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Assessment of DNA damage in Down Syndrome patients by means of a new, optimised single cell gel electrophoresis technique

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Assessment of DNA damage in Down Syndrome patients by means of a new, optimised single cell gel electrophoresis technique

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Abstract

Single cell gel electrophoresis (SCGE), also known as comet assay is a widely used method to detect DNA damage. Its use is nonetheless subjected to some pitfalls, due to differences in experimental set-up, to operator-dependent variability and to quantification of the comets, which is usually accomplished by visual scoring or by image-analysis software. Biological variability in the extent of DNA damage must be taken into account particularly regarding in vivo studies. In the present paper we propose an improved methodology where major features are: a) cryopreservation of lymphocytes collected at different time points and simultaneous analysis in a single run; b) use of an internal control on each slide; c) development of a custom-made software with semi - automated image analysis in order to overcome operator dependent variability. Cryopreservation was accomplished by storing lymphocytes in liquid nitrogen in a solution commonly used for preserving vital cells to be reinfused. We found that this procedure did not alter DNA after 2 and 4 months of storage. The use of quality control from a batch of aliquoted lymphocytes from a healthy donor on each slide, enabled to highlight possible experimental anomalies as well as verify inter-experimental variability. Moreover, by using a newly developed software able to automatically recognise comets we minimised operator-dependent variability in the scoring process. This improved methodology is proposed for longitudinal in vivo studies and in the present work its application made it possible to assess a significant increase of DNA in pediatric Down Syndrome patients compared to healthy controls of the same age.

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... In parallel, samples collected in EDTA vacutainer were immediately treated with a cryopreserving solution (human albumin at 5% and 20% and Dimethyl sulfoxide; 3:1:1), quickly frozen in dry ice and stored at −80°C in order to maintain cellular integrity of peripheral blood mononuclear cells (PBMCs). Whole blood cryopreservation methodology was adapted from a previous protocol on isolated cells [30] and was carefully optimized in order to verify stability of the cryopreserved cells over time. Data produced a viability of recovered cells well over 80% and flow cytometric distribution of major endpoints was reproducible over a period of 6 months. ...
... Oxidative DNA damage was conducted on cryopreserved cells as described above in according method of Tiano et al. [30]. Results are reported as percentage of DNA in the comet tail or tail intensity for each cell expressed as means of the median at different experimental points. ...
Article
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Objectives: Physical exercise significantly impacts the biochemistry of the organism. Ubiquinone is a key component of the mitochondrial respiratory chain and ubiquinol, its reduced and active form, is an emerging molecule in sport nutrition. The aim of this study was to evaluate the effect of ubiquinol supplementation on biochemical and oxidative stress indexes after an intense bout of exercise. Methods: 21 male young athletes (26 + 5 years of age) were randomized in two groups according to a double blind cross-over study, either supplemented with ubiquinol (200 mg/day) or placebo for 1 month. Blood was withdrawn before and after a single bout of intense exercise (40 min run at 85% maxHR). Physical performance, hematochemical parameters, ubiquinone/ubiquinol plasma content, intracellular reactive oxygen species (ROS) level, mitochondrial membrane depolarization, paraoxonase activity and oxidative DNA damage were analyzed. Results: A single bout of intense exercise produced a significant increase in most hematochemical indexes, in particular CK and Mb while, on the contrary, normalized coenzyme Q10 plasma content decreased significantly in all subjects. Ubiquinol supplementation prevented exercise-induced CoQ deprivation and decrease in paraoxonase activity. Moreover at a cellular level, in peripheral blood mononuclear cells, ubiquinol supplementation was associated with a significant decrease in cytosolic ROS while mitochondrial membrane potential and oxidative DNA damage remained unchanged. Discussion: Data highlights a very rapid dynamic of CoQ depletion following intense exercise underlying an increased demand by the organism. Ubiquinol supplementation minimized exercise-induced depletion and enhanced plasma and cellular antioxidant levels but it was not able to improve physical performance indexes or markers of muscular damage.
... Cellular integrity for DNA analysis was ensured by using a cryopreservation medium. (14) Flow cytometric investigations for the Mitochondrial Membrane Potential (MMP) and intracellular ROS levels were performed immediately. ...
