Associations between XRCC1 and ERCC2 polymorphisms and DNA damage in peripheral blood lymphocyte among coke oven workers

A wide variety of base damages and single-strand breaks formed by reactive oxygen species during metabolic activation of polycyclic aromatic hydrocarbons (PAHs) have been recognized to be involved in PAH carcinogenesis. In this study, alkaline comet assay was used to detect the DNA damage in peripheral blood lymphocytes among 143 coke-oven workers and 50 non-coke-oven workers, and the effects of genetic polymorphisms of XRCC1 and ERCC2 genes on DNA damage were evaluated. The olive tail moment was significantly higher in coke-oven workers than in non-coke-oven workers (2.6, 95% CI=2.1–3.3 versus 1.0, 95% CI=0.8–1.2, p<0.01), and significant correlation between ln-transformed urinary 1-OHP and ln-transformed olive tail moment was found in total population (n=193, Pearson's r=0.393, p<0.001) and in coke-oven workers (n=143, Pearson's r=0.224, p=0.007). The olive tail moment was significantly higher in coke-oven workers with GA genotype of G27466A polymorphism of XRCC1 than those with GG genotype (4.6, 95% CI=2.5–8.7 versus 2.4, 95% CI=1.9–2.9, p<0.01 with adjustment for covariates). No significant associations between C26304T, G28152A and G36189A polymorphisms of XRCC1 and G23591A and A35931C polymorphisms of ERCC2 and olive tail moment were found in both groups. The study showed that the alkaline comet assay is a suitable biomarker in the detection of DNA damage among coke-oven workers and it suggested that the A allele of G27466A polymorphism of XRCC1 may be associated with decreased DNA repair capacity toward PAH-induced base damage and strand breaks.     

Effects of physical exercise training in DNA damage and repair activity in humans with different genetic polymorphisms of hOGG1 (Ser326Cys)

The main purpose of this pilot study was to investigate the possible influence of genetic polymorphisms of the hOGG1 (Ser326Cys) gene in DNA damage and repair activity by 8‐oxoguanine DNA glycosylase 1 (OGG1 enzyme) in response to 16 weeks of combined physical exercise training. Thirty‐two healthy Caucasian men (40–74 years old) were enrolled in this study. All the subjects were submitted to a training of 16 weeks of combined physical exercise. The subjects with Ser/Ser genotype were considered as wild‐type group (WTG), and Ser/Cys and Cys/Cys genotype were analysed together as mutant group (MG). We used comet assay in conjunction with formamidopyrimidine DNA glycoslyase (FPG) to analyse both strand breaks and FPG‐sensitive sites. DNA repair activity were also analysed with the comet assay technique. Our results showed no differences between DNA damage (both strand breaks and FPG‐sensitive sites) and repair activity (OGG1) between genotype groups (in the pre‐training condition). Regarding the possible influence of genotype in the response to 16 weeks of physical exercise training, the results revealed a decrease in DNA strand breaks in both groups, a decrease in FPG‐sensitive sites and an increase in total antioxidant capacity in the WTG, but no changes were found in MG. No significant changes in DNA repair activity was observed in both genotype groups with physical exercise training. This preliminary study suggests the possibility of different responses in DNA damage to the physical exercise training, considering the hOGG1 Ser326Cys polymorphism. Copyright © 2015 John Wiley & Sons, Ltd.     

Assessment of DNA sensitivity in peripheral blood leukocytes after occupational exposure to microwave radiation: the alkaline comet assay and chromatid breakage assay

