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.     

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.     

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.     

Epigenetic patterns of two gene promoters (TNF-α and PON) in stroke considering obesity condition and dietary intake

Some causal bases of stroke remain unclear, but the nutritional effects on the epigenetic regulation of different genes may be involved. The aim was to assess the impact of epigenetic processes of human tumor necrosis factor (TNF-α) and paraoxonase (PON) promoters in the susceptibility to stroke when considering body composition and dietary intake. Twenty-four patients (12 non-stroke/12 stroke) were matched by sex (12 male/12 female), age (mean 70?±?12 years old), and BMI (12 normal-weight/12 obese; mean 28.1?±?6.7 kg/m²). Blood cell DNA was isolated and DNA methylation levels of TNF-α (?186 to +349 bp) and PON (?231 to +250 bp) promoters were analyzed by the Sequenom EpiTYPER approach. Histone modifications (H3K9ac and H3K4me3) were analyzed also by chromatin immunoprecipitation in a region of TNF-α (?297 to ?185). Total TNF-α promoter methylation was lower in stroke patients (p?<?0.001) and showed no interaction with body composition (p?=?0.807). TNF-α and PON total methylation levels correlated each other (r?=?0.44; p?=?0.031), especially in stroke patients (r?=?0.72; p?=?0.008). The +309 CpG methylation site from TNF-α promoter was related to body weight (p?=?0.027) and the region containing three CpGs (from ?170 to ?162 bp) to the percentage of lipid intake and dietary indexes (p?<?0.05) in non-stroke patients. The methylation of PON +15 and +241 CpGs was related to body weight (p?=?0.021), waist circumference (p?=?0.020), and energy intake (p?=?0.018), whereas +214 was associated to the quality of the diet (p?<?0.05) in non-stroke patients. When comparing stroke vs non-stroke patients regarding the histone modifications analyzed at TNF-α promoter, no changes were found, although a significant association was identified between circulating TNF-α level and H3K9ac with H3K4me3. TNF-α and PON promoter methylation levels could be involved in the susceptibility to stroke and obesity outcome, respectively. The dietary intake and body composition may influence this epigenetic regulation in non-stroke patients.     

Optimization of the alkaline comet assay for easy repair capacity quantification of oxidative DNA damage in PBMC from human volunteers using aphidicolin block

Assessment of DNA repair capacity (DRC) upon ex vivo challenge of peripheral blood mononuclear cells (PBMC) with oxidative damage inducing agents, as evaluated by the comet assay, is widely used as biomarker to assess the antioxidant status in human studies. Here, the alkaline comet assay was now optimized for easy and time saving detection of repair capacity upon oxidative stress-induced DNA damage using the DNA polymerase inhibitor aphidicolin (APC) to block repair of hydrogen peroxide (H₂O₂) induced DNA damage. Addition of a DMSO-containing DNA damage stop solution was found suitable to replace washing steps for H₂O₂ removal before APC block. Cell treatment with APC at 6 μM did not impact baseline DNA damage but could reliably block DNA repair after H₂O₂ challenge in both fresh and cryopreserved samples thus omitting the use of a starting time point control. Under the conditions used, frozen cells, with or without an additional 4 h rest, showed the same repair capacity as their fresh counterpart. The intra assay coefficient of variation (CV) was 3.3%. To provide proof of principle, the modified assay was applied to cryopreserved PBMC from 19 participants of a short-term Brassica diet intervention study investigating potential health promoting effects of the food intervention. Then, a 33% increase in DRC (p ≤ 0.01) could be shown in samples after intervention (mean ± SD: 5.82 ± 1) as compared to baseline (mean ± SD: 4.38 ± 1.21). Individual samples from baseline and intervention showed an inter-individual CV of 27.65% (baseline) and 17.26% (intervention). Taken together this modified comet assay protocol allows the facilitated detection of DNA repair in fresh or cryopreserved human PBMC samples with a good sensitivity and reliability and could be useful in human studies addressing the antioxidant status and repair capacity of PBMC.     

