0001 vs. healthy elderly). There was a reduction in TAC in healthy elderly compared with the young (359.99 +/- 54.82 vs. 471.47 +/- 94.29 mumol/l trolox equivalents, p < 0.0001), but there was no further reduction in NIDDM patients. Similarly, glutathione was reduced to the same degree in healthy elderly and NIDDM patients (0.29 +/- 0.09, 0.30 +/- 0.11 vs. 0.54 +/- 0.19 mumol/l in young volunteers, p < 0.0001). Vitamin E concentrations were comparable in all groups (26.34 +/- 5.39 young volunteers, 31.50 +/- 8.23 healthy elderly and 30.98 +/- 9.03 mumol/l NIDDM patients), but after correction for serum cholesterol there was a significant reduction in the diabetic group compared with the young, but not with the elderly (5.54 +/- 1.55 vs. 6.67 +/- 1.86 vs. 6.31 +/- 1.85 (mumol/l)/(mmol/l), p < 0.01). We have demonstrated an age-dependent reduction in total antioxidant capacity and glutathione defence and an age-independent increase in LHP in elderly patients with NIDDM. Reduced concentrations of vitamin E were demonstrated in NIDDM patients compared with young, but not elderly, volunteers. Increased oxidative damage occurs independently of age in NIDDM patients despite comparable antioxidant defences in this age group. (+info)