Ali Esat Karakaya

Assessment of DNA strand breakage by comet assay in diabetic patients and the role of antioxidant supplementation

Diabetes patients often show increased production of reactive oxidative species (ROS) together with vascular complications. The presence of these ROS may lead to increased DNA damage in peripheral blood lymphocytes that may be revealed by the comet assay. To test whether DNA is damaged in diabetes, peripheral blood samples were taken from 30 control individuals and 63 diabetic patients (15 insulin dependent (IDDM) and 48 non-insulin dependent (NIDDM)) and the alkaline comet assay was used to evaluate background levels of DNA damage. Significant differences were detected between control and diabetic patients in terms of frequencies of damaged cells. The extend of DNA migration was greater in NIDDM patients by comparison with IDDM patients which might indicate that IDDM patients are handling more oxidative damage on a regular basis. Smoker individuals had higher frequencies of cells with migration by comparison with the non-smokers in both groups. Also, clear differences between patients on placebo and on Vitamin E supplementation for 12 weeks were observed on the basis of the extend of DNA migration during single cell gel electrophoresis.

The micronucleus assay in exfoliated buccal cells: application to occupational exposure to polycyclic aromatic hydrocarbons

Many polycyclic aromatic hydrocarbons (PAHs) have been identified as cancer-inducing chemicals for animals and/or humans. Also, there is sufficient evidence that exposures in the occupational settings are carcinogenic or probably carcinogenic to human. Engine exhaust and used engine oils are major PAH sources in engine repair workshops and traffic. Analysis of micronucleus (MN) in exfoliated buccal cells is a sensitive method for monitoring genetic damage in human populations. In our study, we used three different occupational groups (Group 1; engine repair workers, Group 2; taxi drivers, Group 3; traffic police) and two controls (Control I for Group 1 and Control II for Group 2 and Group 3) for the exposed groups. We analysed MN frequencies in exfoliated buccal cells and compared the exposed groups (Group 1; n=34, Group 2; n=17, Group 3; n=15) and subjects not occupationally exposed to PAH (Control I; n=28, Control II; n=20). The mean (+/-S.D.) MN (%) frequencies in exfoliated buccal cells from Group 1 and Control I were 0.07+/-0.05 and 0. 05+/-0.04, respectively (p>0.05; Table 2). The mean (+/-S.D.) MN (%) frequencies in exfoliated buccal cells from Group 2, 3 and Control II were 0.12+/-0.05, 0.10+/-0.05 and 0.03+/-0.03, respectively (p<0. 0001, p<0.05; Table 2) Smokers and nonsmokers do not differ with respect to the incidence of MN in all groups.

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.

Comparison of genotoxicity of sevoflurane and isoflurane in human lymphocytes studied in vivo using the comet assay.

In the present paper, we report data on the possible genotoxic properties of two inhalation anaesthetics--sevoflurane (SVF) and isoflurane (ISF) - in peripheral blood lymphocytes of patients before, during and after anaesthesia as compared to an unexposed control group. Both anaesthetics were evaluated for genotoxic activity using the comet assay. The exposed groups consisted of 24 ASA grades 1-2 unpremedicated patients (aged 20-66 years, anaesthetized 115-162 min for elective lower abdominal surgery), while the control group consisted of 12 healthy individuals. After induction of anaesthesia (thiopenthone sodium 5-7 mg/kg, fentanyl citrate 0.1mg and vecuronium bromide 0.1mg/kg), anaesthesia was maintained with inhalation of SVF 1-1.5% (n=12) or ISF 1-1.5% (n=12) in oxygen-air mixture. Venous blood samples were obtained before the induction of anaesthesia, at 60 and 120 min of anaesthesia and on the first, third and fifth days following anaesthesia. The comet assay detects DNA damage which includes strand breaks and alkaline labile sites induced directly by genotoxic agents as well as DNA degradation due to cell death. One hundred cells from each sample were examined and graded as no tailed, short and long tailed nuclei. The mean comet response was not different between controls and patients before anaesthesia. However, similar significant increases were observed in the mean comet response in blood sampled from patients at 60 (36.5+/-11.2, 37.8+/-12.1), or 120 min (53.1+/-17.1, 50.0+/-12.2) of anaesthesia and on the first day (37.8+/-15.1, 35.2+/-15.7) after anaesthesia in SVF and ISF treated groups, respectively. Removal of the DNA damage was observed after the third day of anaesthesia and the repair was completed within 5 days. The DNA damage detected in lymphocytes of patients during anaesthesia with SVF or ISF showed similar results as demonstrated by an increased mean comet migration at 120 min of anaesthesia and the cells were able to repair the induced DNA damage completely on the fifth postoperative day.

