Semra Şardaş

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.

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.

SISTER CHROMATID EXCHANGES IN LYMPHOCYTES OF NURSES HANDLING ANTINEOPLASTIC DRUGS

The effects of handling antineoplastic drugs were examined in a group of 23 nurses working in the hematology and oncology departments of different university hospitals in Ankara and in a group of 50 unexposed controls. The cytogenetic repercussions of exposure were assessed by examining sister chromatid exchanges (SCEs) in circulating lymphocytes which result from the breakage and rejoining of DNA at apparently homologous sites on the 2 chromatids of a single chromosome. A significant increased frequency of SCE is observed in nurses in daily contact with antineoplastics (n = 23, mean SCEs/cell +/- SE 6.5 +/- 0.2) as compared to a group of controls (n = 50, mean SCEs/cell 5.2 +/- 0.2). The nurses who smoked also had a higher SCE frequency (n = 15, mean SCEs/cell 7.0 +/- 0.3) than non-smokers, (n = 8, mean SCEs/cell 5.5 +/- 0.3). A significant increase (P less than 0.001) in the mean number of SCE was found for non-smoking nurses as compared to non-smoking controls (n = 27, mean SCEs/cell 4.1 +/- 0.2).

Assessment of smoking-induced DNA damage in lymphocytes of smoking mothers of newborn infants using the alkaline single-cell gel electrophoresis technique

The single-cell gel electrophoretic (SCGE) technique for detecting the presence of DNA strand breaks and alkali-labile damage in individual cells was used to examine the effect on newborn infants of maternal exposure to cigarette smoke. The levels of DNA damage in the lymphocytes of 21 newborns of mothers with different smoking habits were compared to those in 10 newborn infants whose mothers had never smoked and 8 newborns whose mothers were passively exposed for at least 40 h per week in the workplace and home. DNA damage was undetected in lymphocytes of newborns of passively exposed mothers or newborns with mothers of low smoking habit by conditions allowing 40 min DNA unwinding and 40 min electrophoresis. Presumably longer times were needed for lower levels of damage to be detected by SCGE. The mean length of DNA migration in lymphocytes between the newborns of smoking mothers did not show any significance but the percentage of damaged cells increased with the frequency of smoking when assessed by non-parametric Mann-Whitney U test. The results of SCGE were compared with our results published in the same individuals of sister-chromatid exchange (SCE) frequency. The results show similar trends with mean measures of DNA damage increasing with frequency and long history of maternal smoking. These observations encourage the application of SCGE as a sensitive and useful technique for quantitating DNA damage in individual cells.

Increased frequency of sister chromatid exchanges in the peripheral lymphocytes of cigarette smokers

The incidence of sister chromatid exchanges (SCEs) in the peripheral lymphocytes of 23 smokers was significantly higher than that in 27 non-smokers (6.5 +/- 0.3 compared with 4.1 +/- 0.2 per cell, mean +/- SEM). Both the duration of smoking and the number of cigarettes smoked per day appeared to influence SCE frequency. Significantly higher frequencies of SCEs were observed in those who smoked more than 10 cigarettes per day than in those who smoked less than 10 cigarettes per day. The frequency of SCEs in those who had habitually smoked for over 10 years was significantly greater than that in those who had smoked for less than 10 years.

Chromosomal aberrations and alkaline comet assay in families with habitual abortion

Within the pathology of human reproduction, genetic abnormalities play an important role in spontaneous abortions. This paper describes the morphologic, karyotypic features of a consecutive series of singleton spontaneous abortions collected as part of this study and also reports the application of the alkaline comet assay to assess levels of DNA damage in 31 couples comprised of 13 control couples and a patient group of 18 couples with a history of more than one fetal loss. For the cytogenetic analyses, the conventional lymphocyte culture method was applied to all subjects. In this analysis, two women with habitual abortion were determined to carry balanced chromosomal translocation. The alkaline comet assay (single cell gel electrophoresis technique) was applied also to lymphocytes. The comparison of the results of alkaline comet assay in patient and control individuals showed a significant difference in the number of damaged cells. The cells were evaluated according to their grades of damage as: normal (undamaged-no migration), limited migration, (at low damage level) and extensive migration (comet imaged cells-with increasing numbers of breaks, DNA pieces migrate freely into the tail forming a comet image). The frequency of limited migrated and extensive migrated cells in the women in the patient group were higher than in the women in the control group (p<0.001). However, all above parameters were equal for husbands in both the control and patient group (p>0.05).