Ecogenotoxicological studies for an early toxicity screening and monitoring in Epinephalus chlorostigma and Scamberomorus commerson

The study was planned to investigate DNA fragmentation in fish to screen aquatic toxicity and in Epinephalus chlorostigma and Scamberomorus commerson collected from Red sea near Jizan, Saudi Arabia from three locations “(Corniche North park: “16.92161, 42.54631; Jizan Port: 16.874, 42.54952” N and Jizan Economic City: 17.26589, 42.34738“ ”)“ were used as a case study for the application of comet assay. The study area of the Red Sea is polluted due to anthropogenic activities and the disposal of wastes from multiple sources. Comet and micronucleus assays were used to detect genotoxicity in these fish species harvested from three sites. The concentration of Pb, Cr, Zn, Mn, Cu, Cd, Sn, and Hg was higher in the water samples collected from the polluted site compared to the non-polluted site of the Red sea. Comet assay for S. commerson showed significant (p < 0.05) genetic damage about 44.33 ± 3.03% DNA in comet tail at site S1. It was subsequently reduced to 31.71 ± 3.52% and 22.11 ± 2.52% at sites S2 and S3. E. chlorostigma also showed significant DNA in comet tail as 17.34 ± 2.19%, 11.87 ± 3.01%, and 36.41 ± 3.98% at site S1-S3, respectively. Significant (p < 0.05) DNA damage was observed in the fishes procured from non-polluted locations and upstream locations. The micronucleus induction in E. chlorostigma was recorded as 23.20 ± 4.19 and 2.20 ± 0.58%, respectively, non-polluted and polluted sites. S. commerson exhibited significant differences between polluted and non-polluted sites (44.80 ± 3.73 and 8.20 ± 2.20‰) polluted and upstream (44.80 ± 3.73 and 20.60 ± 4.02‰), respectively. A significant difference was obtained between E. chlorostigma and S. commerson for nuclear abnormalities S. commerson showed higher frequencies for nuclear deformities than E. chlorostigma. S. commerson showed substantial micronucleus induction frequencies collected from an area of low pollution intensity (upstream). This study showed that E. clorostigma and S. commerson could be successfully used as a bioindicator to determine the health of the Red Sea through the most specific assays such as comet and micronucleus tests as an early warning and to devise the monitoring strategies to ensure a safe supply of fish for human consumption.     

Aortic Pulse Wave Velocity is Associated with Measures of Subclinical Target Organ Damage in Patients with Mild Hypertension

We evaluated the temporal association between aortic arterial stiffness and subclinical target organ damage, including renal function decline, left ventricular geometric remodeling, and left ventricular diastolic dysfunction in patients with mild hypertension. Automatic pulse wave velocity (PWV) measuring system was applied to examine carotid-femoral PWV (CFPWV) reflecting aortic arterial stiffness in 644 essential hypertensive patients. Clinical data were collected, and cardiac structure and function were assessed by echocardiography. CFPWV was significantly and positively associated with left ventricular mass index (r = 0.153, P = 0.018), relative wall thickness (r = 0.235, P < 0.001), and left atrial diameter (r = 0.192, P = 0.003), and negatively with E/A ratio (r = ?0.361, P < 0.001) and creatinine clearance (r = ?0.248, P < 0.001). Logistic regression analysis demonstrated that CFPWV remained significantly correlated with renal function decline (P = 0.011), left ventricular diastolic dysfunction (P = 0.009) and left ventricular geometric remodeling (P = 0.020). Higher CFPWV was independently associated with greater burden of subclinical disease in renal impairment, left ventricular geometric remodeling and diastolic dysfunction.     