Chromosomal damage in peripheral blood lymphocytes of traffic policemen and taxi drivers exposed to urban air pollution

Urban air contains a diversity of chemical compounds, some of which are genotoxins. An increased risk of cancer has also been reported in occupations with heavy exposure to traffic-related pollution. The aim of this study was to assess the cytogenetic effects of urban air pollution by analyzing the chromosomal aberration (CA) frequencies in lymphocytes and to estimate the polycyclic aromatic hydrocarbons (PAHs) exposure by measuring urinary 1-hydroxypyrene (1-OHP) levels. A total of 15 traffic policemen and 17 taxi drivers working in the city of Ankara were the exposed groups and 23 healthy men working in the office departments were the control group. The overall mean +/- S.D. values of 1-OHP excretions of traffic policemen, taxi drivers and control subjects were 0.59 +/- 0.40 micromol/mol creatinine, 0.32 +/- 0.25 micromol/mol creatinine and 0.57 +/- 0.36 micromol/mol creatinine, respectively. Urinary 1-OHP levels of non-smoking policemen were significantly greater than those of nonsmoking control subjects (p < 0.05). The overall mean +/- S.D. values for CA frequencies (%) from policemen, taxi drivers and control group were 1.29 +/- 1.59, 1.81 +/- 1.79, and 0.26 +/- 0.73, respectively. There was a significantly greater frequency of CAs in exposed groups relative to the matched control population (p < 0.05; p < 0.01). Age, sex and smoking habits have not influenced the cytogenetic end-point in this study. Our results demonstrate that occupational exposure to urban air pollutants leads to a significant induction of cytogenetic damage in peripheral lymphocytes of traffic policemen and taxi drivers.

Use of alkaline Comet assay (single cell gel electrophoresis technique) to detect DNA damages in lymphocytes of operating room personnel occupationally exposed to anaesthetic gases

Here, we report the possible in vivo induction DNA damage by exposure to various waste anaesthetic gases such as halothane, nitrous oxide and isoflurane. The alkaline comet assay (single cell gel electrophoresis technique) was carried out on 66 operating room personnel (anaesthetists [doctors]; anaesthesia nurses and anaesthesia unit technicians) currently employed at the Ankara Hospital in Turkey. A significant increase in the number of lymphocytes with DNA migration was observed in operating room personnel as compared to controls. Also, the extent of damage in exposed smokers were significantly higher than exposed nonsmokers. This study supports the existence of an association between DNA damage and occupational exposure to inhalation anaesthetics.

Sister-chromatid exchanges in operating room personnel

Sister-chromatid exchange (SCE) analysis was carried out in 67 operating room personnel (anaesthetists M.D.; anaesthesia nurses and anaesthesia unit technicians) exposed to waste anaesthetic gases such as halothane, nitrous oxide and isoflurane and in 50 healthy unexposed controls. The SCE frequencies were increased significantly in operating room personnel as compared to controls. A significant increase in SCEs was found in non-smoking operating room personnel as compared to non-smoking controls. This study supports the existence of an association between occupational exposure to mutagens and an increase in SCEs in lymphocytes.

Cytogenetic analysis of buccal cells from shoe- workers and pathology and anatomy laboratory workers exposed to n-hexane, toluene, methyl ethyl ketone and formaldehyde

