Jan Błaszczyk

Evaluation of oxidative stress markers in pathogenesis of primary open-angle glaucoma

Primary open-angle glaucoma (POAG) is the leading cause of blindness in the industrial countries. It is reported that oxidative stress might be an important risk factor in the pathogenesis of POAG. Forty subjects including 20 patients with open-angle glaucoma (9 men and 12 women, mean age 61.8±12.1yr) and 20 controls without glaucoma symptoms (9 men and 12 women, mean age 58.1±17.7yr) were enrolled in our study. The main aim of the work was to evaluate oxidative stress markers in the pathogenesis of open-angle glaucoma. In our work the activity of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) as well as the total antioxidant status (TAS) was estimated. An alkaline comet assay was used to measure DNA damage of strand breaks (SB), oxidized purines as glicosylo-formamido-glicosylase (Fpg) sites, and oxidized pirmidines as endonuclease III (Nth) sites. We measured endogenous as well as exogenous DNA damage after 10μM hydrogen peroxide treatment (H(2)O(2)). We did not observe any statistical changes of DNA strand break lesion in examined POAG patients according to healthy subjects (P>0.05). However, either endogenous (P<0.01) or exogenous (P<0.001) levels of oxidative DNA damage in POAG patients were found to be statistically higher than controls. A significant decrease of antioxidant enzymes: CAT (P<0.001), SOD (P<0.05), and GPX (P<0.001) and a non-statistical decrease of TAS status (P>0.05) in glaucoma patients according to controls were also indicated. In conclusion our data revealed that oxidative stress had a pathogenic role in primary open-angle glaucoma. Therefore, we suggested that the modulation of a pro-oxidant/antioxidant status might be a relevant target for glaucoma prevention and therapy.

Alterations in the erythrocyte plasma membranes in patients with alcohol-induced liver cirrhosis – preliminary results

Introduction Conformations of membrane proteins, membrane fluidity of erythrocytes in patients with AILC were studied with the use of electron paramagnetic resonance and spectrophotometric methods. The concentration of substances reacting with thiobarbituric acid was also determined. The aim of the study was to recognize the nature, level and causes of changes in the structure of erythrocytary membrane observed in erythrocytes of patients compared to erythrocytes from healthy controls. Material and methods Spin labels: MSL and ISL binding covalently to thiol groups of membrane cytoskeleton proteins were used to analyse modifications occurring in erythrocytary membrane proteins. Doxyl derivatives of fatty acids: 5-DS, 12-DS and 16-DS binding hydrophobically to erythrocytary membrane were used as spin labels for the analysis of erythrocyte membrane lipid fluidity. Results Modification of membrane cytoskeleton proteins and increase of membrane lipids fluidity were observed in erythrocytes of the investigated patients. An increase of the concentration of substances reacting with thiobarbituric acid was also confirmed in the erythrocytes of AILC patients. Conclusions Observed disorders in the structure of erythrocyte cytoskeleton proteins in patients, which might developed as a consequence of oxidative stress may be conformation changes in the structure of proteins which affect membrane cytoskeleton. The differences in the structure of membrane proteins could be associated with an increase in membrane lipids fluidity. Increased fluidity of erythrocyte membrane may be a result of disorders in protein-lipid interaction or membrane lipid peroxidation activity.

Impaired nucleotide excision repair pathway as a possible factor in pathogenesis of head and neck cancer.

Tobacco smoking is one of the major risk factors in pathogenesis of head and neck squamous cell carcinomas (HNSCC). Many of the chemical compounds present in tobacco are well-known carcinogens which form adducts with DNA. Cells remove these adducts mainly by the nucleotide excision repair pathway (NER). NER also eliminates a broad spectrum of pyrimidine dimers (CPD) and photo-products (6-4PP) induced by UV-radiation or DNA cross-links after cisplatin anti-cancer treatment. In this study DNA damage and repair was examined in peripheral blood lymphocytes obtained from 20 HNSCC patients and 20 healthy controls as well as HTB-43 larynx and SSC-25 tongue cancer cell lines. DNA repair kinetics in the examined cells after cisplatin or UV-radiation treatment were investigated using alkaline comet assay during 240min of post-treatment incubation. MTT assay was used to analyse cell viability and the Annexin V-FITC kit specific for kinase-3 was employed to determine apoptosis after treating the cells with UV-radiation at dose range from 0.5 to 60J/m(2). NER capability was assessed in vitro with cell extracts by the use of a bacterial plasmid irradiated with UV-light as a substrate for the repair. The results show that lymphocytes from HNSCC patients and HTB-43 or SSC-25 cancer cells were more sensitive to genotoxic treatment with UV-radiation and displayed impaired DNA repair. Also evidenced was a higher rate of apoptosis induction after UV-radiation treatment of lymphocytes from the HNSCC patients and the HTB-43 cancer cells than after treatment of those from healthy donors. Finally, our results showed that there was a significant decrease in NER capacity in HTB-43 or SSC-25 cancer cells as well as in peripheral blood lymphocytes of HNSCC patients compared to controls. In conclusion, we suggest that the impaired NER pathway might be a critical factor in pathogenesis of head and neck cancer.