Analysis of apoptosis and DNA damage in bovine cumulus cells after exposure in vitro to different zinc concentrations

The purpose of this study was to investigate the effect of Zn (zinc) concentration on CCs (cumulus cells) during in vitro maturation. For this purpose, DNA integrity of CCs by addition of different Zn concentrations [0 (control); 0.7 μg/ml (Zn1); 1.1 μg/ml (Zn2) and 1.5 μg/ml (Zn3)] to the culture medium was evaluated by comet assay. In addition, early apoptosis was analysed by annexin staining assay. CCs treated with Zn showed a significant decrease in the DNA damage in a dose‐dependent manner. Comet assay analysed for TM (tail moment) was significantly higher in cells cultured without Zn (control, P<0.01) with respect to cells treated with Zn (control: 5.24±16.05; Zn1: 1.13±5.31; Zn2: 0.10±0.36; Zn3: 0.017±0.06). All treatments were statistically different from the control (P=0.014 for Zn1; P<0.01 for Zn2 and Zn3). The frequency of apoptotic cells was higher in the control group (control: 0.142±0.07; Zn1: 0.109±0.0328; Zn2:0.102±0.013; Zn3: 0.0577±0.019). Statistical differences were found between control and Zn1 (P=0.0308), control and Zn2 (P=0.0077), control and Zn3 (P<0.0001), Zn1 and Zn3 (P<0.001) and Zn2 and Zn3 (P=0.0004). No differences were found between Zn1 and Zn2. In conclusion, low Zn concentrations increase DNA damage and apoptosis in CCs cultured in vitro. However, adequate Zn concentrations ‘protect’ the integrity of DNA molecule and diminish the percentage of apoptotic CC.     

Nanosecond electric pulses induce DNA breaks in cisplatin-sensitive and -resistant human ovarian cancer cells

Human ovarian cancer cells COC1 and COC1/DDP (cisplatin-resistant subline) were exposed to 6 kV/cm nanosecond electric pulses (nsEP) with a pulse length of 8, 16 or 24 ns. The potential in a subcellular unit was calculated using a multilayer dielectric spherical model, and area under the voltage–time curves (AUC) integrated with a lower limit of 0.2 V. Cell viability was determined, and double-stand and total DNA breaks detected with the neutral and alkaline comet assays. nsEP evoked a higher voltage and AUC in nucleoplasm, and the levels in COC1 cells was just above those in COC1/DDP cells. Comets only appeared in the alkaline assay demonstrating single-stand DNA break. Fewer DNA break (16.51% vs. 35.13% at 24 ns, p = 0.0150) and more survival (22.42% vs. 13.19% at 24 ns, p = 0.0015) occurred in COC1/DDP cells despite an equal electric energy and almost equal cell sizes. 24-ns EP led to higher rates of cell-death and comet. The comet rate correlated with cell-death fraction in either cell line (r = 0.5701, p = 0.0135; r = 0.5110, p = 0.0302). There was no a correlation between the tail length, tail moment or Olive tail moment and cell-death rate. The data showed that response of chemosensitive cells differed from that of chemoresistant cells and DNA damage contributed to percent of cell death.     

Potato virus X induces DNA damage in leaf nuclei of the host plant Nicotiana tabacum L. var. xanthi

We employed the comet assay (single cell gel electrophoresis) to evaluate induced DNA damage in nuclei isolated from tobacco leaves (Nicotiana tabacum var. xanthi) inoculated with Potato virus X (PVX). The highest DNA damage, expressed by the tail moment value, was observed in the inoculated leaves and decreased in the 1^st to 4^th systemic leaves. DNA damage increased with the time after the inoculation (from day 3 to day 21) and was higher in nuclei isolated from a part of the leaf at the petiole compared to nuclei isolated from the leaf tip. A Pearson moment correlation (r = 0.94) between the induced DNA damage and the PVX titres expressed by ELISA absorbance values was observed. The PVX infection did not induce a significant increase in the rate of somatic mutations evaluated by appearance of dark green, yellow, and double green/yellow sectors on the heterozygous pale green leaves of N. tabacum var. xanthi.     