DNA sensitivity in peripheral blood leukocytes of radar-facility workers daily exposed to microwave radiation and an unexposed control subjects was investigated. The study was carried out on clinically healthy male workers employed on radar equipment and antenna system service within a microwave field of 10 μW/cm²–20 mW/cm² with frequency range of 1,250–1,350 MHz. The control group consisted of subjects of similar age. The evaluation of DNA damage and sensitivity was performed using alkaline comet assay and chromatid breakage assay (bleomycin-sensitivity assay). The levels of DNA damage in exposed subjects determined by alkaline comet assay were increased compared to control group and showed inter-individual variations. After short exposure of cultured lymphocytes to bleomycin cells of subjects occupationally exposed to microwave (MW) radiation responded with high numbers of chromatid breaks. Almost three times higher number of bleomycin-induced chromatid breaks in cultured peripheral blood lymphocytes were determined in comparison with control group. The difference in break per cell (b/c) values recorded between smokers and non-smokers was statistically significant in the exposed group. Regression analyses showed significant positive correlation between the results obtained with two different methods. Considering the correlation coefficients, the number of metaphase with breaks was a better predictor of the comet assay parameters compared to b/c ratio. The best correlation was found between tail moment and number of chromatid with breaks. Our results indicate that MW radiation represents a potential DNA-damaging hazard using the alkaline comet assay and chromatid breakage assay as sensitive biomarkers of individual cancer susceptibility.     

Comparison of three oxidative stress biomarkers in a sample of healthy adults

Oxidative stress is a potentially important aetiological factor for many chronic diseases, including cardiovascular disease, neurodegenerative disease and cancer, yet studies often find inconsistent results. The associations between three of the most widely used biomarkers of oxidative stress, i.e. F₂-isoprostanes for lipid peroxidation and 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxo-dG) and the comet assay with FPG for oxidative DNA damage, were compared in a sample of 135 healthy African-American and white adults. Modest associations were observed between F₂-isoprostanes and the comet assay (r?=?0.22, p?=?0.01), but there were no significant correlations between 8-oxo-dG and the comet assay (r?=??0.09) or F₂-IsoP (r?=??0.04). These results are informative for researchers seeking to compare results pertaining to oxidative stress across studies and/or assessment methods in healthy disease-free populations. The development and use of oxidative stress biomarkers is a promising field; however, additional validation studies are necessary to establish accuracy and comparability across oxidative stress biomarkers.     

Base excision repair capacity in chronic renal failure patients undergoing hemodialysis treatment

The aim of this study was to determine if the differences observed in the levels of DNA damage in a group of patients suffering from chronic renal failure are due to differences in the repair capability. DNA damage was initially measured with the comet assay in 106 hemodialysis patients. A selected group of 21 patients representing high (ten patients) and low (11 patients) levels of DNA damage were obtained for determination of base excision repair capacity. This was measured in an in vitro assay where protein extracts from lymphocytes were incubated with a substrate of DNA containing 8‐oxoguanine, and the rate of incision was measured with the comet assay. Patients with high levels of genomic damage showed, as an average, significantly lower repair capacity (12·73 ± 1·84) in comparison with patients with low levels of genomic damage (18·13 ± 1·13). Nevertheless, the correlation coefficient between repair ability and levels of genomic damage was found to be only close to the significance value (r:?0·423, p: 0·056). Although DNA damage was clearly related to time on hemodialysis, base excision repair capacity was not. This is one of the few studies providing information on the repair capacity of chronic renal failure patients undergoing hemodialysis. As a summary, our results would indicate that DNA damage levels are in part associated to the repair capacity of the patients, and this repair capacity is not associated with the duration of hemodialysis treatment. Copyright © 2013 John Wiley & Sons, Ltd.     

Assessment of peripheral DNA damage by alkaline comet assay in maintenance hemodialysis subjects with hepatitis C infection