... This software has several advantages over commercial software, allowing a semi-automatic analysis which greatly reduces operator-dependent variability. (14) Results are reported as percentage of DNA in the comet tail or tail intensity (TI) for each cell expressed as means of the median at different experimental points. ...
Article
Full-text available
Reactive oxygen species not only cause damage but also have a physiological role in the protection against pathogens and in cell signalling. Mitochondrial nutrients, such as coenzyme Q10 and α-lipoic acid, beside their acknowledged antioxidant activities, show interesting features in relation to their redox state and consequent biological activity. In this study, we tested whether oral supplementation with 200 mg/day of coenzyme Q10 alone or in association with 200 mg/die of α-lipoic acid for 15 days on 16 healthy subjects was able to modulate the oxidative status into different compartments (plasma and cells), in basal condition and following an oxidative insult in peripheral blood lymphocytes exposed in vitro to H2O2. Data have shown that tested compounds produced antioxidant and bioenergetic effects improving oxidative status of the lipid compartment and mitochondrial functionality in peripheral blood lymphocytes. Simultaneously, an increased intracellular reactive oxygen species level was observed, although they did not lead to enhanced DNA oxidative damage. Coenzyme Q10 and α-lipoic acid produced beneficial effects also steering intracellular redox poise toward a pro-oxidant environment. In contrast with other antioxidant molecules, pro-oxidant activities of tested mitochondrial nutrients and consequent oxidant mediated signalling, could have important implications in promoting adaptive response to oxidative stress.
... Observations were performed at a magnification of 200×. A total of 200 Comet images for each treatment at each time point were acquired in triplicate and subsequently image processed using a custom made analytical software able automatically detect comet and calculate the major DNA damage index: Tail length (TL), Tail moment (TM), and Tail intensity (TI) [50,51]. ...
Article
Full-text available
The extensive employment of copper-based fungicides has increased copper concentration in vineyard soils. The present study’s objectives were to monitor copper concentration in two vineyard soils during two cropping seasons and study the ecotoxicological effects on the earthworm Eisenia fetida. Total, soluble, and bioavailable copper fractions were measured at the end of two cropping seasons and different depths in two vineyards of central Italy, characterised by three anticryptogamic control methods: copper compounds, chitosan, and combined treatments of them. A laboratory experiment to assess the effects on Eisenia fetida was conducted with soil samples collected in the vineyards with a mean copper concentration of 60 mg/kg and two higher concentrations of 90 and 150 mg/kg. Results showed low levels of total copper concentration in the first 20 cm of soils, regardless of antifungal treatment, highlighting prudent management of the vineyards under study, but the soluble fractions showed a significant increase in all samples during the two cropping seasons. At the dose of 150 mg/kg, earthworms suffer during the first two days, showing weight loss and DNA damage, but they are able to recover until day 28, showing no permanent harm at this copper concentration in soil.
... Electrophoresis was conducted at 11 V cm − 1 for 20 min at 4 • C. Slides were washed in H 2 O, neutralised in buffer (0.4 M Tris-HCl pH 7.5), dehydrated in 75 % methanol (Valverde et al., 1999;Mincarelli et al., 2016), stained with Sybr Gold and then imaged using Lionheart FX Automated Microscope (Biotek, U.S.A.) at 200 × 200 magnification. Comet images were acquired in triplicate and processed to calculate the major DNA damage index: Tail length (TL), Tail moment (TM), and Tail intensity (TI) (Tiano et al., 2005;Orlando et al., 2018). ...