Relationships between PON1 Q192R polymorphism and clinical outcome of antiplatelet treatment after percutaneous coronary intervention: a meta-analysis

This meta-analysis was performed to assess the relationships between the PON1 Q192R (rs662 T>C) polymorphism and the clinical outcome of antiplatelet treatment after percutaneous coronary intervention (PCI). A range of electronic databases were searched: Web of Science (1945–2013), the Cochrane Library Database (Issue 12, 2013), PubMed (1966–2013), EMBASE (1980–2013), CINAHL (1982–2013) and the Chinese Biomedical Database (CBM) (1982–2013) without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. The crude odds ratio (OR) with their 95 % confidence interval (CI) were calculated. Six clinical cohort studies with a total number of 5,189 patients undergoing PCI for coronary heart disease were included. Our meta-analysis revealed that the PON1 Q192R polymorphism was correlated with an increased risk of major adverse cardiovascular events (MACE) in patients receiving antiplatelet treatment after PCI (C allele vs. T allele: OR = 1.22, 95 % CI 1.04–1.43, P = 0.014; CT+CC vs. TT: OR = 1.38, 95 % CI 1.03–1.86, P = 0.029; CC vs. TT: OR = 1.45, 95 % CI 1.05–1.99, P = 0.024; respectively), especially among Asians. Furthermore, we found significantly positive correlations between the PON1 Q192R polymorphism and the incidence of stent thrombosis in patients receiving antiplatelet treatment after PCI (C allele vs. T allele: OR = 1.42, 95 % CI 1.08–1.87, P = 0.011; CT+CC vs. TT: OR = 1.93, 95 % CI 1.01–3.67, P = 0.046; CC vs. TT: OR = 2.18, 95 % CI 1.09–4.35, P = 0.027; respectively). Our meta-analysis of clinical cohort studies provides evidence that the PON1 Q192R polymorphism may increase the risk of MACE and stent thrombosis in patients receiving antiplatelet treatment after PCI.     

The Added and Interpretative Value of CGM-Derived Parameters in Type 1 Diabetes Depends on the Level of Glycemic Control

ObjectiveIn type 1 diabetes mellitus (T1DM) management, continuous glucose monitoring (CGM)-derived parameters can provide additional insights, with time in range (TIR) and other parameters reflecting glycemic control and variability being put forward. This study aimed to examine the added and interpretative value of the CGM-derived indices TIR and coefficient of variation (CV%) in T1DM patients stratified according to their level of glycemic control by means of HbA1C.MethodsT1DM patients with a minimum disease duration of 10 years and without known macrovascular disease were enrolled. Patients were equipped with a blinded CGM device for 7 days. TIR and time spent in hypoglycemia and hyperglycemia were determined, and CV% was used as a parameter for glycemic variability. Pearson (r) and Spearman correlations (r_s) and a regression analysis were used to examine associations.ResultsNinety-five patients (age: 45 ± 10 years; HbA1C level: 7.7% ± 0.8% [61 ± 7 mmol/mol]) were included (mean blood glucose [MBG]: 159 ± 31 mg/dL; TIR: 55.8% ± 14.9%; CV%: 43.5% ± 7.8%) and labeled as having good (HbA1C level ≤7% [≤53 mmol/mol]; n = 20), moderate (7%-8%; n = 44), or poor (>8% [>64 mmol/mol]; n = 31) glycemic control. HbA1C was significantly associated with MBG (r_s = 0.48, P < .001) and time spent in hyperglycemia (total: r_s = 0.52; level 2: r = 0.46; P < .001) but not with time spent in hypoglycemia and CV%, even after an analysis of the HbA1C subgroups. Similarly, TIR was negatively associated with HbA1C (r = −0.53; P < .001), MBG (r_s = −0.81; P < .001), and time spent in hyperglycemia (total: r_s = −0.90; level 2: r_s = −0.84; P < .001) but not with time in hypoglycemia. The subgroup analyses, however, showed that TIR was associated with shorter time spent in level-2 hypoglycemia in patients with good (r_s = −0.60; P = .007) and moderate (r_s = −0.25; P = .047) glycemic control. In contrast, CV% was strongly positively associated with time in hypoglycemia (total: r_s = 0.78; level 2: r_s = 0.76; P < .001) but not with TIR or time in hyperglycemia in the entire cohort, although the subgroup analyses showed that TIR was negatively associated with CV% in patients with good glycemic control (r = −0.81, P < .001) and positively associated in patients with poor glycemic control (r = +0.47; P < .01).ConclusionThe CGM-derived metrics TIR and CV% are related to clinically important situations, TIR being strongly dependent on hyperglycemia and CV% being reflective of hypoglycemic risk. However, the interpretation and applicability of TIR and CV% and their relationship depends on the level of glycemic control of the individual patient, with CV% generally adding less clinically relevant information in those with poor control. This illustrates the need for further research and evaluation of composite measures of glycemic control in T1DM.     