Despite the high prevalence of hepatitis C infection among hemodialysis subjects, there is no information concerning the DNA damage of hepatitis C (+) hemodialysis subjects. We aimed to find out if there is any additional effect of hepatitis C infection on peripheral DNA damage in maintenance hemodialysis subjects. Fifteen hepatitis C (+) and 22 hepatitis C (-) hemodialysis subjects, 21 hepatitis C subjects without renal disease, and 22 healthy controls were enrolled. Peripheral DNA damage was assayed using alkaline comet assay. Median DNA damage levels of the study groups were as follows: hepatitis C (+) maintenance hemodialysis subjects, 88 (0-232); hepatitis C (-) maintenance hemodialysis subjects, 58 (0-228); hepatitis C (+) subjects without renal disease, 112 (44-252); controls, 26 (0-72). DNA damage level was significantly higher among hepatitis C (+) subjects without renal disease than hepatitis C (-) maintenance hemodialysis subjects and healthy controls (both p<0.05/6). Both maintenance hemodialysis subjects with and without HCV infection had significantly higher DNA damage level than healthy controls (both p<0.05/6). DNA damage level was comparable between hepatitis C (+) subjects without renal disease and HCV (+) hemodialysis subjects, and between hemodialysis subjects with and without hepatitis C infection (all p>0.05/6). Linear regression analysis revealed that hepatitis C infection was the only independent factor in predicting the peripheral DNA damage (p<0.05, beta=0.395). Each one of end-stage renal disease and hepatitis C infection significantly increases DNA damage level. However, in hemodialysis subjects, hepatitis C infection does not cause significant additional increase in DNA damage level, and it may be partly due to protective effect of hemodialysis on hepatitis C infection.     

Simvastatin treatment prevents oxidative damage to DNA in whole blood leukocytes of dyslipidemic type 2 diabetic patients

Type 2 diabetes (T2D) is associated with increased oxidative stress as indicated by elevated levels of lipid peroxidation and protein oxidation products. Since reactive oxygen species (ROS) can cause damage to biological macromolecules including DNA, this study investigated oxidative damage to DNA using the alkaline (pH > 13) comet assay in peripheral whole blood leukocytes sampled from 15 dyslipidemic T2D patients treated with simvastatin (20 mg/day), 15 dyslipidemic T2D patients not treated with simvastatin, 20 non‐dyslipidemic T2D patients, and 20 healthy individuals (controls). Our results showed a greater DNA migration in terms of damage index (DI) (p < 0.01) in the dyslipidemic T2D patients not treated with statin (DI = 67.70 ± 10.89) when compared to the dyslipidemic T2D patients under statin treatment (DI = 47.56 ± 7.02), non‐dyslipidemic T2D patients (DI = 52.25 ± 9.14), and controls (DI = 13.20 ± 6.40). Plasma malondialdehyde (MDA) and C‐reactive protein (CRP) levels were also increased and total antioxidant reactivity (TAR) and paraoxonase activity (PON1) decreased in non‐dyslipidemic T2D patients and dyslipidemic T2D non‐treated with simvastatin. We also found that DI was inversely correlated with TAR (r = ?0.61, p < 0.05) and PON1 (r = ?0.67, p < 0.01). In addition, there was a significant positive correlation between DI and CRP (r = 0.80, p < 0.01). Our results therefore indicate that simvastatin treatment plays a protective role on oxidative damage to DNA in dyslipidemic T2D patients probably reflecting a general decrease in oxidative stress in these patients. Copyright © 2010 John Wiley & Sons, Ltd.     

DNA damage evaluated by the comet assay in lymphocytes of children with Cs internal contamination caused by the Chernobyl accident

The comet assay is one of the most versatile and popular tools for evaluating DNA damage. Its sensitivity to low dose radiation has been tested in vitro, but there are limited data showing its application and sensitivity in chronic exposure situations. The influence of the internal contamination caused by the Chernobyl accident on the level of DNA damage was evaluated by the comet assay on lymphocytes of 56 Ukrainian children. The study was performed during 2003 on children with demonstrable ¹³⁷Cs internal contamination caused by food consumption. The children were selected for the study immediately after a ¹³⁷Cs whole body counter measurement of internal contamination. The minimal detectable amount of ¹³⁷Cs was 75 Bq. The control group included 29 children without detectable internal contamination, while in the exposed group 27 children with measured activity between 80 and 4037 Bq and committed effective dose between 54 and 3155 μSv were included. Blood samples were taken by a finger prick. The alkaline version of the comet assay was used, in combination with silver stained comets and arbitrary units (AU), for comet measurement. Factors such as disease, medical treatment, surface contamination of children's living location, etc., were considered in the study. Non-significant differences (p > 0.05) in DNA damage in control (9.0 ± 5.7 AU) versus exposed (8.5 ± 4.8 AU) groups were found. These results suggest that low doses of ¹³⁷Cs internal contamination are not able to produce detectable DNA damage under the conditions used for the comet assay in this study. Further studies considering effects of high exposure should be performed on chronically exposed people using this assay.     

