Damage In Peripheral Blood Lymphocytes Research Articles

Expression and Polymorphisms of Gene 8-Oxoguanine Glycosylase 1 and the Level of Oxidative DNA Damage in Peripheral Blood Lymphocytes of Patients with Alzheimer's Disease

The purpose of this study was to determine the level of 8-oxo-2'-deoxyguanosine (8-oxo2dG) and expression of three isoforms of 8-oxoguanine glycosylase 1 (OGG1), OGG1-1a, 1b, and 1c, and OGG1 protein and Ser326Cys and Arg46Gln polymorphisms of the OGG1 gene, in peripheral blood lymphocytes of patients with Alzheimer's disease (AD) and healthy controls. The study was performed in 41 AD patients and 51 healthy subjects. The level of 8-oxo2dG was determined by high performance liquid chromatography/electrochemical; expression of OGG1-1a, 1b, and 1c by real-time quantitative polymerase chain reaction; and OGG1 protein by Western blotting. The polymerase chain reaction-restriction fragment length polymorphism analysis was conducted to analyze the Ser326Cys and Arg46Gln polymorphisms. It was found that AD patients and controls have three isoforms, OGG1-1a, 1b, and 1c. The OGG1-1c isoform seems to be associated with early stage of AD, while an increase in the expression of the OGG1-1b isoform and levels of OGG1 protein appears to be similarly related to the progression of AD. All of the studied OGG1 isoforms show a decreased expression in advanced AD. The CG Ser326Cys genotype seems to have a tendency to decrease 8-oxo2dG via control of repair mechanisms. The Arg46Gln polymorphism is not associated with the pathogenesis of AD. It appears that the OGG1-1a, 1b, and 1c isoforms are involved in the pathogenesis of AD.

Primary DNA Damage Assessed With the Comet Assay and Comparison to the Absorbed Dose of Diagnostic X-rays in Children

The aim of this work is to assess DNA damage in peripheral blood lymphocytes of children prior to and following airway X-ray examinations of the chest using the alkaline comet assay and to compare data with the measured absorbed dose. Twenty children with pulmonary diseases, between the ages of 5 and 14 years, are assessed. Absorbed dose measurements are conducted for posterior-anterior projection on the forehead, thyroid gland, gonads, chest, and back. Doses are measured using thermoluminescent and radiophotoluminescent dosimetry systems. Differences between tail lengths, tail intensity, and tail moments as well as for the long-tailed nuclei before and after exposures are statistically significant and are dependent on the individual. The results demonstrate the usefulness of the comet assay as a measure of X-ray damage to lymphocytes in a clinical setting. Doses measured with both dosimeters show satisfactory agreement (0.01 mSv) and are suitable for dosimetric measurements in X-ray diagnostics.

Abstract: P806 EVALUATION OF PROTEIN C HIGHLY SENSITIVE CHILDREN AND ADOLESCENTS IN MUNICIPALITY OF ITAPETININGA-BRAZIL

DNA damage caused by the accumulation of bio-products generated in the biotransformation of ethanol to acetaldehyde mediated by the CYP2E1 enzyme has been studied. To evaluate DNA damage in peripheral blood lymphocytes and the possible association with polymorphisms in the promoter region of the CYP2E1 gene, we performed a case-control study including 75 alcoholics and 59 individuals who consume alcohol socially. Alcoholics were previously diagnosed by the Psychosocial Care Center – Alcohol and Drugs (CAPS A/D) in the city of Goiania, Goias state, Central Brazil. DNA damage was evaluated by comet assay. The analysis of the rs3813867, rs2031920, and rs2031921 polymorphisms in the promoter region of CYP2E1 gene was performed by Sanger sequencing. Men older than 35 years old were the most common alcoholics. We found increased DNA damage in the case group, compared to the control group (p < 0.001). Alcoholics who were heterozygous in the rs3813867, rs2031920, and rs2031921 polymorphisms showed higher DNA damage (tail length and olive tail moment), compared to individuals with the homozygous non-mutated allele. Previous studies have shown that polymorphisms in the promoter region of the CYP2E1 gene could cause higher CYP2E1 transcriptional activity, increasing enzyme activity compared with nondrinkers, indicating that the presence of the mutated allele (heterozygous or homozygous) may be associated with higher alcohol metabolic rates and therefore show increased acetaldehyde levels after alcohol consumption, which then can exert its carcinogenic effect.