Article
Earthworms and microbial communities are essential non-target soil organisms that are useful to assess the collateral impact of pesticides. The present paper reports three laboratory experiments performed to investigate the effects of sub-lethal doses of two insecticides, a biologically-derived (spinosad) and a synthetic organophosphate (chlorpyrifos), on earthworm Eisenia foetida and microorganisms in organic soil. The effects were studied in terms of behaviour, reproduction, survival, and DNA damage (comet assay) in earthworms, and Next Generation Sequencing-Illumina was employed to detect the changes in the microbial community. In addition, the influence of earthworms on the degradation kinetics of insecticides and on microbial diversity was evaluated. The weights, reproductive activity and behaviour of earthworms were particularly compromised and followed a dose-dependent trend in chlorpyrifos trials, where the insecticide's degradation wasn't affected by the presence of Eisenia foetida. However, earthworms contributed to spinosad's metabolisation without significantly impacting their health. Early DNA damage was estimated in earthworms exposed to chlorpyrifos, while the impact of spinosad was significant only at the end of the toxicity test. The analysis on the microbial community indicated the buffering effect earthworms had on the bacterial communities starting from earliest sampling until the end of the trial, as well as bacterial community members' degradation response to pesticides over time.
... In this view abnormal expression of trisomic genes, in association with responses to environmental stimuli might alter the expression of disomic genes as well. A pivotal role in these processes is played by reactive oxygen species both in relation to the acknowledged condition of oxidative stress experienced by DS [9,16,18] patients and to their activity as mediators of gene expression [17]. Oxidative stress is known to occur in DS from very early stages [3]: already during embryonic development mitochondrial dysfunction has been reported also as a marker of oxidative damage [7]. ...
Article
Down syndrome (DS) is a chromosomal abnormality (trisomy 21) associated with mental retardation and Alzheimer-like dementia, characteristic change of the individual's phenotype and premature ageing. Oxidative stress is known to play a major role in this pathology since a gene dose effect leads to elevated ratio of superoxide dismutase to catalase/glutathione peroxidase compared to controls in all age categories suggesting that oxidative imbalance contributes to the clinical manifestation of DS. Hyperuricemia is another feature of DS that has an interesting relationship with oxidative stress since uric acid represents an important free radical scavenger. However its formation is connected to the conversion of Xanthine dehydrogenase (XDH) to Xanthine oxidase (XO) which leads to concomitant production of free radicals. Here we report that plasma samples from DS patients in pediatric age, despite an increased total antioxidant capacity, largely due to elevated Uric acid content (UA), present significantly elevated markers of oxidative damage such as increased allantoin levels. Moreover DS plasma samples do not differ from healthy control ones in terms of Coenzyme Q10 and susceptibility to peroxidative stimuli. On the contrary, lymphocyte and platelet CoQ10 content was significantly lower in DS patients, a fact that might underlie oxidative imbalance at a cellular level.
... This software has several advantages over commercial software that allows a semiautomatic analysis greatly reducing operator-dependent variability. More details on the software are described in [23]. For each comet, data relative to tail length (TL), tail migration (TMi), percentage tail DNA (TI), and tail moment (TM) were recorded. ...
Article
Numerous sunscreens contain titanium dioxide (TiO(2)) because of its ability to reflect, scatter, and absorb UV radiation, thus preventing sunlight-related skin disorders. Since TiO(2) is well known to generate reactive oxygen species (ROS) under photoexcitation, it is chemically modified when used in sunscreens. In the present study, five modified TiO(2) particles, specifically developed and marketed for sunscreens, were tested using different in vitro models, including cultured human skin fibroblasts (HuDe), to investigate their possible photocatalytic effects following UVA exposure. The results obtained show that the type of modification and crystal form determine their ability to (a) induce photobleaching of the DPPH radical, (b) photodegrade deoxyribose, (c) reduce cell viability, (d) increase/decrease DNA damage, and (e) increase/decrease intracellular ROS. This research concludes that some modified TiO(2) particles still retain photocatalytic activity under the experimental conditions employed, especially those in which the anatase crystal form of TiO(2) is present. The penetration of TiO(2) nanosized particles into the viable epidermis of skin is still under debate; thus, the results presented here contribute to gaining further knowledge on the potential effects of TiO(2) particles at the cellular level, in the worst possible case that they do penetrate.
... Supplementation with ubiquinol-10 reduces oxidative markers in plasma [50] and support the rationale for CoQ 10 supplementation in DS. We have also shown significantly increased levels of DNA damage in peripheral blood leucocytes of DS children compared to healthy controls of the same age [51,52]. The protective effect of CoQ 10 against oxidative DNA damage in lymphocytes from healthy volunteers has already been reported in the literature [53,54] and interpreted as being associated with antioxidant activity or modulation of DNA repair enzymes. ...