Effects of VEGF levels on anti-VEGF therapy for patients with idiopathic choroidal neovascularization

The objective of this study is to investigate the levels of vascular endothelial growth factor (VEGF) and other cytokines in aqueous humor of patients with idiopathic choroidal neovascularization (CNV) and their effects together with central retinal thickness (CRT) on the response to intravitreal injection of anti-VEGF antibody ranibizumab. This clinical study recruited 32 eyes from 32 patients with CNV under or besides fovea. VEGF, interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1 levels were detected in aqueous humor (0.1 ml) sampled during intravitreal injection. Aqueous humor controls were from nine cataract patients without any systemic disorders. The VEGF levels in aqueous humor were negatively related (r = ?0.373, p = 0.035) to CRT, which was positively related (r = 0.743, p < 0.001) to the number of injections. The VEGF levels before treatment and during the third injection in four patients with three or more injections were 13.42 ± 8.50 and 5.75 ± 3.68 (p = 0.055), respectively. The average best corrected visual acuity (BCVA) before and 12 months after treatment were 57.03 ± 16.15 and 75.16 ± 11.78 (p < 0.001), and the average CRT before and 12 months after treatment were 352.09 ± 84.15 and 251.13 ± 63.96 (p < 0.001), respectively. The visual improvement was negatively related (r = ?0.815, p < 0.001) to the visual baseline, and the vision 12 months after treatment was positively related (r = 0.581, p < 0.001) to that before treatment. No severe ocular or systemic complication appeared during treatment and follow-ups for all the patients. Intravitreal injection of anti-VEGF antibody ranibizumab is safe and effective for the treatment of idiopathic CNV through decreasing CRT. The patients with larger CRT baseline need more injections of ranibizumab.     

Does dietary arsenic and mercury affect cutaneous bleeding time and blood lipids in humans?

Fish species may contain considerable amounts of trace elements, such as selenium (Se), arsenic (As), and mercury (Hg). The present study investigated the relationships between dietary intake of these elements and cutaneous bleeding time and blood lipids in 32 healthy volunteers. For 6 wk, one group (n=11) consumed approx 250 g Se-rich fish daily, providing them with an average Se intake of 115±31 μg Se/d, Hg intake of 18±8 μg/d, and As intake of 806±405 μg/d, all values analyzed in 4-d duplicate food collections. To study the effect of Se alone, one group (n=11) included Se-rich bread in their normal diet, giving them a Se intake (135±25 μg/d) that was comparable to the fish group. A control group (n=10) ate their normal diet, providing 77±25 gmg Se/d, 3.1±2.5 μg Hg/d, and 101±33 μg As/d. The dietary As load strongly correlated both with bleeding times and changes in bleeding times (r=0.48,p<0.01 andr=0.54,p<0.002, respectively). Dietary Hg showed a positive correlation with LDL-cholesterol (r=0.55,p<0.01), whereas dietary Hg in the fish group showed a strong negative relationship with HDL-cholesterol (r=?0.76,p<0.01). Selenium seemed to have only a modest effect on bleeding time. Our results suggest that mercury and arsenic from fish may be factors contributing to or modifying some of the known effects of fish ingestion.     