Prevention of DNA damage by polyphenol‐rich food

Epidemiological and experimental data suggest that an increased intake of plant food rich in polyphenols can reduce the risk of cancers. DNA damage is an important step in the initiation of carcinogenesis. Here we report data on the effect of polyphenol rich food such as apples and apple juice on DNA damage in colon epithelial cells from animal studies and in peripheral blood lymphocytes (PBL) from human intervention studies.1,2‐Dimethylhydrazine‐induced DNA strand breaks in colon epithelial cells in rats were significantly inhibited by consumption of cloudy and clear apple juice. In a human intervention study, consumption of 1 kg apples caused no changes in endogenous DNA strand breaks, in Fpg‐sensitive sites (oxidative purine damage) or in the capacity to protect DNA against damage induced by hydrogen peroxide in PBL. But, a statistically significant decrease in the levels of endonuclease III sensitive sites (oxidative pyrimidine damage) and an increase in capacity to protect DNA against strand breaks induced by iron chloride were identified. In contrast to the effects of apples, a human intervention with 2, 5 or 8 servings of fruit and vegetables/day had no effect on DNA damage in PBL. Thus, we have identified apples as another fruit to add to the already known types of fruit (kiwi) and vegetables (tomato) with antigenotoxic properties and partly characterized their antigenotoxic mechanism in humans.Supported by the Deutsche Forschungsgemeinschaft and the Federal Ministry of Research and Education, Germany

High protein-high red meat versus high carbohydrate weight loss diets do not differ in effect on genome stability and cell death in lymphocytes of overweight men

The importance of diet in DNA damage prevention is well established; however, the comparison of weight loss diets with different micronutrient and macronutrient profiles on genome stability in peripheral blood lymphocytes (PBLs) has not been studied. This study tested the hypothesis that genome stability in PBLs of overweight men who consume a high protein-high red meat (HP) weight loss diet is different from that of overweight men who consume a high carbohydrate (HC) weight loss diet. Thirty-three male subjects were randomly assigned to an HP or HC isocaloric energy-restricted dietary intervention for 12 weeks intensive weight loss and weight maintenance up to 52 weeks. Blood samples were collected at 0, 12 and 52 weeks. DNA damage in PBLs was assessed using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Average weight loss after 12 weeks was 9.3 +/- 0.7 kg for both diets, with no further change at 52 weeks. Two-way analysis of variance showed no time or diet effect on micronucleus frequency (chromosome loss/breaks). There was a significant trend with time (P = 0.03) but not diet, for reduction of nuclear buds (gene amplification). There was a positive trend with time for increased nucleoplasmic bridges (chromosome rearrangement) (P = 0.051). Necrosis and apoptosis both significantly decreased with time (P = 0.037 and P = 0.007, respectively) with no diet effect. There was no significant effect of time or diet for nuclear division index, a biomarker of immune response. The results suggest that the effect of the HP weight loss diet on DNA damage measured using the CBMN-Cyt assay in PBLs was not different from that observed for the HC weight loss diet.

Micronuclei frequency in peripheral blood lymphocytes of thyroid cancer patients after radioiodine therapy and its relationship with metastasis

In most cancers peripheral blood lymphocytes exhibit DNA damage. In the case of thyroid cancer the micronucleus (MN) assay has been used to assess DNA damage before and after exposure to iodine-131 ( 131I). The aim of our study was to use this method to assess DNA damage in peripheral blood lymphocytes of thyroid cancer patients and search for its relationship with metastasis as well as 131I exposure. A significant increase in micronuclei frequency was observed in peripheral blood lymphocytes of 54 thyroid cancer patients in comparison to 38 controls ( p = 0.000). Further analysis revealed significant elevation in micronuclei index from 48.5 MN/1000 BN cells (range: 25.1–111.2, n = 25) in patients without metastasis to 68.1 MN/1000 BN cells (range: 26.2–135.5, n = 29, p = 0.001) in group of patients with metastasis to one or more sites. There was no clear correlation between the micronuclei frequency and the therapeutic 131I dose ranging from 0.41 to 31.5 GBq with the exposure interval of <1 to 126 months. In addition, age and sex did not show any influence on micronuclei frequency in either patients or control population. These findings are indicative of increased basal DNA damage in thyroid cancer patients before treatment. Radioiodine treatment did not increase DNA damage measured by the micronuclei frequency for the interval between the last radioiodine dose administered and analysis of blood sample. However a significant increase of peripheral blood lymphocytes micronuclei was observed in thyroid cancer patients with metastasis.