Article
Structural changes and abnormal function of mitochondria have been documented in Down's syndrome (DS) cells, patients, and animal models. DS cells in culture exhibit a wide array of functional mitochondrial abnormalities including reduced mitochondrial membrane potential, reduced ATP production, and decreased oxido-reductase activity. New research has also brought to central stage the prominent role of oxidative stress in this condition. This review focuses on recent advances in the field with a particular emphasis on novel translational approaches involving the utilization of coenzyme Q(10) (CoQ(10) ) to treat a variety of clinical phenotypes associated with DS that are linked to increased oxidative stress and energy deficits. CoQ(10) has already provided promising results in several different conditions associated with altered energy metabolism and oxidative stress in the CNS. Two studies conducted in Ancona investigated the effect of CoQ(10) treatment on DNA damage in DS patients. Although the effect of CoQ(10) was evidenced only at single cell level, the treatment affected the distribution of cells according to their content in oxidized bases. In fact, it produced a strong negative correlation linking cellular CoQ(10) content and the amount of oxidized purines. Results suggest that the effect of CoQ(10) treatment in DS not only reflects antioxidant efficacy, but likely modulates DNA repair mechanisms.
... Many studies regarding increased DNA damage in DS are in agreement with the observations of the present study. [8,11,41] Musalmah et al., (2006) and Ponte et al., [25,42] reported an increase in DNA damage in lymphocytes of individuals with DS compared with controls, which was reduced by more than 50% after the addition of Vitamin E to the cell culture. As Vitamin E is a powerful antioxidant, they hypothesized that the increased DNA damage in DS resulted from increased oxidative stress with a protective effect of Vitamin E on chromosomal damage. ...
Article
Oxidative stress plays a major role in the pathogenesis of leukemia-prone diseases such as Fanconi anemia (FA) and Down syndrome (DS). To explore the oxidative stress state in children with DS and FA by estimating the levels of antioxidants (e.g., malondialdehyde [MDA], total antioxidant capacity, and superoxide dismutase [SOD] activity) and DNA damage, and to evaluate of the effect of antioxidant treatment on these patients. The study included 32 children clinically diagnosed with (15 patients) and FA (17 patients) in addition to 17 controls matched for age and sex. MDA, total antioxidant capacity, SOD activity, and DNA damage were measured. Antioxidants including Vitamin A, E, and C were given to the patients according to the recommended daily allowance for 6 months. Clinical follow-up and re-evaluation were conducted for all patients. Laboratory tests including complete blood count, karyotyping, DNA damage, and oxidative stress were re-evaluated. Statistical analysis was performed using statistical computer program Statistical Package for the Social Sciences version 14.0. Children with FA and DS had elevated levels of oxidative stress and more DNA damage than controls. Oxidative stress parameters and DNA damage improved in FA and DS patients after antioxidant administration. Early administration of antioxidants to FA and DS patients is recommended for slowing of the disease course with symptoms amelioration and improvement of general health.
... The formation of thymine dimer (T^T) within human genomic DNA has been detected by immuno-coupled PCR (IC-PCR) (Karakoula et al., 2003). DNA damage such as SSBs, DSBs and oxidative DNA damage caused by UVR, ultrasound electromagnetic frequency radiation etc. may be detected by comet assay (Olive et al., 1990;Tiano et al., 2005;Heepchantree et al., 2006). Recently a modified version of comet assay i.e., apo/necro-comet assay has been developed that differentiate viable, apoptotic and necrotic cells and also correlate the DNA fragmentation pattern (Moreley et al., 2006). ...