Determination of DNA damage induced by oxidative stress in hyperlipidemic patients

In the present paper, we report data on the genotoxic properties of hydrogen peroxide in polymorphonuclear neutrophils (PMNLs) separated from normolipidemic and type II/a hyperlipidemic patients. In all, 15 hyperlipidemic patients (11 female, 4 male, mean age 54.6±10.25 years) were involved in the study, and 7 normolipidemic patients (5 female, 2 male, mean age 53.4±8.07 years) served as controls. Using the comet assay, there was a significant difference in the degree of DNA damage between the two groups. The visual score characteristic of the degree of DNA damage was 350.97±31.31 in the hyperlipidemic group, while it was 289.5±29.49 in the control group (P<0.001). In the hyperlipidemic patients, a positive correlation was found between the degree of DNA damage and the basic oxidation of PMNLs (r=0.517), and the superoxide anion production of the cells stimulated with phorbolmiristate acetate (PMA) (r=0.326) and formyl-Met–Leu–Phe (FMLP) (r=0.525) as well. There was a negative correlation between DNA damage and HDL-associated antioxidant paraoxonase (PON) activity (r=−0.469), and the PON/HDL ratio (r=−0.631). No correlation was found between the degree of DNA damage and the plasma concentration of nitric oxide (NO) (r=0.098) and thiobarbituric acid-reactive substances (TBARS) (r=0.061) in hyperlipidemic patients. Our results show that in hyperlipidemic patients there is an increase in lymphocyte DNA damage caused by oxidative stress when compared to normolipidemic individuals as demonstrated by comet assay. Decreased antioxidant capacity in hyperlipidemic patients may play a significant role in this process.     

Low IGF-1 levels are associated with cardiovascular risk factors in haemodialysis patients

Cardiovascular diseases (CVD) constitute a significant risk and may, in part, explain the high morbidity and mortality rates among haemodialysis (HD) patients. Several studies have implicated reduced insulin like growth factor (IGF-1) levels in the development of CVD. However, it is not clear whether IGF-1, and its relationship with other hormones such as leptin, insulin, and growth hormone (GH), as well as anthropometric variables may explain the high incidence of vascular complications in chronic kidney disease (CKD) patients. This study was designed to measure total serum IGF-1, leptin, insulin and GH levels in CKD patients and in age-matched control subjects and to elucidate the relationship between IGF-1 and GH, leptin, and insulin as well as other known aetiological risk factors for CVD including blood pressure, body mass index (BMI), and age. The study consisted of 50 CKD patients [36 M and 14 F; mean age; 41.8 ± 10.3 years) on maintenance haemodialysis and 50 healthy control subjects (36 M and 14 F; mean age 41.6 ± 10.2 years) matched for age and sex. None of the subject among patients and controls reported either smoking or history of diabetes mellitus. The circulating levels of IGF-1 were significantly lower (P < 0.001) in both male and female patients compared to the control subjects. Moreover, IGF-1 was strongly and inversely correlated with both systolic blood pressure (SBP) (r = −0.360; P < 0.01) and diastolic blood pressure (DBP) (r = −0.512; P < 0.001) in the CKD group, and when the two groups were combined SBP (r = −0.396; P < 0.001) and DBP (r = −0.296; P < 0.01). When adjusted for age, the correlation was more significant, however, when adjusted for BMI no significant correlation was observed between IGF-1 and blood pressure. IGF-1 was inversely correlated with age (r = −0.367; P < 0.01) and BMI (r = −0.310; P < 0.05) in the control group, but not the patient group. In controls and patients, respectively, a positive correlation between leptin and BMI (r = 0.358; P < 0.01; r = 0.640, P < 0.001) was observed. The results show that circulating levels of IGF-1 were significantly lower in CKD patients as compared to healthy normal subjects and were inversely correlated with SBP and DBP independent of age, but not BMI indicative of a strong relationship between cardiovascular risk factors and low IGF-1 levels. Although, the data do not clearly indicate low IGF-1 levels as a cause or an effect of these cardiovascular risk factors, they do point to an interesting relationship between low IGF-1 levels and increased cardiovascular risk factors among CKD patients as compared to age-matched healthy control subjects.     