Sensitivity to NNKOAc is associated with renal cancer risk

Cigarette smoking has been investigated as a major risk factor for renal cell carcinoma (RCC). 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most abundant carcinogenic N-nitrosamines present in cigarette smoke. However, the association between repair capacity of NNK-induced DNA damage and RCC risk remains unknown. We used the comet assay to assess whether sensitivity to a NNK precursor 4-[(acetoxymethyl) nitrosamino]-1-(3-pyridyl)-1-butanone (NNKOAc) induced DNA damage, which partly reflects host sensitivity to NNK, was associated with increased risk of RCC in a population-based case-control study. The study included 95 RCC cases and 188 matched controls. Epidemiologic data were collected via in-person interview. Baseline and NNK-induced DNA damage in peripheral blood lymphocytes were measured using the comet assay and quantified by the Olive tail moment. The NNKOAc-induced median Olive tail moments were significantly higher in cases than in controls (2.27 versus 1.76, P = 0.002). Using the 75th percentile Olive tail moments of the controls as the cutoff point, we found that higher levels of NNKOAc-induced DNA damage were associated with a significantly increased risk of RCC [odds ratio, 2.06; 95% confidence interval, 1.17-3.61]. In quartile analysis, there was a dose-response association between NNKOAc-induced damage and risk of RCC (P for trend, 0.006). Our data strongly suggest that higher levels of NNKOAc-induced damage are associated with higher risks of RCC. Future studies with larger sample sizes are warranted to further investigate whether repair of NNKOAc-induced damage, as quantified by the comet assay, could be used as a predictive marker for RCC risk.

Association of maternal polymorphisms in folate metabolizing genes with chromosome damage and risk of Down syndrome offspring

We analyzed the role of six common polymorphisms in folate metabolizing genes as possible risk factors for having a child with Down syndrome (DS) in 94 Italian mothers of a DS child (MDS) and 113 matched control mothers, both aged less than 35 years at conception. Investigated polymorphisms include methylenetetrahydrofolate reductase ( MTHFR) 677C > T and 1298A > C, methionine synthase ( MTR) 2756A > G, methionine synthase reductase ( MTRR) 66A > G, and thymidylate synthase ( TYMS) 28 bp repeat and 1494del6. We also measured the amount of chromosome damage in peripheral blood lymphocytes of 42 MDS and 41 matched controls, by means of the micronucleus assay, and searched for association between this cytogenetic endpoint and any of the studied polymorphisms. Micronuclei in peripheral blood lymphocytes have been analyzed several years after conception: the mean age at sampling was 45.6 ± 11.4 years for MDS and 47.95 ± 6.9 years for controls. The combined MTHFR 677TT/ MTR 2756AA genotype was associated with increased DS risk ( P = 0.034), and the combined MTHFR 1298AC/ TYMS 2R/2R genotype with reduced risk ( P = 0.003). Moreover, we observed a significant increased frequency of micronucleated lymphocytes in MDS as compared to controls ( P < 0.0001) and, in the total population, a significant correlation between micronucleated cells and both MTHFR 677C > T ( P = 0.031) and 1298A > C ( P = 0.047) polymorphisms.

Reduction of the genomic damage level in haemodialysis patients by folic acid and vitamin B12 supplementation

Cancer incidence and genomic damage of peripheral lymphocytes are elevated in patients with end-stage renal failure. Among other uraemic toxins, homocysteine (Hcy) levels are increased in most of these patients. In healthy individuals, plasma Hcy correlates with the degree of genomic damage observed in peripheral blood lymphocytes (PBL). The accumulation of Hcy can be reduced by supplementation with folic acid and vitamin B12. The aim of this study was to analyse whether this supplementation can also lower the genomic damage in PBL of haemodialysis patients. This may ultimately help to reduce cancer incidence in renal patients. In a prospective study with 27 patients, we analysed the genomic damage in dialysis patients before and at different time points after the initiation of folate/vitamin B12 supplementation. Genomic damage was measured by the frequency of micronuclei, a subset of chromosomal aberrations, in PBL. Supplementation with folic acid and vitamin B12 (more markedly with both) reduced the micronucleus frequency in PBL of dialysis patients. This was not mediated by altered lymphocyte proliferation capacity or changes in DNA cytosine-methylation. Plasma-Hcy was lowered more efficiently by the combined folic acid/vitamin B12 supplementation, and lymphocyte DNA of this group exhibited a nonsignificant trend for a reduction of 1,N(6)-etheno-2'-deoxyadenosine, a marker for oxidative stress. A reduction of the genomic damage in PBL can be achieved in dialysis patients by supplementation with folic acid and vitamin B12. This may be mediated by Hcy reduction.

