Significance of the O6-methylguanine-DNA methyltransferase and glutathione S-transferase activity in the sera of patients with malignant and benign ovarian tumors.

OBJECTIVE: To demonstrate O6-methylguanine-DNA methyltransferase (MGMT) and glutathione S-transferase (GST) activities by analyzing the sera separately obtained from patients with malignant ovarian tumors, benign ovarian tumors, and healthy individuals. STUDY DESIGN: Fourty-nine patients with ovarian cancer, nine patients with benign tumors, and 22 healthy women were included in this study. Blood samples were obtained from all the subjects in the malignant-tumor, benign-tumor, and control groups. Patients with malignant tumors underwent second and third phlebotomies one week following the surgery and after the chemotherapy regimen, respectively. MGMT, GST, and protein levels were measured for each serum sample. GST activity of the samples was measured by the method of Habig et al. using l-chloro-2-4 dinitrobenzene (CDNB) as substrate. MGMT activity was measured by the transfer of radio labelled methyl groups from a prepared MG-DNA substrate to the enzyme fraction of serum. Protein concentration was measured by biuret method. RESULTS: Our work demonstrated that untreated patients with malignant ovarian tumors revealed significantly greater MGMT and GST activities in their sera than did both healthy individuals and patients with benign ovarian tumors, while no significant difference was found between the healthy group and the patients with benign ovarian tumors with respect to their sera MGMT and GST activities. GST activity following chemotherapy was significantly lower than the postoperative values preceding chemotherapy. A relationship between sera MGMT and GST activities, tumor histology and pathology was not found in this study. CONCLUSION: Our work suggests the fact that detection of sera MGMT and GST activities is important in diagnostic and therapeutic approaches during the course of ovarian cancer.

DNA damage and antioxidant defense in peripheral leukocytes of patients with Type I diabetes mellitus.

We determined relationship among DNA damage, nitric oxide (NO) and antioxidant defense in leukocytes of patients with Type 1 DM. DNA damage was evaluated as strand breakage and formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites by the comet assay in DNA from leukocytes of the subjects. Nitrite level, as a product of NO, superoxide dismutase (SOD) activity and glutathione peroxidase (G-Px) activity of the leukocytes were measured by spectrophotometric kits. Serum glucose level and glycosylated haemoglobin (HbA(1c)) were higher in the patients, as expected. Differences in measured parameters between controls and patients were assessed in men and women separately. There was no significant difference between patient and control groups in neither men nor women for nitrite level. Strand breakage and Fpg-sensitive sites were found to be increased, SOD and G-Px activities of the leukocytes were found to be decreased in both men and women of patient group as compared to their respective controls. Significant correlations were determined between strand breakage and HbA(1c) (r = 0.37, P<0.05); Fpg-sensitive sites and HbA(1c) (r = 0.59, P<0.01); Fpg-sensitive sites and glucose (r = 0.45, P<0.02); Fpg-sensitive sites and SOD (r = -0.48, P<0.02); HbA(1c) and SOD (r = -0.50, P<0.02). In conclusion, impaired antioxidant defense in leukocytes of patients with Type 1 DM may be one of the responsible mechanisms for increased DNA damage in those patients.

Evaluation of O6-methylguanine DNA methyltransferase activity in patients with gastric cancer.

O6-Methylguanine DNA methyltransferase (O6-MGMT) reverses DNA alkylation damage produced alkylating agents. O6-MGMT is also a major determinant of cellular resistance to adjuvant chemotherapy with alkylating drugs. O6-MGMT activity was measured in samples from patients with gastric cancer, including tumor, adjacent normal appearing mucosa, and peripheral blood leukocytes (PBL). O6-MGMT activity of PBL from healthy individuals was evaluated as control. There was no significant difference between controls and patients for O6-MGMT activity in PBL. O6-MGMT activity was significantly increased in tumor tissue. Tumor O6-MGMT activity was found to be independent from tumor subgroups and tumor grade. A positive correlation was determined between O6-MGMT activity in tumor and in circulating PBL. The results indicate that O6-MGMT, a defense protein against alkylating agent-mediated carcinogenesis, increased in gastric tumors. This may explain the low response rate to drug combinations, including chloroethylnitrosoureas, exhibited by patients with gastric cancer.

Effect of oxidative stress on glutathione pathway in red blood cells from patients with insulin-dependent diabetes mellitus.