Analysis of oxidative DNA damage and HPRT mutations in humans after hyperbaric oxygen treatment

DNA damage induced by reactive oxygen species (ROS) seems to play an important role in the induction of mutations and cancer. We have recently shown that hyperbaric oxygen (HBO) treatment of volunteers (i.e., exposure to 100% oxygen at a pressure of 2.5 ATA) induces DNA damage detected in leukocytes with the comet assay. Using formamidopyrimidine-DNA glycosylase (FPG protein) we provided indirect evidence for the induction of oxidative DNA base damage. We now comparatively evaluated FPG-sensitive sites with the comet assay and 7,8-dihydro-8-oxo-deoxyguanosine (8-OHdG) with HPLC analysis after a single HBO. As 8-OHguanine (8-OHgua) is one of the major DNA modifications induced by ROS and a pre-mutagenic lesion, we looked for HBO-induced mutations at the HPRT locus with the T cell cloning test. We also determined the genotypes for glutathione transferases (GST) and tested a possible influence of the GSTM1 and GSTT1 genotypes on the sensitivity of subjects against HBO-induced genotoxicity. Our results indicate that despite a clear induction of FPG-sensitive sites no increased levels of 8-OHdG and no induction of HPRT mutations was detected in lymphocytes after HBO. Furthermore, the DNA effects in the comet assay and the mutant frequencies in the HPRT test seem to be unrelated to the GST genotypes of the test subjects.     

Evaluation of oxidative DNA damage and antioxidant defense in patients with nasal polyps

Abstract

Objectives

The presence of inflammatory cells indicates the development of epithelial cell injury in nasal polyposis (NP) and the potential for production of high levels of reactive oxygen and nitrogen species. The aim of our study was to clarify the role of oxidative stress and antioxidant status in the deterioration accompanying NP.

Methods

Twenty patients (11 men) aged 47.2 ± 17.0 years with nasal polyps were included in the study. Twenty healthy subjects (7 men) aged 48.2 ± 15.3 years formed the control group. The erythrocyte activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and plasma nitric oxide (NO) concentrations were measured. An alkaline comet assay was used to determine the extent of blood lymphocyte DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites.

Results

A significant increase of NO (P < 0.05) and non-significant decreases of SOD (P > 0.05), CAT (P > 0.05), and GPx (P > 0.05) were seen in NP patients compared to healthy controls. The level of blood lymphocyte oxidative DNA damage in NP patients was significantly higher compared to the control group (P = 0.01).

Discussion

The blood lymphocyte DNA damage level increased in patients with NP. Elevated DNA damage may be related to overproduction of reactive oxygen and nitrogen species and/or decreased antioxidant protection.     

Plasma homocysteine and DNA damage profiles in normal and obese subjects in the Pakistani population

Dependence of plasma total homocysteine (tHcy) and DNA damage profiles on melanodialdehyde (MDA), oxidative stress, liver function tests (LFT), and lipids was studied in non-obese and obese subjects in the Pakistani population. Development of obesity is influenced by both genetic, biochemical and environmental factors. Plasma homocysteine (Hcy) and DNA damage profiles play a pivotal role in its progression. We studied 160 obesity patients and 160 lean subjects. Leukocytes were evaluated for DNA damage by comet assay and blood plasma for biochemical properties using commercial kits. Plasma Hcy level and DNA damage were strongly correlated with triglycerides (P < 0.000), LDL-cholesterol (P < 0.001), systolic blood pressure (P < 0.001), cholesterol (P < 0.004), MDA (P < 0.004) and total oxygen stress (P < 0.004) in obese individuals. Both Hyc and DNA damage were negatively associated with total anti-oxidant response and globulin. Both Hcy profile and DNA damage may have a role in the endothelium damage even in the normal range and are related to triglycerides, ALT, MDA, TOS, HDL- and LDL-cholesterol in the Pakistani population.     