Levodopa therapy reduces DNA damage in peripheral blood cells of patients with Parkinson’s disease

Oxidative stress seems to play a major role in the pathogenesis of neurodegeneration. In Parkinson's disease (PD) patients, the dopaminergic neurons are subjected to oxidative stress resulting from reduced levels of antioxidant defenses such as glutathione and high amount of intracellular iron. Levodopa (LD) is widely used for the symptomatic treatment of PD, but its role in oxidative damage control is still unclear. The aim of this study was to analyze the presence of DNA damage in peripheral blood lymphocytes (PBL) of PD patients, during a washout and a controlled LD dosage and to evaluate the oxidative damage fluctuation after LD intake. The standard and the Fpg-modified version of Comet assay were applied in analyzing DNA damage in PBL from blood samples of nine PD patients and nine matched controls. Due to the limited number of patients we cannot reach definite conclusions even if our data confirm the accumulation of DNA lesions in PD patients; these lesions decrease after LD intake.

Association between polycyclic aromatic hydrocarbons exposure levels and nucleoplasmic bridge and nuclear bud frequencies in coke-oven workers

To seek new effect biomarkers as to evaluating the chromosomal damage in peripheral blood lymphocytes in coke-oven workers who were exposed to polycyclic aromatic hydrocarbons (PAHs). One hundred and fifty-eight coke-oven workers and 69 controls were recruited in this study. Nucleoplasmic bridges and nuclear buds were counted as indicators of chromosomal damage in terms of cytokinesis-block micronucleus (CBMN) test. Occupational history, age, sex, smoking and alcohol using status of all subjects were collected by questionnaire. Frequencies of nucleoplasmic bridge in coke-oven workers were (9.41 +/- 3.73)% per hundred, and the frequencies of nuclear buds were (7.13 +/- 4.01)% per hundred, which were significantly higher (P < 0.01) than those of controls (1.88 +/- 1.49)% per hundred and (2.20 +/- 1.73)% per hundred respectively. The dose-effect relationships between nucleoplasmic bridges or nuclear buds and PAHs exposure levels were identified. Compared with male coke-oven workers, female workers had less nucleoplasmic bridges or nuclear buds. No effects of age, smoking and alcohol using were found on nucleoplasmic bridges or nuclear buds among coke-oven workers. Nucleoplasmic bridges and nuclear buds might be effect biomarkers in coke-oven workers.

Benfotiamine reduces genomic damage in peripheral lymphocytes of hemodialysis patients

Hemodialysis patients have an elevated genomic damage in peripheral blood lymphocytes (PBLs) and an increased cancer incidence, possibly due to accumulation of uremic toxins like advanced glycation end products (AGEs). Because the vitamin B1 prodrug benfotiamine reduces AGE levels in experimental diabetes, and dialysis patients often suffer from vitamin B1 deficiency, we conducted two consecutive studies supplementing hemodialysis patients with benfotiamine. In both studies, genomic damage was measured as micronucleus frequency of PBLs before and at three time-points after initiation of benfotiamine supplementation. AGE-associated fluorescence in plasma, and in the second study additionally, the antioxidative capacity of plasma was analyzed. Benfotiamine significantly lowered the genomic damage of PBLs in hemodialysis patients of both studies independent of changes in plasma AGE levels. The second study gave a hint to the mechanism, as the antioxidative capacity of the plasma of the treated patients clearly increased, which might ameliorate the DNA damage.