People employed in the shoe manufacture and repair industry are at an increased risk for cancer, the strongest evidence being for nasal cancer and leukaemia. A possible causal role for formaldehyde is likely for cancer of the buccal cavity and nasopharynx. Exfoliated buccal cells are good source of tissue for monitoring human exposure to inhaled and ingested occupational and environmental genotoxicants. To assess the cytogenetic damage related to occupational exposure to airborne chemicals during shoe-making and the processes in pathology and anatomy laboratories, the micronuclei (MN) count per 3000 cells was measured in buccal smears from shoe-workers (group I, n = 22) exposed to mainly n-hexane, toluene and methyl ethyl ketone (MEK) and from anatomy and pathology staff (group II, n = 28) exposed to formaldehyde (FA). Eighteen male university staff were used as controls. The mean time-weighted average (TWA) concentrations of n-hexane, toluene and MEK in 10 small shoe workshops were 58.07 p.p.m., 26.62 p.p.m. and 11.39 p.p.m., respectively. The measured air concentrations of FA in the breathing zone of the anatomy and pathology laboratory workers were between 2 and 4 p.p.m. Levels of 2,5-hexadione (2,5-HD) and hippuric acid (HA), metabolic markers of n-hexane and toluene exposure, respectively, were significantly higher in the urine of workers in group I than in control subjects (p < 0.001 and p < 0.01, respectively). The mean (±SD) MN frequencies in buccal mucosa cells from workers in group I, group II and controls were 0.62±0.45%, 0.71±0.56% and 0.33±0.30%, respectively (p < 0.05 and p < 0.05 compared with controls for group I and group II, respectively). The effects of smoking, age and duration of exposure on the frequency of micronucleated buccal cells from workers in all three groups studied were also evaluated. Overall, the results suggest that occupational exposure to organic solvents, mainly n-hexane, toluene, MEK and FA, may cause cytogenetic damage in buccal cells and that use of exfoliated buccal cells seems to be appropriate to measure exposure to organic solvents.

Cytogenetic biomonitoring of workers exposed to bitumen fumes

Bitumen samples and fumes consist essentially of polycyclic hydrocarbons (PAH) and their derivatives, some of which are known to be carcinogenic or co-carcinogenic in animals. The level of total PAH is low when compared with coal-tar products. There is very limited data on possible health risk from exposure to bitumen fumes in workers. In this study, sister-chromatid exchange (SCE), micronuclei (MN) and high frequency of SCE cells (HFCs) were determined for 28 workers exposed to bitumen fumes and 28 control subjects. Urinary 1-hydroxypyrene (1-OHP) excretion was used as a biomarker of occupational exposure to PAH. The mean value of 1-OHP excretion of workers was 0.78+/-0.46 micromol/mol creatinine and for controls 0.52+/-0.44 micromol/mol creatinine (p<0.05). The mean values of SCE per cell and the frequency ( per thousand) of MN in peripheral lymphocytes from the workers and controls were 5.13+/-0. 64, 4.71+/-0.67, and 2.25+/-0.42, 1.79+/-0.32 respectively (p<0.05, p<0.0001). The mean value of HFCs for workers and controls were 7. 85+/-2.3 and 7.05+/-3.16, respectively (p>0.05). Our data reveal that bitumen fumes during road paving operations are absorbed by workers and that bitumen fume exposure is able to significantly induce cytogenetic damage in peripheral lymphocytes of workers after controlling some possible confounding factors, such as age, sex and smoking habits.

Evaluation of micronuclei in exfoliated urothelial cells and urinary thioether excretion of smokers

Mutagens are present in large quantities in the urine of cigarette smokers, thus, their urothelial cells may represent a possible target for absorbed and excreted mutagens. Our aim is to validate the micronucleus (MN) test in exfoliated urothelial cells obtained from urine samples of cigarette smokers. The urinary thioether (UT) test is also carried out on the same individuals in order to find out whether there is any correlation between these two end-points. The mean (+/- SE) MN frequency and UT determination is 1.93 (+/- 0.11)% and 9.71 (+/- 1.61) mmol SH/mol creatinine for 23 smokers, and 0.66 (+/- 0.05)% and 4.20 (+/- 0.56) mmol SH/mol creatinine for 20 nonsmokers. Our results show a higher frequency of micronucleated cells (p < 0.001) and higher excretion of UTs (p < 0.05) in smokers as compared to nonsmokers. Concentrations of UTs and MN frequencies increased with tobacco consumption. The MN frequencies showed only a marginal increase, not significant (p > 0.05), after passive smoking compared to nonsmoking values. There was no significant correlation between MN frequencies and UTs, either in smokers (r = 0.164, p > 0.05) or in nonsmokers (r = -0.018, p > 0.05). Our data demonstrate tobacco-induced chromosome damage in bladder tissue consistent with increased risk of cancer at this site among smokers.