Genotoxic and Mutagenic Assessment of Hexavalent Chromium in Fish Following In Vivo Chronic Exposure

Chromium is a well-documented carcinogen. To evaluate the genotoxic potential of hexavalent chromium on an aquatic bio-system, freshwater murrel fish (Channa punctatus) were exposed to potassium dichromate. The 96-h LC₅₀ for potassium dichromate was 61.80 mg/L for the test fish in a static system. On the basis of the 96-h LC₅₀, fish were exposed to sublethal concentrations of the test chemical. Fish exposed to the test chemical were sampled on days 1, 7, 14, 21, and 28 post-exposure and blood and gill cells were collected. Significantly (p < .05) higher DNA damage in both lymphocyte and gillcells and micronuclei formation in whole blood was observed at different test concentrations and sampling times of the test chemical as compared to control fish. The mean% tail DNA in the comet tail assay showed a concentration-dependent increase and the maximum% tail DNA was observed on day 7 of exposure in both cells. A similar trend was also observed in micronuclei induction in blood with maximum induction on day 21. Hexavalent chromium showed genotoxic potential in chronic exposure of C. punctatus, and the micronucleus test and the comet assay are the methods for sensitive and rapid detection of the genetic effects.     

Detection of oxidative DNA damage in isolated marine bivalve hemocytes using the comet assay and formamidopyrimidine glycosylase (Fpg)

Organisms in polluted areas can be exposed to complex mixtures of chemicals; however, exposure to genotoxic contaminants can be particularly devastating. DNA damage can lead to necrosis, apoptosis, or heritable mutations, and therefore has the potential to impact populations as well as individuals. Single cell gel electrophoresis (the comet assay) is a simple and sensitive technique used to examine DNA damage in single cells. The lesion-specific DNA repair enzyme formamidopyrimidine glycoslyase (Fpg) can be used in conjunction with the comet assay to detect 8-oxoguanine and other damaged bases, which are products of oxidative damage. Fpg was used to detect oxidative DNA damage in experiments where isolated oyster (Crassostrea virginica) and clam (Mercenaria mercenaria) hemocytes were exposed to hydrogen peroxide. Standard enzyme buffers used with Fpg and the comet assay produced unacceptably high amounts of DNA damage in the marine bivalve hemocytes used in this study necessitating a modification of existing methods. A sodium chloride based reaction buffer was successfully used. Oxidative DNA damage can be detected in isolated oyster and clam hemocytes using Fpg and the comet assay when the sodium chloride reaction buffer and protocols outlined here are employed. The use of DNA repair enzymes, such as Fpg, in conjunction with the comet assay expands the usefulness and sensitivity of this assay, and provides important insights into the mechanisms of DNA damage.     

Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay

Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to ?80 °C—a laborious process. A recent publication (Al‐Salmani et al. Free Rad Biol Med 2011; 51: 719–725) describes a simple method in which small volumes of whole blood are frozen to ?20 or ?80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H₂O₂, and so a common test of antioxidant status (resistance to strand breakage by H₂O₂) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H₂O₂. In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H₂O₂ sensitivity and endogenous damage—both reflecting the antioxidant status of the cells—correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory. Copyright © 2013 John Wiley & Sons, Ltd.     