Chromosomal aberrations under basal conditions and after treatment with X-ray in human lymphocytes as related to the GSTM1 genotype

The frequency of chromosomal aberrations (CAs) was evaluated in blood lymphocytes from 18 healthy subjects. Basal CA frequencies were not significantly different in GSTM1 positive and GSTM1 null subjects (P>0.05), whereas they were considerably higher in smokers than in non-smokers. After 1 Gy dose of X-ray challenge of blood samples, CA frequencies were significantly higher in GSTM1 null subjects, compared to GSTM1 positive subjects (P<0.005), and in smokers, compared to non-smokers. These effects are ascribed to the influence of GSTM1 genotype and of smoking status on DNA repair capacities. As the induction of CAs are associated with carcinogenesis, the challenge assay is able to detect enhanced susceptibility for CA caused by genetic predisposition of DNA repair deficiency.

The effect of smoking on sister chromatid exchange rate of newborn infants born to smoking mothers.

Sister chromatid exchange (SCE) frequencies in lymphocytes of 21 smoking mothers and their 21 newborns were compared to those that of 10-infants whose mothers had never smoked and to those of 8 infants whose mothers were passive smokers and reported high exposure to tobacco smoke by living or working with smokers. Mothers in the first group also smoked throughout their pregnancy. Results confirm our earlier study on smoking effects reported for adults. Additionally, we saw that neonates have consistently lower SCE frequencies than adults.

Evaluation of genotoxic potential of styrene in furniture workers using unsaturated polyester resins

Styrene is a widely used chemical, mostly in making synthetic rubber, resins, polyesters, plastics and insulators. Increasing attention has been focused on this compound since experiments using cytogenetic end-points have implicated styrene as a potential carcinogen and mutagen. In order to perform biological monitoring of genotoxic exposure to styrene monomer, we evaluated the urinary thioether (UT) excretion, and sister chromatid exchanges (SCEs) and micronuclei (MN) in peripheral lymphocytes from 53 furniture workers employed in small workplaces where polyester resin lamination processings were done and from 41 matched control subjects. The mean air concentration of styrene in the breathing zone of workers was 30.3 ppm. As a metabolic marker for styrene exposure, mandelic acid + phenylglyoxylic acid was measured in the urine and the mean value was 207 mg/g creatinine. The mean +/- SD value of UT excretions of workers was 4.43 +/- 3.42 mmol SH-/mol creatinine and also mean UT for controls was found to be a 2.75 +/- 1.78 mmol SH-/mol creatinine. The mean +/- SD/cell values of SCE frequency in peripheral lymphocytes from the workers and controls were 6.20 +/- 1.56 and 5.23 +/- 1.23, respectively. The mean +/- SD frequencies (%o) of MN in the exposed and control groups were 1.98 +/- 0.50 and 2.09 +/- 0.35, respectively. Significant effects of work-related exposure were detected in the UT excretion and SCEs analyzed in peripheral blood lymphocytes (p < 0.05 and p < 0.01, respectively). The MN frequency in lymphocytes from the styrene-exposed group did not differ from that in the controls (p > 0.05). Effect of smoking, age and duration of exposure on the genotoxicity parameters analyzed were also evaluated. In conclusion, although our data do not demonstrate a dose-response relationship, they do suggest that styrene exposure was evident and that this styrene exposure may contribute to the observed genotoxic damage in furniture workers.

Sister-chromatid exchanges in epileptic patients on anticonvulsant therapy

Sister-chromatid exchanges (SCE) have been studied to analyze genotoxic effects in epileptic patients on anticonvulsant therapy. All patients had an increased frequency of SCE per metaphase (p < 0.001) compared to controls, indicating a genotoxic effect of anticonvulsant drugs.