Plasma antioxidative activity during atorvastatin and fluvastatin therapy used in coronary heart disease primary prevention.

We estimated the effect of atorvastatin and fluvastatin on plasma antioxidative activity used in coronary heart disease (CHD) primary prevention. Anti‐oxidative activity of blood plasma was determined by Bartosz et al. method [Curr. Top. Biophys. (1998)22:11–13], based on reduction of preformed cation radical of 2,2,azinobis(3‐ethylbenzothiazoline‐6‐sulphonic acid) by blood plasma. The study comprised 35 patients with CHD risk who were randomly divided into two groups. The atorvastatin group comprised 17 patients who were administered the drug orally in a daily dose of 10 mg and the fluvastatin group consisted of 18 patients on an oral dose of 40 mg once daily. The control group comprised 12 healthy subjects with no drug administration. Blood samples were collected from cubital vein before and after 6‐week therapy. Significantly (P < 0.05) increased – in comparison with the initial values – antioxidative activity of blood plasma was found in atorvastatin and fluvastatin groups after 6‐week therapy. Moreover, the increase in antioxidative plasma activity in atorvastatin group was significantly higher in comparison with the fluvastatin group. The results of our study have demonstrated that atorvastatin and fluvastatin have an additional mechanism independent of the effect on cholesterol concentration. Thus, we presume that administration of these statins in CHD risk patients may have a beneficial effect.

Estimation of cell membrane properties and erythrocyte red-ox balance in patients with metabolic syndrome

Metabolic syndrome (MS) is associated with occurrence of the many cardiovascular risk factors such as atherogenic dyslipidemia, visceral fat distribution, arterial hypertension and pro-thrombotic and pro-inflammatory status. In our study the effect of disorders that appear in MS on red-ox balance and erythrocyte cell membrane properties were estimated. The study comprised 50 patients with diagnosed MS and in 25 healthy subjects. Content of thiobarbituric acid reactive substances (TBARS) and catalase, superoxide dismutase and glutathione peroxidase activity were estimated in red blood cells. Moreover, conformation status of membrane proteins, membrane fluidity and osmotic fragility were evaluated. MS was found to manifest: (1) the increase of the concentration of TBARS in erythrocytes with no statistically significant differences in antioxidant enzymes activity, (2) disorders in the structure of erythrocyte cytoskeleton proteins, (3) the increase in membrane lipids fluidity at the depth of 5th and 12th carbon atom of fatty acid hydrocarbon chain and significantly decreased fluidity at the depth of 16th carbon atom, (4) increased erythrocyte osmotic fragility.

Do catalase and glutathione peroxidase protect blood platelets from lipid peroxidation in multiple sclerosis

Purpose: Catalase (cat) and glutathione peroxidase (GSH-Px) activities and thiobarbituric acid reactive substances (TBARS) concentration in blood platelets were determined in patients with multiple sclerosis (MS). Methods: The study was carried out in a group of 36 patients, men and women, aged 21-50 years old. They were divided into groups dependently on the degree of motor disability and duration of the disease. The control group included 15 healthy individuals of similar age to the study group. The activity of catalase was estimated according to the method by Beers and Sizer. Glutathione peroxidase activity was determined by the method of Sedlak and Lindsay, modified by Little and O’Brien. The concentration of thiobarbituric acid reactive substances in blood platelets was carried out using the method by Placer et al. Results: We observed a lower level of TBARS concentration in platelets of MS patients than in control group with an enhanced activities of both antioxidative enzymes on the basis of disability degree and duration of the disease. Conclusions: Catalase and glutathione peroxidase protect blood platelets from lipid peroxidation process in multiple sclerosis patients and may play a role in the course of the disease. It may also suggest involvement of lipid peroxidation in the activity of multiple sclerosis.