Chapter
The correct execution of the DNA replication process is crucially important for the maintenance of genome integrity of the cell. Several types of sources, both endogenous and exogenous, can give rise to DNA damage. The most potent carcinogenic forms of UV-induced DNA lesions are CPDs, 6-4PPs and their Dewar isomers. The incidence of UVR, IR and certain genotoxic chemicals may result in single as well as double DNA strand breaks (DSBs) leading to arrest of DNA replication fork that may impede with normal cellular capability and functional integrity, reduction of RNA synthesis, arrest of cell cycle progression, mutagenesis, tumorigenesis and apoptosis. A number of detection strategies have been devised to detect different types of DNA lesions. To overcome the above malfunctions, nature has invented certain specific repair mechanisms such as photoreactivation, excision repair, mismatch repair (MMR), double strand break (DSB) repair and certain other mechanisms like damage tolerance (dimer bypass), prokaryotic SOS response and cell cycle checkpoint activation in organisms. The defects in repair machinery may alter the configuration of cell structure, lipids, proteins and nucleic acids and can cause a number of human diseases.
... Furthermore, by incubating the lysed cells in RNase and proteinase K, the sensitivity of the assay could be increased Stephens, 1997, 1998). DNA damage in the patients with Down syndrome has been assessed by using new optimized comet assay (Tiano et al., 2005). Recently, a modified version of comet assay i.e. apo/necro-comet-assay has been devised that differentiate viable, apoptotic and necrotic cells and determines the viability status of individual cells. ...
Article
Even under the best of circumstances, DNA is constantly subjected to chemical modifications. Several types of DNA damage such as SSB (single strand break), DSB (double strand break), CPDs (cyclobutane pyrimidine dimers), 6-4PPs (6-4 photoproducts) and their Dewar valence isomers have been identified that result from alkylating agents, hydrolytic deamination, free radicals and reactive oxygen species formed by various photochemical processes including UV radiation. There are a number of strategies such as PCR (polymerase chain reaction), comet, halo, TUNEL (Terminal deoxyribonucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling) assay, HPLC-Electrospray tandem mass spectrometry, FISH (Fluorescence in situ hybridization), FCM (Flow cytometry), annexin V labeling, immunological assays including immunofluorescent and chemiluminescence thymine dimer detection, immunohistochemical assay, Enzyme-linked immunosorbent assay (ELISA), Radio immunoassay (RIA), Gas chromatography-mass spectrometry and electrochemical methods, that are commonly used to detect DNA damage in various organisms. The main aim of this review is to present a brief account of the above mentioned DNA damage detection strategies for the convenience of interested readers.
... We are currently evaluating the results of a clinical trial conducted in patients affected by Down syndrome treated with CoQ 10 . The aim of this study is to measure the extent of DNA damage, and the effect of CoQ 10 administration, by means of a new, optimized, single-cell gel electrophoresis technique [30]. ...
Article
Full-text available
For a number of years, coenzyme Q (CoQ10 in humans) was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in plasma, and extensively investigated its antioxidant role. These two functions constitute the basis on which research supporting the clinical use of CoQ10 is founded. Also at the inner mitochondrial membrane level, coenzyme Q is recognized as an obligatory co-factor for the function of uncoupling proteins and a modulator of the transition pore. Furthermore, recent data reveal that CoQ10 affects expression of genes involved in human cell signalling, metabolism, and transport and some of the effects of exogenously administered CoQ10 may be due to this property. Coenzyme Q is the only lipid soluble antioxidant synthesized endogenously. In its reduced form, CoQH2, ubiquinol, inhibits protein and DNA oxidation but it is the effect on lipid peroxidation that has been most deeply studied. Ubiquinol inhibits the peroxidation of cell membrane lipids and also that of lipoprotein lipids present in the circulation. Dietary supplementation with CoQ10 results in increased levels of ubiquinol-10 within circulating lipoproteins and increased resistance of human low-density lipoproteins to the initiation of lipid peroxidation. Moreover, CoQ10 has a direct anti-atherogenic effect, which has been demonstrated in apolipoprotein E-deficient mice fed with a high-fat diet. In this model, supplementation with CoQ10 at pharmacological doses was capable of decreasing the absolute concentration of lipid hydroperoxides in atherosclerotic lesions and of minimizing the size of atherosclerotic lesions in the whole aorta. Whether these protective effects are only due to the antioxidant properties of coenzyme Q remains to be established; recent data point out that CoQ10 could have a direct effect on endothelial function. In patients with stable moderate CHF, oral CoQ10 supplementation was shown to ameliorate cardiac contractility and endothelial dysfunction. Recent data from our laboratory showed a strong correlation between endothelium bound extra cellular SOD (ecSOD) and flow-dependent endothelial-mediated dilation, a functional parameter commonly used as a biomarker of vascular function. The study also highlighted that supplementation with CoQ10 that significantly affects endothelium-bound ecSOD activity. Furthermore, we showed a significant correlation between increase in endothelial bound ecSOD activity and improvement in FMD after CoQ10 supplementation. The effect was more pronounced in patients with low basal values of ecSOD. Finally, we summarize the findings, also from our laboratory, on the implications of CoQ10 in seminal fluid integrity and sperm cell motility.
