A. Süha Yalçin

Different kinds of reactive oxygen and nitrogen species were detected in colon and breast tumors

Several studies have shown the involvement of reactive oxygen species (ROS; O2*-, hypochlorite, hydroxyl radical, hydrogen peroxide) in carcinogenesis. With certain pathologies, nitric oxide (NO) is formed and can interact with superoxide radical (O2*-) resulting in the propagation of the highly reactive species, peroxynitrite. In order to study the molecular mechanisms underlying the ability of reactive oxygen and nitrogen species (RONS) to mediate carcinogenesis, we have measured ROS, NO, and peroxynitrite content of cancerous tissues obtained from colon and breast carcinoma cases by chemiluminescence technique. All ROS were significantly increased in cancerous colon tissues with hypochlorite making the most important contribution and suggesting the role of inflammatory cells. NO was also increased and the peroxynitrite concentration was higher in cancerous samples. For breast carcinoma cases, only O2*- was significantly increased. Hypochlorite was not detected excluding the contribution of inflammatory cells. NO concentrations were not significantly different, therefore, ROS might originate by change in the redox state of the tissue.

Lipid peroxidation and osmotic fragility of red blood cells in sleep-apnea patients

BACKGROUND Obstructive sleep apnea (OSA) refers to the occurrence of episodes of complete or partial pharyngeal obstruction with oxyhemoglobin desaturation during sleep. These hypoxia/reoxygenation episodes may cause generation of reactive oxygen species. Reactive oxygen species are toxic to biomembranes and may lead to the peroxidation of lipids. We tested the hypothesis that obstructive sleep apnea is linked to increased oxidative stress and lipid peroxidation. In order to identify target tissue/cell damage, we studied the osmotic fragility of red blood cells. METHODS Six subjects polysomnographically diagnosed as obstructive sleep apnea syndrome and 10 controls were included. After all subjects gave written informed consent, blood samples were collected in the morning between 08:00 and 09:00 a.m. following polysomnography. Blood samples were immediately transferred to the laboratory. Glutathione, lipid peroxidation and osmotic fragility of red blood cells were measured manually. RESULTS Mean glutathione and lipid peroxidation concentrations of patients were not different than those of control subjects (105.6+/-38.6 U/g Hb and 3.1+/-2.3 nmol MDA/l vs. 100.6+/-62.1 U/g Hb and 3.2+/-2.8 nmol MDA/l, respectively). In both groups, osmotic fragility of red blood cells was not changed. CONCLUSION The present study failed to support the hypothesis that obstructive sleep apnea is linked with increased oxidative stress and lipid peroxidation.

Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment.

Whey proteins were isolated from whey powder by a combination of gel exclusion chromatography and protease (pepsin or trypsin) treatment. Whey solution (6g/dl) was applied to Sephadex G-200 column chromatography and three fractions were obtained. Gel electrophoresis (SDS-PAGE) was used to identify the fractions; the first one contained immunoglobulins and bovine serum albumin, the second contained beta-lactoglobulin and alpha-lactalbumin whereas the third fraction contained small peptides. We have also subjected the whey filtrate to proteases (pepsin and trypsin). Treatment with proteases showed that beta-lactoglobulin can be obtained after hydrolysis of the second fraction with pepsin. When the whey filtrate was treated with pepsin and then applied to Sephadex G-200 column chromatography three fractions were obtained; the first one was bovine serum albumin, the second was beta-lactoglobulin and the third fraction contained small peptides. After trypsin treatment only two fractions were obtained; the first one was serum albumin and the second fraction was an alpha-lactalbumin rich fraction. We have determined the antioxidant activity of the fractions using an assay based on the measurement of superoxide radical scavenging activity. Our results showed that among the three fractions, the first fraction had the highest superoxide radical scavenging activity. Also, protease treatment of the second fraction resulted in an increase in the antioxidant activity.