Recently, increased oxidative stress and impaired antioxidant defense have been suggested as a contributory factor for initiation and progression of complications in diabetes. Although glutathione (GSH) and the enzymes included by glutathione redox cycle have an important role for protection of cells against free radical-mediated damage, they may be susceptible to oxidation themselves. We examined the susceptibility of the GSH pathway to oxidation and inactivation in subjects with well-controlled and poorly controlled insulin-dependent diabetes mellitus (IDDM) versus controls and the effect of glycemic control on this susceptibility. Red blood cells (RBCs) were isolated, RBC level of GSH, activity of glutathione peroxidase (G-Px), and glutathione reductase (G-Red) were measured at the baseline and after a 2-hour incubation with hydrogen peroxide. Significant decreases were observed in the GSH level and in the activity of GSH peroxidase and GSH reductase in all the groups after the incubation with hydrogen peroxide. Maximum decrease was observed in the poorly controlled diabetic group for all parameters. This result indicates that the GSH pathway is susceptible to oxidation; and this susceptibility increases in poorly controlled diabetics. Therefore, insufficient antioxidant defense by the GSH pathway may be one of the factors responsible for development of complications in patients with IDDM.

Assessment of DNA base oxidation and glutathione level in patients with type 2 diabetes.

The first aim of the present study was to examine the relationship between reduced glutathione (GSH) level, a powerful cellular antioxidant, and oxidative damage to DNA; and secondly, to see the effect of glycemic control on oxidative DNA damage in type 2 diabetics. We determined GSH level and, using the comet assay, formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites which indicates oxidised guanine in freshly isolated blood from age-matched type 2 diabetics and controls. We found significant differences between men and women in the control group for both GSH and Fpg-sensitive sites. Therefore, we compared the controls and type 2 diabetics separately in men and women. GSH level of whole blood was found to be lower, Fpg-sensitive sites in leukocytes was found to be higher in the both type 2 diabetic men and women, as compared with their respective controls. When the diabetic group was divided into two groups as well-controlled diabetics and poorly-controlled diabetics with respect to glycosylated haemoglobine levels, it was found that Fpg-sensitive sites was significantly higher in the poorly-controlled diabetics than in the well-controlled diabetics in both the men and women. GSH level was lower in the poorly-controlled diabetics but not significantly. Fpg-sensitive sites were found to be moderately correlated with both glycosylated haemoglobine and GSH, and weakly correlated with glucose. Data indicate that decreased GSH level may be a contributory factor for enhanced oxidative DNA damage in type 2 diabetics; and chronic hyperglycemia derived from poorly-controlled diabetic conditions may induce oxidative DNA damage in these patients.

Susceptibility of glutatione and glutathione-related antioxidant activity to hydrogen peroxide in patients with type 2 diabetes: effect of glycemic control.

OBJECTIVES: The aim of the present study was to examine the susceptibility of glutathione (GSH) and glutathione related antioxidant enzymes to oxidation in type 2 diabetic patients with and without glycemic control. DESIGN AND METHODS: Erythrocyte glutathione level and activities of glutathione peroxidase (G-Px), glutathione reductase (G-Red) and glutathione S-transferase (GST) in controls, well controlled and poorly controlled type 2 diabetics were measured by spectrophotometric assays before and after the incubation in vitro with H2O2. RESULTS: GSH level, G-Px and G-Red activities decreased but GST activity increased in the erythrocytes from all the groups after the incubation with H2O2. Percentage of decrease in GSH was independent from glycemic control, whereas the percentage of decreases in G-Px and G-Red was related to glycemic control. The percentage of increase in GST was found to be independent from diabetes. CONCLUSIONS: GSH and GSH-related antioxidant enzymes in human erythrocytes are susceptible to oxidation, particularly, G-Px and G-Red which were found to be more susceptible to oxidation in erythrocytes from poorly controlled type 2 diabetic patients.

Superoxide dismutase activity and glutathione system in erythrocytes of men with Behchet's disease.

In order to clarify whether erythrocyte superoxide dismutase (SOD) activity and glutathione system including reduced glutathione (GSH), glutathione peroxidase (G-Px), glutathione reductase (G-Red), glutathione S-transferase (GST) are impaired in men with Behchet's disease (BD) at the first diagnosed time, erythrocyte SOD activity, GSH level, activities of G-Px, G-Red and GST were determined in men with new diagnosed BD. Erythrocyte GSH level, G-Px and G-Red activities were found to be lower, SOD activity was found to be higher in the patients as compared the controls. There was no significant difference between patients and controls for GST activity. Significant positive correlations between GSH and G-Px, GSH and G-Red; significant negative correlations between GSH and SOD, G-Px and SOD, G-Red and SOD were determined. It was concluded that erythrocyte SOD activity and glutathione system are altered in men with new diagnosed BD. It was concluded that these alterations may be a contributory factor for tissue damage associated with BD.