... These include any in vitro or ex vivo cell exposure and key stages of the Comet protocol such as slide preparation, cell lysis and electrophoresis conditions (including homogeneity of the agarose layers, electrical field inhomogeneity inside the tank, buffer variations), and also comet analysis (5). In attempts to reduce/minimize such variation, several clinical and human biomonitoring studies have included supposed 'internal' standards in which untreated or treated 'reference' cells were analysed alongside the test cells as 'negative' and 'positive' controls, respectively (16)(17)(18)(19). However, in all these studies the reference cells are present in separate gels to the test cells, so it is more appropriate to consider these as 'external' or 'parallel' standards, rather than true internal standards, as they will not account for inter-gel variations. ...
Article
The Comet assay (CA) is a sensitive/simple measure of genotoxicity. However, many features of CA contribute variability. To minimize these, we have introduced internal standard materials consisting of 'reference' cells which have their DNA substituted with BrdU. Using a fluorescent anti-BrdU antibody, plus an additional barrier filter, comets derived from these cells could be readily distinguished from the 'test'-cell comets, present in the same gel. In experiments to evaluate the reference cell comets as external and internal standards , the reference and test cells were present in separate gels on the same slide or mixed together in the same gel, respectively, before their co-exposure to X-irradiation. Using the reference cell comets as internal standards led to substantial reductions in the coefficient of variation (CoV) for intra-and inter-experimental measures of comet formation and DNA damage repair; only minor reductions in CoV were noted when the reference and test cell comets were in separate gels. These studies indicate that differences between individual gels appreciably contribute to CA variation. Further studies using the reference cells as internal standards allowed greater significance to be obtained between groups of replicate samples. Ultimately, we anticipate that development will deliver robust quality assurance materials for CA.
... After washing in H 2 O, neutralization in Tris buffer (pH 7.5) for 5 min, and dehydration in 50% methanol, the slides were dried at 50°C. DNA on each slide was stained with 15 μL ethidium bromide (20 μg/mL), and the comets were analyzed using fluorescence microscopy using a dedicated imaging software developed as previously reported [25]. For each comet, data relative to tail length (TL), tail migration (TMi), percent tail DNA (TI), and tail moment (TM) were recorded. ...
Article
Full-text available
Reactive oxygen species (ROS) production in the skin is among the highest compared to other organs, and a clear correlation exists between ROS production and skin aging. Many attempts are underway to reduce oxidative stress in the skin by topical treatment or supplementation with antioxidants/cosmeceuticals, and cultures of human dermal fibroblasts (HDF) are widely used for these studies. Here, we examined the influence of oxygen tension on cell aging in HDF and how this impacted ROS production, the enzymatic and nonenzymatic antioxidant response system, and the efficacy of this defense system in limiting DNA damage and in modulating gene expression of proteins involved in the extracellular matrix, linked to skin aging. We investigated a selection of parameters that represent and reflect the behavior of cellular responses to aging and oxygen tension. Serial passaging of HDF under normoxia (21%) and hypoxia (5%) leads to cell aging as confirmed by β-galactosidase activity, p16 expression, and proliferation rate. However, in HDF under 21% O2, markers of aging were significantly increased compared to those under 5% O2 at matched cell passages despite having lower levels of intracellular ROS and higher levels of CoQ10, total GSH, SOD1, SOD3, and mitochondrial superoxide anion. miRNA-181a, which is known to be upregulated in HDF senescence, was also analyzed, and indeed, its expression was significantly increased in old cells at 21% O2 compared to those at 5% O2. Upregulation of MMP1 and downregulation of COL1A1 along with increased DNA damage were also observed under 21% O2 vs 5% O2. The data highlight that chronic exposure to atmospheric 21% O2 is able to trigger hormetic adaptive responses in HDF that however fail, in the long term, to prevent cellular aging. This information could be useful in further investigating molecular mechanisms involved in adaptation of skin fibroblasts to oxidative stress and may provide useful hints in addressing antiaging strategies.