Increased erythrocyte susceptibility to lipid peroxidation in human Parkinson's disease

Recent evidence suggests that among the factors that lead to neurodegenerative changes in Parkinsons disease are stimulation of lipid peroxidation and deficiency of glutathione and glutathione peroxidase in substantia nigra. We have investigated the effect of neurodegenerative changes on plasma and erythrocytes of patients with Parkinsons disease and compared the results with those of age-matched controls. Both plasma lipid peroxide levels and erythrocyte susceptibility to lipid peroxidation were significantly increased in Parkinsons disease. Erythrocyte fragility tests revealed that in 35% of the patients there was increased fragility. In addition, erythrocyte catalase activities were not changed whereas glutathione levels and glutathione peroxidase activities were decreased in Parkinsons disease. Our results suggest that erythrocyte membrane integrity may be impaired in Parkinsons disease.

Effects of N-acetylcysteine on myocardial ischemia-reperfusion injury in bypass surgery

Myocardial ischemia–reperfusion injury may complicate coronary artery bypass grafting (CABG) operations. N-Acertylcysteine (NAC) had antioxidant and microcirculatory effects, and inhibits neutrophil aggregation. The aim of this study was to determine the effects of NAC in limiting myocardial ischemia–reperfusion injury in CABG operations. Twenty patients undergoing elective coronary bypass operation with cardiopulmonary bypass were enrolled and randomly assigned to two groups: a control group operated with a routine CABG protocol, and one where NAC was administered intravenously during the operation (NAC group). Blood samples from coronary sinus for tumor necrosis factor-α assay, myocardial biopsy specimens for chemiluminescent luminol, and lucigenin measurements of reactive oxygen species were taken. The luminol (specific for •OH, H2O2, and HOCl− radicals) and lucigenin (specific for O2•−) levels and the difference ratios after reperfusion were significantly lower in the NAC group. Tumor necrosis factor-α levels increased in the control group but, in contrast, a significant decrease was detected in the NAC group (P < 0.01). Creatine kinase-MB levels at 6 and 12 hours were singnificantly lower in the NAC group (P = 0.02). N-Acetylcysteine has potential effects to limit ischemia reperfusion injury during CABG operations. We believe that its effects on clinical outcome may be more apparent in patients prone to ischemia–reperfusion injury.

Effect of Mitochondrial Electron Transport Chain Inhibitors on Superoxide Radical Generation in Rat Hippocampal and Striatal Slices

In this study, we have compared the generation of superoxide radical in rat hippocampal and striatal slices in the presence of specific mitochondrial electron transport chain (ETC) inhibitors (complexes I and III) under control and depolarization conditions [incubation in artificial cerebrospinal fluid (ACSF) or depolarizing ACSF (dACSF), respectively]. Superoxide radical generation was increased in both ACSF- and dACSF-incubated hippocampal and striatal slices when rotenone and antimycin A were added to the incubation medium. The increase in superoxide radical was dependent on the concentration of ETC inhibitors under control, but not depolarization conditions. Rotenone was found to be more effective than antimycin A in producing superoxide radical from hippocampal and striatal slices. Our results also showed that hippocampal slices were more sensitive to ETC inhibitors compared with striatal slices. Thus, different regions of the brain seem to differ in their capacity to generate free radicals and vulnerability to oxidative stress conditions. This difference should be considered in developing therapeutic modalities against oxidative stress-related disorders and neurodegeneration.

The effect of vitamin E therapy on plasma and erythrocyte lipid peroxidation in chronic hemodialysis patients.

The effect of vitamin E therapy on plasma and erythrocyte (RBC) lipid peroxidation was investigated in patients undergoing chronic hemodialysis. Before vitamin E therapy, both plasma and RBC lipid peroxidation values of chronic hemodialysis patients were significantly higher than those of healthy controls. Treatment with vitamin E (300 mg/day) for 1 month resulted in a significant decrease of lipid peroxidation. Vitamin E therapy may be a promising approach to prevent peroxidation of membrane lipids in chronic renal failure.

