Ela Kadioglu

Detection of oxidative DNA damage in lymphocytes of patients with Alzheimer's disease

Oxidative damage to DNA may play an important role in both normal ageing and in neurodegenerative diseases. The deleterious consequences of excessive oxidations and the pathophysiological role of reactive oxygen species have been intensively studied in Alzheimers disease. Although the role of oxidative stress in the aetiology of Alzheimers disease is still not clear, the detection of an increased damage status in the cells of patients could have important therapeutic implications. The levels of oxidative damage in peripheral lymphocytes of 24 Alzheimers disease patients and of 21 age-matched controls were determined by comet assay applied to freshly isolated blood samples with oxidative lesion-specific DNA repair endonucleases (endonuclease III for oxidized pyrimidines, formamidopyrimidine glycosylase for oxidized purines). It was demonstrated that Alzheimers disease is associated with elevated levels of oxidized pyrimidines and purines (p<0.0001) as compared with age-matched control subjects. It was also demonstrated that the comet assay is useful as a biomarker of oxidative DNA damage when used with oxidative lesion-specific enzymes.

The role of GSTM1, GSTT1, GSTP1, and OGG1 polymorphisms in type 2 diabetes mellitus risk: a case-control study in a Turkish population.

The aim of the present study was to investigate the role of some polymorphisms in GSTs (GSTM1, GSTT1 and GSTP1) which are very important protective mechanisms against oxidative stress and in OGG1 gene which is important in DNA repair, against the risk of type 2 diabetes mellitus (T2DM). 127 T2DM and 127 control subjects were included in the study. DNA was extracted from whole blood. Analyses of GSTM1 and GSTT1 gene polymorphisms were performed by allele specific PCR and those of GSTP1 Ile105Val and OGG1 Ser326Cys by PCR-RFLP. Our data showed that GSTM1 null genotype frequency had a 2-6 times statistically significant increase in a patient group (OR=3.841, 95% CI=2.280-6.469, p<0.001) but no significance with GSTT1 null/positive and GSTP1 Ile105Val genotypes was observed. When T2DM patients with OGG1 Ser326Cys polymorphism were compared with patients with a wild genotype, a 2-3 times statistically significant increase has been observed (OR 1.858, 95% CI=1.099-3.141, p=0.021). The combined effect of GSTM1 null and OGG1 variant genotype frequencies has shown to be statistically significant. Similarly, the risk of T2DM was statistically increased with GSTM1 null (OR=3.841, 95% CI=2.28-6.469), GSTT1 null+GSTP1 (H+M) (OR=4.118, 95% CI=1.327-12.778) and GSTM1 null+OGG1 (H+M) (OR=3.322, 95% CI=1.898-5.816) and GSTT1 null+OGG1 (H+M) (OR=2.179, 95% CI=1.083-4.386) as compared to the control group. According to our study results, it has been observed that the combined evaluation of GSTM1-GSTT1-GSTP1 and OGG1 Ser326Cys gene polymorphisms can be used as candidate genes in the etiology of T2DM, especially in the development of T2DM.

Genoprotective role of vitamin E and selenium in rabbits anaesthetized with sevoflurane

In this study, genotoxic effects of repeated sevoflurane anaesthesia were investigated in rabbits with or without antioxidant supplementation. Twenty-one New Zealand male rabbits were included in the study and randomized into three groups as: placebo treated (Group I), vitamin E supplemented (Group II) and selenium supplemented (Group III). Vitamin E and selenium were given intraperitoneally for 15 days before anaesthesia treatment. Anaesthesia was administered using 3% sevoflurane in 4 L/min oxygen for a 3-hour period and continued for 3 days. Blood samples were collected before anaesthesia (Sample 1), after the first, second and third days of sevoflurane administration (Sample 2, Sample 3 and Sample 4 respectively) and the last samples were taken 5 days after the last sevoflurane administration (Sample 5). Genotoxic damage was examined using the comet assay. The degree of damage is assessed by grading the cells into three categories of no migration (NM), low migration (LM) and high migration (HM) depending on the fraction of DNA pulled out into the tail under the influence of the electric field. The number of comets in each sample was calculated (1 × number of comets in category NM + 2 × number of comets in category LM + 3 ×number of comets in category HM) and expressed as the total comet score (TCS), which summarizes the damage frequencies. In Group I, a significant increase in the mean TCSs was observed for Samples 3 and 4 as compared with Sample 1. However, there were no significant differences between Samples 1, 2 and 5. The mean TCS of Sample 4 was significantly higher than Sample 1, 2 and 3 in Group II. Group III demonstrated no significant mean TCSs for any experimental conditions. Statistical differences were also observed between the groups with significant P values. This experimental study points out the presence of DNA damage with repeated sevoflurane anaesthesia and the genoprotective role of antioxidant supplementation on DNA damage in mononuclear leukocytes of rabbits by highly sensitive comet assay.

Assessment of genotoxic damage in nurses occupationally exposed to anaesthetic gases or antineoplastic drugs by the comet assay.