Levels of peripheral blood cell DNA damage in insulin dependent diabetes mellitus human subjects

Increased production of reactive oxygen species (ROS) in vivo can lead to cellular biomolecule damage. Such damage has been suggested to contribute to the pathogenesis of insulin dependent diabetes mellitus (IDDM). In this study, we used the alkaline comet assay to measure DNA damage (single-stranded DNA breaks and alkali-labile sites) in freshly isolated whole blood, lymphocytes, monocytes, and neutrophils from 23 subjects with IDDM and 32 age- and sex-matched controls. Analysis of the results showed elevated levels of DNA damage (expressed as % comet tail DNA) in the lymphocyte (4.10+/-0. 47, 3.22+/-0.22), monocyte (4.28+/-0.47, 3.49+/-0.18), and whole blood (4.93+/-0.51, 4.51+/-0.23) fractions from IDDM subjects compared to controls, respectively, but the increases observed were not statistically significant. However, we found significantly elevated basal levels of DNA damage in the neutrophil fraction (8. 38+/-0.64, 4.07+/-0.23; p<0.001, Mann-Whitney U test) in IDDM subjects compared to controls. Given these novel neutrophil findings, we extended the study to include a total of 50 IDDM subjects and 50 age- and sex-matched control subjects and determined basal levels of DNA damage in the neutrophils of all 100 subjects. We found significantly elevated mean levels of DNA damage (8.40+/-0.83, 4. 34+/-0.27; p<0.001, Mann-Whitney U test) in the neutrophils from the IDDM subjects when compared to controls. Our results show that even with acceptable glycaemic control there is a significantly elevated level of DNA damage within diabetic neutrophils in vivo.     

Deoxyribonucleic acid damage study in primary amenorrhea by comet assay and karyotyping

AIM:

This study aims at evaluating the chromosomal abnormalities and deoxyribonucleic acid (DNA) damage in cases with primary amenorrhea by karyotyping and comet assay.

STUDY DESIGN:

A total of 30 cases of primary amenorrhea were recruited. Secondary sexual characters were assessed by Tanner staging. Chromosomal analysis was performed by conventional phytohemagglutinin stimulated lymphocyte cell culture technique. Alkaline version of comet assay was used to evaluate DNA damage.

RESULTS:

The chromosomal pattern of 20 subjects (66.7%) was found to be normal (46,XX). Two subjects had 46,XY pattern and eight subjects had Turner syndrome (45,X or 45,X/46,XX). The comet parameters were found to be increased among subjects with 45,X monosomy, when compared to the rest of the study group and also in subjects with Tanner stage 1 when compared to stage 2.

CONCLUSION:

Comet assay revealed increased DNA damage in cases with 45,X monosomy, compared with subjects with 46,XX and 46,XY karyotype, which correlated with clinical features.     

Interindividual differences in initial DNA repair capacity when evaluating H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced DNA damage in extended-term cultures of human lymphocytes using the comet assay

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H₂O₂)-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H₂O₂, and there was a significant decrease in the H₂O₂-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H₂O₂-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H₂O₂-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.     

Influence of age on the association of retinol-binding protein 4 with metabolic syndrome

Objective: The relationships of retinol‐binding protein 4 (RBP4) with insulin sensitivity and body fat distribution have been investigated in a few recent studies with conflicting results. This may have been due to differences in ages of the subjects in the different studies. The aim of this study was to investigate whether the association of RBP4 and insulin sensitivity and percent trunk fat are influenced by age. Methods and Procedures: Cross‐sectional analyses of 48 young subjects and 55 elderly subjects. Insulin sensitivity was determined by a hyperinsulinemic–euglycemic clamp. Body fat distribution was determined by a dual‐energy X‐ray absorptiometry (DXA). Results: In the young subjects, RBP4 levels were associated with insulin sensitivity (r = ?0.30, P = 0.04), percent trunk fat (r = 0.54, P < 0.001), triglycerides (r = 0.44, P = 0.003), low‐density lipoprotein (r = 0.38, P = 0.01). In contrast, in the elderly subjects there was no correlation between RBP4 levels and insulin sensitivity (r = ?0.18, P = 0.20), percent trunk fat (r = 0.00, P = 0.10), triglycerides (r = 0.25, P = 0.10), and low‐density lipoprotein (r = ?0.11, P = 0.47). Discussion: The associations of RBP4 with insulin sensitivity, percent trunk fat, and lipid levels are influenced by age.