Reduced Levels of Brain Derived Neurotrophic Factor (BDNF) in the Serum of Diabetic Retinopathy Patients and in the Retina of Diabetic Rats

Diabetic retinopathy (DR) is widely recognized as a neurovascular disease. Retina, being a neuronal tissue of the eye, produces neurotrophic factors for its maintenance. However, diabetes dysregulates their levels and thereby may damage the retina. Among neurotrophins, brain derived neurotrophic factor (BDNF) is the most abundant in the retina. In this study, we investigated the level of BDNF in the serum of patients with DR and also in the serum and retina of streptozotocin-induced diabetic rats. The level of BDNF was significantly decreased in the serum of proliferative diabetic retinopathy patients as compared to that of non-diabetic healthy controls (25.5 ± 8.5–10.0 ± 8.1 ng/ml, p < 0.001) as well as compared to that of diabetic patients with no retinopathy (21.8 ± 4.7–10.0 ± 8.1 ng/ml, p < 0.001), as measured by ELISA techniques. The levels of BDNF in the serum and retina of diabetic rats were also significantly reduced compared to that of non-diabetic controls (p < 0.05). In addition, the expression level of tropomyosin-related kinase B (TrkB) was significantly decreased in diabetic rat retina compared to that of non-diabetic controls as determined by Western blotting technique. Caspase-3 activity was increased in diabetic rat retina after 3 weeks of diabetes and remained elevated until 10 weeks, which negatively correlated with the level of BDNF (r = ?0.544, p = 0.013). Our results indicate that reduced levels of BDNF in diabetes may cause apoptosis and neurodegeneration early in diabetic retina, which may lead to neuro-vascular damage later in DR.     

Influence of chronic exposure to low doses of γ-radiation and <Superscript>90</Superscript>Sr on the level of DNA breaks and cell sensitivity to hydrogen peroxide in the mouse spleen

Using comet assay, a statistically significant increase (p < 0.05) in the level of DNA breaks in spleen cells was revealed in male CBA/lac mice exposed to γ-radiation (1.7 mGy/day) or ⁹⁰Sr (150–250 Bq/day) for 210 days. The level of DNA breaks also increased under combined exposure to both γ-radiation and ⁹⁰Sr (p < 0.05), but to a lesser degree than under exposure to each of these factors alone. Upon additional in vitro treatment of spleen cells with hydrogen peroxide, the relative increase in the level of DNA breaks was smaller in cells of irradiated mice than in the control. The ratio of the level of DNA breaks after hydrogen peroxide treatment to that before this treatment in control mice was 4.2 ± 0.9, compared to 1.4 ± 0.6 in γ-irradiated mice, 1.9 ± 0.8 in ⁹⁰Sr-irradiated mice, and 2.3 ± 0.8 in mice exposed to both γ- and ⁹⁰Sr-irradiation.     

Assessment of DNA damage in sperm after repeated non‐invasive sampling in zebrafish Danio rerio

Repeated non‐invasive sampling of zebrafish Danio rerio sperm was conducted, sperm counts were obtained and a method for measurement of DNA damage in sperm was developed and validated (single‐cell gel electrophoresis, comet, assay). DNA damage in sperm increased with concentration of hydrogen peroxide (H₂O₂, 0–200 µM), and in vitro exposure of sperm to 200 µM H₂O₂ produced 88·7 ± 3·9% tail DNA compared to unexposed controls [12 ± 0·7% tail DNA (mean ± s.e ., n = 3)]. Frequency of sperm sampling (sampled every 2, 4 or 7 days) did not affect DNA damage in sperm, but sperm counts decreased 57 and 22% for fish sampled every 2 or 4 days, respectively.     

Insulin resistance is associated with DNA damage in peripheral blood cells in non-diabetic patients with genotype 1 chronic hepatitis C

Abstract

Background. In chronic liver diseases of different etiologies, including viral hepatitis, genotoxic effects of oxidative stress have been shown, both in clinical and in experimental conditions, suggesting that this mechanism may contribute to the evolution of the disease. Aim. To evaluate DNA damage in the peripheral blood of untreated non-diabetic patients with chronic hepatitis C and control subjects, and its correlation with demographic, anthropometric, biochemical, and histological parameters in the patient sample. Patients and methods. This study comprised 100 subjects of both genders, 60 of whom were treatment-naïve patients with positive serology for genotype 1 hepatitis C. The remaining 40 were blood donors with negative serology for hepatitis who were used as control subjects, and matched by gender, age, weight, and BMI. DNA damage was determined using the comet assay in the total peripheral blood. Results. The DNA damage evaluated by the comet assay revealed higher values in the group of patients with hepatitis compared with that in the control group. The relationships of the comet assay with the studied variables were assessed using multivariate analysis; significant correlations were only identified with insulin (r = 0.343, p = 0.008) and Homeostasis Model Assessment Insulin Resistance (HOMA-IR) (r = 0.331, p = 0.011). Conclusion. Patients with genotype 1 chronic hepatitis C have higher rates of DNA damage, as determined by comet assay and this alteration is correlated with the HOMA index of insulin resistance.     