BQ-123, a specific endothelin (ETA) receptor antagonist, prevents ischemia-reperfusion injury in kidney transplantation

We studied the effects of the specific endothelin (ETA) receptor antagonist, BQ-123, on reperfusion injury in a rat model of kidney transplantation. First, Sprague-Dawley rats were divided into three groups: a sham nephrectomy (SNEPH), an autotransplantation (AUTO-Tx), and an allotransplantation (ALLO-Tx) group. In a fourth group, ALLO-Tx+BQ, allografts were flushed with 20 μg BQ-123 containing cold Ringers lactate before transplantation. For the allograft groups, kidneys from white Wistar albino rats were transplanted into allogeneic Sprague Dawley recipients. Grafts were allowed 120 min of reperfusion after 40 min of cold ischemia. ET-1,2 plasma concentrations in the renal venous blood, and kidney tissue prostaglandin (PG) E2 and leukotriene (LT) B4 levels were studied. Diene conjugates (DC), hydroxyalkanals (HAA), hydroxyalkenals (HAE) and malondialdehyde (MDA) levels, as the products of lipid peroxidation, and protein carbonyls (PC) and protein sulphydryls (PS), as the parameters of protein oxidation, were also analyzed in the kidney tissue. Plasma ET concentrations increased significantly in the AUTO-Tx and ALLO-Tx groups (P<0.05 and P<0.01, respectively) but this increase was reversed in the ALLO-Tx+BQ group. None of the lipid peroxidation products except DCs (P<0.05) increased in the AUTO-Tx group, whereas they all increased in the ALLO-Tx group (P<0.01). Protein oxidation parameters also changed significantly (P<0.01) in the ALLO-Tx group but did not in the AUTO-Tx group (P<0.05). The differences in PGE2 and LTB4 levels were not significant. Histopathologic examination revealed prominent glomerular and tubular injury in the AUTO-Tx and ALLO-Tx groups but less in the ALLO-Tx+BQ group. In the last group, all parameters of lipid peroxidation (P<0.001 for all) and PCs decreased, and PSs were preserved (P<0.001 for both) when compared with the AUTO-Tx and ALLO-Tx groups. We conclude that BQ-123, in addition to inhibiting the binding of ET-1,2 to the ETA receptor, may also inhibit the release and/or synthesis of ET-1,2 and prevent reperfusion injury in kidney transplantation.

Evaluation of oxidant stress in chronic hemodialysis patients: use of different parameters

Patients with chronic renal failure, particularly those undergoing regular dialysis treatment (RDT) are candidates for free radical damage. It is difficult to quantitate free radicals because of their short half-lives and reactive nature. Therefore, indirect methods measuring products of lipid peroxidation and protein oxidation are preferred. The present study displays a profile of lipid peroxidation and protein oxidation parameters, which are more sensitive and specific than the widely used method measuring thiobarbituric acid reactive substances (TBARS), adapted to the plasma and erythrocyte samples of RDT patients. We have observed increased levels of plasma and erythrocyte lipid peroxidation and also demonstrated increased protein oxidation in erythrocyte membranes of RDT patients.

Low dose MK-801 protects against iron-induced oxidative changes in a rat model of focal epilepsy

We have used chemiluminescence measurements to examine the relationship between free radical formation and excitotoxicity in a post-traumatic epilepsy model. For this purpose, seven days after injecting iron in rat brain cortices, we measured luminol- and lucigenin-enhanced chemiluminescence in different brain regions (ipsilateral cortex, contralateral cortex, hypothalamus and hippocampus). In all brain regions (except contralateral cortices) both luminol- and lucigenin-enhanced chemiluminescence were increased in iron-injected group compared to saline-injected control group. These increases returned to control values in iron-injected rats pretreated with MK-801. Our results suggest that both free radicals and excitatory amino acids play important roles in the development of post-traumatic epilepsy and that MK-801 has protective effects against iron-induced chemiluminescence formation.