Oxidative stress, DNA damage, and antioxidant enzyme activity induced by hexavalent chromium in Sprague‐Dawley rats

Chromium is a widespread industrial compound. The soluble hexavalent chromium Cr (VI) is an environmental contaminant widely recognized as carcinogen, mutagen, and teratogen toward humans and animals. The fate of chromium in the environment is dependent on its oxidation state. The reduction of Cr (VI) to Cr (III) results in the formation of reactive intermediates leading to oxidative tissue damage and cellular injury. In the present investigation, Potassium dichromate was given intraperitoneally to Sprague-Dawley rats for 5 days with the doses of 2.5, 5.0, 7.5, and 10 mg/kg body weight per day. Oxidative stress including the level of reactive oxygen species (ROS), the extent of lipid peroxidation and the activity of antioxidant enzymes in both liver and kidney was determined. DNA damage in peripheral blood lymphocytes was determined by single-cell gel electrophoresis (comet assay). The results indicated that administration of Cr (VI) had caused a significant increase of ROS level in both liver and kidney after 5 days of exposure, accompanied with a dose-dependent increase in superoxide dismutase and catalase activities. The malondialdehyde content in liver and kidney was elevated as compared with the control animals. Dose- and time-dependent effects were observed on DNA damage after 24, 48, 72, and 96 h posttreatment. The results obtained from the present study showed that Cr (VI) could induce dose- and time-dependent effects on DNA damage, both liver and kidney show defense against chromium-induced oxidative stress by enhancing their antioxidant enzyme activity. However, liver was found to exhibit more antioxidant defense than the kidney.

Intravenous iron exacerbates oxidative DNA damage in peripheral blood lymphocytes in chronic hemodialysis patients.

Patients undergoing maintenance hemodialysis have elevated markers of oxidative stress, but the reasons for this are not fully understood. Intravenous administration of iron, which many of these patients receive, may provoke the generation of bioactive iron, which enhances oxidative stress and lipid peroxidation. In this study, 110 hemodialysis patients were randomly assigned to five groups that were administered single intravenous doses of iron sucrose, ranging from 20 to 500 mg. A time- and dosage-dependent rise in lymphocyte 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in lymphocyte DNA, a marker of oxidative DNA damage, with a significant increase at 2 h after intravenous iron of > or = 200 mg (P < 0.05). Four weeks later, patients were randomly assigned to weekly iron sucrose (100 mg of elemental iron) or saline for 12 wk, and 89 patients completed the study. Mean lymphocyte 8-OHdG content was significantly higher in patients receiving intravenous iron compared with control subjects (P < 0.05), especially in those with ferritin levels > 500 microg/L. In addition, flow cytometric techniques revealed increased production of reactive oxygen species in lymphocytes among those treated with intravenous iron. Treatment with intravenous iron but not saline was also associated with decreased plasma ascorbate and alpha-tocopherol levels and increased oxidized glutathione/reduced glutathione ratio (P < 0.05). In summary, intravenous iron sucrose provokes oxidative damage to peripheral blood lymphocyte DNA in hemodialysis patients, especially among those with high levels of ferritin.

481 Cytokinesis Blocked Micronucleus (Cbmn) Assay Is Predictive of Gastrointestinal (GI) Sequeale 2 Years Following Radiation Therapy (RT) for Prostate Carcinoma (CAP) Whilst Micronutrients Reduce Their Severity