The potential mutagenic/carcinogenic action of waste anaesthetic gases and antineoplastic drugs in occupationally exposed human populations has been previously reported in several studies. Antineoplastic agents discovered in the first two decades of cancer therapy (1950 to 1970) largely interact with DNA or precursors, inhibiting the synthesis of DNA or causing irreparable damage to DNA itself. Considering the mechanisms of the antineoplastic drugs that are used, it is not surprising that many persons involved in health care, especially nurses, are worried about the health effects of these drugs. Experimental and epidemiological studies suggest that genotoxic effects can arise from inhalation anaesthetics. Due to their widespread use in operating rooms, there is a great concern that operating room personnel as well as patients might be at health risk from anaesthetics. The aim of the present study was to assess the possible genotoxic risk, by the alkaline comet assay, in the peripheral blood lymphocytes of nurses who are handling antineoplastic drugs or are exposed to waste anaesthetic gases.

The role of oxidative DNA damage, DNA repair, GSTM1, SOD2 and OGG1 polymorphisms in individual susceptibility to Barrett’s esophagus:

Determination of the genetic alterations, which play a role in the etiology of Barrett’s esophagus (BE), could help identify high-risk individuals for esophageal adenocarcinoma (EA). The aim of the present study was to investigate the role of oxidative DNA damage, glutathione (GSH) concentration as oxidative stress parameters and DNA repair capacity, GSTM1, SOD1 Ala16Val and OGG1 Ser326Cys genetic polymorphisms as individual susceptibility parameters in the etiology of BE. The study groups comprised BE patients who were clinically diagnosed (n = 40) and a healthy control group (n = 40). Basal DNA damage, pyrimidine and purine base damage after H2O2 induction, H 2O2 sensitivity, DNA repair capacity, oxidized pyrimidine and purine base damage repair were evaluated in peripheral blood lymphocytes with a modified comet assay using specific endonucleases (Endo III and Fpg). Polymerase chain reaction—restriction length polymorphism (PCR-RFLP)-based assays were used for genotyping. The patient group showed elevated levels of basal DNA damage, pyrimidine base damage and H2O2 sensitivity as compared to controls (p < .05). DNA repair capacity, oxidized pyrimidine and purine base damage repair capacity, were not statistically different between patients and controls. GSH concentration was found to be significantly lower in smoking patients than in the controls (p < .05). None of the genetic variations changed the risk of having BE disease. However, patients carrying the variant OGG1 Cys allele showed elevated levels of pyrimidine base damage as compared to patients carrying the wild-type OGG1 Ser (p < .05). The results of this study point to a role of oxidative DNA damage in BE. However, DNA repair capacity, GSTM1, SOD1 Ala16Val and OGG1 Ser326Cys genetic polymorphisms appeared to play no role in the individual susceptibility to this disease.

Determination of DNA damage by alkaline halo and comet assay in patients under sevoflurane anesthesia

In this study, we report data on the possible genotoxic effect of inhalation anesthetic sevoflurane (SVF) by comparing two techniques, comet and alkaline halo assay, in peripheral blood lymphocytes (PBL) of patients before, during, and after anesthesia and in controls. DNA single strand breaks were detected in PBL of malignant breast cancer diagnosed patients (stage II–III), who were undergoing mastectomy. Blood samples were taken before the induction of anesthesia, at 120 min of SVF anesthesia, and on the postoperative fifth day. The nuclear spreading factor (NSF) for each cell was assessed by alkaline halo assay, and the total comet score (TCS) was evaluated by comet assay. A statistically significant increase (P < 0.0001) was observed in the mean NSF at 120 min of anesthesia (38.24 ± 14.14) as compared with samples before anesthesia (12.33 ± 6.14), and the mean NSF was significantly decreased after the postoperative fifth day (17.89 ± 9.44). Similar results were obtained by the comet assay with significant increase (P < 0.0001) in DNA damage at 120 min of anesthesia (79.66 ± 15.28) as compared with samples before anesthesia (36.30 ± 11.39). The DNA damage was almost with the preoperative damage rates after the fifth day of anesthesia (43.40 ± 12.19). In conclusion, the study points out a reversible genotoxic effect of SVF and the similar DNA damage levels obtained by comet and alkaline halo assay indicate that although halo assay has a completely different principle, it can conveniently be utilized for the assessment of DNA single strand breakage in individual mammalian cells with its experimental advantages.