Oxidative stress and increased formation of vasoconstricting F2-isoprostanes in patients with reversible cerebral vasoconstriction syndrome

The pathophysiology of reversible cerebral vasoconstriction syndrome (RCVS) is unknown. Oxidative stress is detrimental to endothelial function and vascular reactivity. We hypothesized that the oxidative stress marker 8-iso-prostaglandin F_2α, which is also a potent vasoconstrictor, might contribute to the pathogenesis of RCVS. Recruited participants included 103 RCVS patients, 53 patients with primary headache with acute severe attacks, and 54 healthy controls. Subjects recruited prior to 2009 were discovery cohort, whereas those after 2009, replication cohort. Urine samples were obtained from all patients at registration and from 79 patients with RCVS again at remission stage. Urine 8-iso-prostaglandin F_2α was analyzed by liquid chromatography-tandem mass spectrometry. Patients with RCVS received magnetic resonance angiography and transcranial color-coded sonography. In RCVS patients, the urine 8-iso-prostaglandin F_2α level was higher than that in the other groups in discovery, replication, and combined cohorts (RCVS, 0.29±0.18; primary headache with acute severe attacks, 0.21±0.19; control, 0.18±0.09 ng/mg creatinine; P<0.001), and it was positively correlated with the flow velocities of major intracranial arteries, especially within the first week of disease onset (middle cerebral artery, Spearman's correlation coefficient [r_s]=0.580, P=0.002; anterior cerebral artery, r_s=0.472, P=0.042; posterior cerebral artery, r_s=0.457, P=0.022; basilar artery, r_s= 0.530, P=0.002). The 8-iso-prostaglandin F_2α level decreased from the ictalto remission stage in RCVS patients (0.31±0.21 vs 0.16±0.10 ng/mg creatinine, P<0.001). 8-Iso-prostaglandin F_2α was higher in patients with RCVS and correlated with the severity of vasoconstrictions. Further studies are required to explore its potential pathogenic role.     

Alterations in serum selenium levels and their relation to troponin I in acute myocardial infarction

Selenium (Se) is an essential trace element with antioxidant function. The aim of the present study was to estimate the alterations of Se serum level during the acute phase of myocardial infarction and its relation to biomarkers of myocardial necrosis. Serum Se levels were measured at admission and after 24 h in 60 consecutive patients with acute coronary syndrome (both with and without ST elevation). Troponin I (TnI) was assessed at admission and then twice daily for 3 days; patients with normal levels were excluded. Fifty-five patients with acute MI (positive TnI) were included into the analysis. During the first day of hospitalization, patients received standard therapy, including acetylsalicylic acid, clopidogrel, and heparin or enoxaparin; all underwent urgent coronary angiography and percutaneous intervention, when appropriate. Mean Se levels at baseline and 24 h later were comparable (67.1 ± 2.1 vs. 67.2 ± 1.8 μg/L, ns). Linear regression has shown significant correlation between baseline Se levels and peak TnI (y = 3.4x ? 116, r ² = 0.13, P = 0.008). Positive correlation was found also between the peak TnI and the difference from baseline to 24 h (y = 2.2x + 115, r ² = 0.08, P = 0.04). Moreover, close negative correlation was observed between baseline Se levels and the difference from baseline to 24 h (y = ?0.9x + 62.7, r ² = 0.55, P<0.001). Our results have shown marked individual changes in Se levels during the acute phase of MI as well as correlation between Se levels and peak TnI. These results suggest that alterations in serum Se may be related to the extent of myocardial infarction.