RT for CaP is associated with GI sequelae including anorectal dysfunction up to 5 years after RT(1). Patients receive the same radiation dose despite variations in normal tissue tolerance(2). The CBMN assay quantifies spontaneous and radiation-induced DNA damage in peripheral blood lymphocytes(3) and has been correlated with GI sequelae after RT for CaP(2,4). The molecular basis of radiation sensitivity and role of dietary micronutrients on GI sequelae is unknown. This prospective study aims to develop biomarkers predictive for GI sequelae after RT for CaP, identifying modifying dietary and genetic factors.Methods:Before RT, venous blood of 21 patients 69(55-79)yrs with CaP, was assayed for (i)CBMN, (ii)plasma homocysteine, B12, folate, Zn, Se, lutein, retinol, α-tocopherol, lycopene and α-carotene and (iii)genotyping. LENT-SOMA GI symptoms(1), anorectal manometry and endoanal ultrasound were evaluated before RT, 1mth, 1 and 2yrs after RT(3). Data analyzed by ANOVA. Relationships among the anorectal function parameters and rectal volumes, CBMN and micronutrient data were examined by linear regression. Results:GI symptoms increased after RT and persisted at 2yrs. This was associated with reductions in (i)basal pressures, (ii)pressures in response to increased intra-abdominal pressure, (iii)rectal compliance, (iv)threshold volumes and (v)internal anal sphincter thickness. Spontaneous CBMN data were directly related to (a) 1 yr total GI symptom scores (r=0.52, p<0.05) and inversely related to squeeze pressures at 1 (r=-0.54, p<0.05) and 2 (r=-0.47, p<0.05) yrs. 1 mth desire to defecate volumes were also inversely related to radiation-induced (3Gy ex-vivo) CBMN index (r=-0.56, p<0.05). Plasma lycopene(r=-0.46, p<0.05) and α-carotene(r=-0.48, p<0.05) were inversely related to 1 yr diarrheal scores. Plasma α-carotene was also directly related to 1 yr threshold volumes (r=0.60, p<0.01). There were direct relationships between (a) plasma homocysteine and 1 yr diarrheal scores (r=0.62, p<0.01), (b) plasma zn and 1 yr basal pressures (r=0.45, p<0.05), (c) plasma retinol and 1 yr threshold volumes (r=0.59, p<0.01). Rectal bleeding scores at 1 mth were directly related to rectal volumes (r=0.48, p<0.05). Conclusion:The CBMN assay predicts GI sequelae 2 yrs after RT for CaP. Diets that include higher intake of foods rich in lycopene, α-carotene, zinc and retinol and which produce a lower homocysteine concentration in plasma, are likely to reduce the late adverse effects of RT for CaP. (1)Yeoh et al Am J Gastroenterol 2004, 2:361-369,(2)Lee et al Int J Radiat Oncol Biol Phys 2003,57:222-229,(3)Fenech, Mut Res 2000,455:81-95,(4)Yeoh et al,Gastroenterol 2007,132:A590

Oxidative stress status in liver mitochondria and lymphocyte DNA damage of atherosclerotic rabbits supplemented with water soluble coenzyme Q10

The effects of the administration of water soluble coenzyme Q10 (25 mg/kg per day) over 30 days, after 50 days feeding on a high-fat diet (3% lard + 1.3% cholesterol), were investigated in the plasma and liver mitochondria of rabbits. Results showed that this atherogenic diet enhanced lipid levels both in plasma and liver mitochondria, reduced plasma and mitochondrial concentrations of retinol and coenzyme Q10, led to higher DNA damage in peripheral blood lymphocytes and reactive oxygen species concentration in liver mitochondria. The treatment of animals with coenzyme Q10 reduced (to the healthy group levels) lipid concentration in liver mitochondria with no effect on plasma lipids, increased mitochondrial levels of alpha-tocopherol, restored mitochondrial coenzyme Q10 and improved alpha-tocopherol levels in plasma. Moreover, coenzyme Q10 supplementation reduced mitochondrial reactive oxygen species levels and decreased DNA damage in peripheral blood lymphocytes. The findings suggest that antioxidant therapy with coenzyme Q10 may be used in the treatment of liver pathologies associated to the intake of high-fat, atherogenic, diets.

DNA damage in peripheral blood lymphocytes of patients with cirrhosis related to alcohol abuse or to hepatitis B and C viruses

Both alcohol abuse and hepatitis B or C virus infections are implicated in the development of hepatocellular carcinoma, but it is still controversial whether the pathogenetic mechanism is epigenetic or genotoxic. Considering that alcohol promotes the generation of reactive oxygen species and both viruses infect peripheral lymphocytes, in this study we investigated the occurrence of DNA fragmentation in peripheral blood lymphocytes from patients with alcoholic cirrhosis and from patients with cirrhosis related to B and C viruses, and analyzed the correlation between the degree of DNA fragmentation and the Child-Pugh score used to assess the degree of hepatic insufficiency. The study population consisted of two groups: group I involved 12 patients with alcoholic cirrhosis; group II involved 25 patients with hepatic B virus or hepatic C virus cirrhosis. The control group involved 20 healthy individuals. The degree of DNA fragmentation in peripheral blood lymphocytes was determined with the alkaline Comet assay that provides two indexes of the frequency of DNA single-strand breaks and alkali-labile sites, the tail length and the tail moment. Mean values of both tail length and tail moment were significantly increased (P<0.001) in lymphocytes from 12 patients with alcoholic cirrhosis and in lymphocytes from 25 patients with HBV or HCV cirrhosis, as compared with average tail length and tail moment values of lymphocytes from 20 healthy individuals. A significant positive correlation was found to exist between the degree of DNA fragmentation present in lymphocytes of each of the 37 patients with alcoholic or viral cirrhosis and the corresponding value of the Child-Pugh score. The occurrence of DNA fragmentation in peripheral blood lymphocytes reflects a direct genotoxic effect of either alcohol or HBV and HCV and suggests that the same genotoxic effect may operate in the liver and contribute to hepatocarcinogenesis.

Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice

Oxidative stress is considered as a possible molecular mechanism involved in lead toxicity. This study was carried out to investigate whether lead acetate could induce oxidative stress in mice, and the following damages as well. Lead acetate was given orally to mice for 4 weeks at doses of 0, 10, 50, 100mg/kg body weight every other day, respectively. Production of reactive oxygen species (ROS) and malondialdehyde (MDA) were measured as indicators of oxidative stress. DNA damage in peripheral blood lymphocytes was determined by comet assay. Ultrastructure alteration was detected using transmission electron microscopy. The alterations of p53, Bax, and Bcl-2 expression were determined by western blotting. The results showed that lead acetate significantly increased the levels of ROS and MDA in mice. Meanwhile, severe DNA damage and ultrastructure alterations were obviously observed. In addition, p53 and Bax expressions increased and the imbalance of Bax/Bcl-2 occurred. Therefore, it strongly suggests that lead may induce oxidative stress and change the expressions of apoptosis-related proteins in mouse liver.

DNA damage in peripheral blood lymphocytes in patients during combined chemotherapy for breast cancer

Combined chemotherapy is used for the treatment of a number of malignancies such as breast cancer. The target of these antineoplastic agents is nuclear DNA, although it is not restricted to malignant cells. The aim of the present study was to assess DNA damage in peripheral blood lymphocytes (PBLs) of breast cancer patients subjected to combined adjuvant chemotherapy (5-fluorouracil, epirubicin and cyclophosphamide, FEC), using a modified comet assay to detect DNA single-strand breaks (SSB) and double-strand breaks (DSB).Forty-one female patients with advanced breast cancer before and after chemotherapy and 60 healthy females participated in the study. Alkaline and neutral comet assays were performed in PBLs according to a standard protocol, and DNA tail moment was measured by a computer-based image analysis system.Breast cancer patients before treatment had higher increased background levels of SSB and DSB as compared to healthy women. During treatment, a significant increase in DNA damage was observed after the 2nd cycle, which persisted until the end of treatment. Eighty days after the end of treatment the percentage of PBLs with SSB and DSB remained elevated, but the magnitude of DNA damage (tail moment) returned to baseline levels. There was no correlation between PBL DNA damage and response to chemotherapy.DNA-SSB and DSB in PBLs are present in cancer patients before treatment and increase significantly after combined chemotherapy. No correlation with response to adjuvant chemotherapy was found. Biomonitoring DNA damage in PBLs of cancer patients could help prevent secondary effects and the potential risks of developing secondary cancers.

Biomonitoring of DNA damage in peripheral blood lymphocytes of subjects with dental restorative fillings

Dental fillings provide a major iatrogenic exposure to xenobiotic compounds due to the high prevalence of surface restorations in developed countries. Experimental data suggest that both amalgams, which contain mercury, and resin-based dental materials cause an impairment of the cellular pro- and anti-oxidant redox balance. The aim of this study was to assess the potential genotoxicity of dental restorative compounds in peripheral blood lymphocytes of young exposed subjects compared with controls. The study examined, by use of the comet assay, 68 carefully selected subjects taking into account the major known confounding factors. In the 44 exposed subjects, the mean numbers of restored surfaces was 3.0 and 3.8 in males and females, respectively. Tail length, percentage of DNA in the tail, tail moment or Olive tail moment were twofold higher in the exposed group than in unexposed controls, with significant differences. No significant difference was observed between amalgam and composite fillings. Furthermore, as shown by multivariate analysis, the association between dental fillings and DNA damage was enhanced by the number of fillings and by the exposure time. Among the lifestyle variables, a moderate physical activity showed a protective effect, being inversely correlated to the DNA damage parameters evaluated. On the whole, the use of DNA-migration allowed us to detect for the first time the potential adverse impact on human health of both kinds of dental filling constituents, the amalgams and the methacrylates. The main mechanism underlying the genotoxicity of dental restorative materials of various nature may be ascribed to the ability of both amalgams and methacrylates to trigger the generation of cellular reactive oxygen species, able to cause oxidative DNA lesions.