Oxidative DNA damage and total antioxidant status in rats during experimental gram-negative sepsis

Sepsis and septic shock remains as leading cause of death in adult intensive care units. It is widely accepted that gram-negative bacteria and their endotoxins cause sepsis and septic shock, predominantly. Enhanced generation of reactive oxygen species may be responsible for tissue injury in septic shock and endotoxemia. The aim of this study was to assess oxidative DNA damage and the total antioxidant status (TAS) in peripheral lymphocytes of rats during different intraperitoneal gram-negative sepsis stages. Adult male Sprague-Dawley rats were divided randomly into four groups. Control group was intraperitoneally inoculated with 2 mL of pyrogene-free saline (Group I, n = 6), and the other rats received an intraperitoneal inoculum with 2 mL of saline containing 2 × 108 CFU of Escherichia coli. The animals were killed at time zero (Group I, n = 6), at 6th (Group II, n = 7), 12th (Group III, n = 7), and 24th (Group IV, n = 7) hour after the E. coli inoculation. Oxidative DNA damage in peripheral lymphocytes of rats was evaluated by modified comet assay (single-cell gel electrophoresis). Formamidopyrimidine DNA glycosylase (Fpg) and Endonuclease III (Endo III) were used to detect oxidized purines and pyrimidines, respectively. Total antioxidant quantification was carried out using ABTS+ (2,2′-Azino-di-[3 ethylbenzthiazoline sulphonate]) radical formation kinetics (Randox kit) in serum samples. Significant elevations of basal levels of strand breaks (SB) in Group IV were observed as compared with Group I, II, and III. There was a significant increase in Fpg sites in Group III as compared with Group I and II. However, there was no significant difference in terms of Endo III sites in any of the groups. Although the TAS was decreased with the stages of sepsis, this moderate decrease was significant in only Group IV as compared with Group I. There was no statistically significant correlation between DNA damage and TAS for any of the groups.

Assessment of Individual Susceptibility to Baseline DNA and Cytogenetic Damage in a Healthy Turkish Population: Evaluation with Lifestyle Factors

BACKGROUND Cytogenetic biomarkers are most frequently used well-established endpoints in human population studies with their sensitivity for measuring exposure to genotoxic agents. They have an important role as early predictors of cancer risk. Identification of individual genotypes of metabolic gene polymorphisms helps to understand the modulation of cancer susceptibility by environmental exposures, such as cigarette smoking and other lifestyle factors. AIM To evaluate individual susceptibility to chemicals, we determined individual DNA damage related to glutathione S-transferase (GST) genotypes (GSTM1, GSTT1, and GSTP1) in a Turkish population. METHODS Peripheral blood lymphocytes (PBL) and DNA samples of 127 subjects were analyzed for the presence of DNA damage, using single-cell gel electrophoresis (the Comet assay), and for cytogenetic parameters (chromosomal aberrations [CAs], bleomycin-induced CA, and a cytokinesis-blocked micronucleus assay), and the polymerase chain reaction/restriction fragment length polymorphism method, respectively. RESULTS Individuals carrying a GSTT1-null allele showed higher frequencies of CA and micronucleus (MN) (p=0.026, p=0.003, respectively), whereas the GSTM1-null and GSTP1 mutant genotypes did not show any differences in cytogenetic parameters. Our findings demonstrated that none of the lifestyle factors (smoking, alcohol drinking, dietary habits, vitamin intake, and physical activity), except for vitamin intake (p=0.002), were significantly associated with the studied cytogenetic parameters. CONCLUSION Our results suggest that the GSTT1 gene polymorphism may influence the baseline cytogenetic frequency in a healthy population.

Determination of DNA damage after exposure to inhalation anesthetics in human peripheral lymphocytes and sperm cells in vitro by comet assay

In this study, genotoxic activities of four halogenated anesthetics (halothane, isoflurane, sevoflurane and desflurane) were investigated in human peripheral blood lymphocytes (PBLs) and sperm cells in vitro by alkaline comet assay. For this purpose, sperm or lymphocyte suspension was exposed to different concentrations (0.1 mM, 1 mM, 10 mM and 100 mM) of anesthetic agents and 1% dimethyl sulfoxide (DMSO) or phosphate-buffered saline (PBS) as controls. The DNA strand breaks as well as alkali-labile sites were measured as percentage tail intensity with comet assay. The results of this study demonstrate that all analyzed drugs were capable of inducing DNA damage on PBLs in a dose-dependent manner in vitro. However, the results in sperm cells were slightly different since we did not observe any genotoxic effect for desflurane in any of the exposure doses, and the genotoxic effect of halothane was not dose dependent. This experimental study points out to the presence of DNA damage after exposure to halogenated anesthetics in both PBLs and sperm cells, although this effect seems to be higher in PBLs.

DNA Damage, Glutathione, and Total Antioxidant Capacity in Anesthesia Nurses

ABSTRACT The possibilities of a potential mutagenic/carcinogenic action of waste anesthetic gases in occupationally exposed anesthesia personnel have been previously reported in several studies. The aim of this study was to assess the DNA damage, reduced glutathione (GSH), and total antioxidant capacity (TAC) in anesthesia nurses. DNA damage was determined with comet assay, GSH levels were measured spectrophotometrically, and TAC was determined by using Randox kit. Anesthesia nurses (n = 40) showed increased DNA damage in terms of mean percentage of the total DNA in the comet tail compared to controls (n = 40) (p < .001). Mean TAC and GSH levels of the anesthesia nurses were significantly lower than that of the controls (p < .001, p < .05, respectively). The results of this study indicate that occupational exposure to anesthetic gases induce DNA damage, which may lead to changes in TAC and GSH levels.