... Subsequently DNA was allowed to unwind for 30 min in alkaline electrophoresis buffer at 4°C in the dark and finally run in electrophoretic chamber (1 V/cm 30 min). Resulting DNA Comets were measured by fluorescence microscopy coupled with a dedicated image analysis software and quantified in terms of percentage of DNA in the tail intensity (TI) Tail length (TL) and Tail moment (TM) (Tiano et al. 2005). ...
Article
Full-text available
The paper reports the results of a laboratory test on the bioaccumulation and toxicological effects of sub-lethal soil concentration of copper, a widely used fungicide in organic farming, on DNA damage, a critical marker increasingly used in ecotoxicology in the earthworm Eisenia andrei. In the same experimental setting we evaluated gene expression of classical biomarker of stress induced by xenobiotic. [Heat Shock Protein 70 (HSP70) and Metallothionein (MET)], as well as genes coding for enzymes involved in detoxification of reactive oxygen species [Superoxide dismutase (SOD) and catalase (CAT)]. Additionally, expression of genes involved in the immune response were investigated: a Toll-like receptor (TLR), a receptor with cytolytic activity named Cytolytic Factor (CCF) and two antimicrobial peptides, fetidin (FET) and lysenin (LYS). Results showed significant time-dependent bioaccumulation of Cu and DNA damage at concentrations remarkably lower than those found in most agricultural soils worldwide. MET was increased as was FET and TLR. The present work gives new insights into the mechanisms of sub-lethal toxicity of copper as an environmental pollutant and in the identification of novel sub-lethal biomarkers of cellular response to the stressor such as immune response genes.
... For each sample, fifteen randomly acquired images were recorded and processed using a custom made software Marche [13] based on Labview programming platform (National Instruments) that enables automatic identification of the comets, greatly reducing operator-dependent variability. A key feature of the software is its ability to identify the comets from the background and to estimate the commonly used DNA-damage in-dexes. ...
Conference Paper
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The Comet Assay is a well-known procedure employed to investigate the DNA damage and can be applied to several research areas such as environmental, medical and health sciences. User dependency and computation time effort represent some of the major drawbacks of the Comet Assay. Starting from this motivation, we applied a Machine Learning (ML) tool for discriminating DNA damage using a standard hand-crafted feature set. The experimental results demonstrate how the ML tool is able to objectively replicate human experts scoring (accuracy detection up to 92%) by solving the related binary task (i.e., controls vs damaged comets).
... Dataset images were annotated using a custom made software Marche [31] based on Labview programming platform (National Instruments). Firstly the software en-155 ables an initial automatic identification of the comets from the back-ground. ...
Article
Full-text available
Background and Objective DNA damage analysis can provide valuable information in several areas ranging from the diagnosis/treatment of a disease to the monitoring of the effects of genetic and environmental influences. The evaluation of the damage is determined by comet scoring, which can be performed by a skilled operator with a manual procedure. However, this approach becomes very time-consuming and the operator dependency results in the subjectivity of the damage quantification and thus in a high inter/intra-operator variability. Methods In this paper, we aim to overcome this issue by introducing a Deep Learning methodology based on Faster R-CNN to completely automatize the overall approach while discovering unseen discriminative patterns in comets. Results The experimental results performed on two real use-case datasets reveal the higher performance (up to mean absolute precision of 0.74) of the proposed methodology against other state-of-the-art approaches. Additionally, the validation procedure performed by expert biologists highlights how the proposed approach is able to unveil true comets, often unseen from the human eye and standard computer vision methodology. Conclusions This work contributes to the biomedical informatics field by the introduction of a novel approach based on established object detection Deep Learning technique for evaluating the DNA damage. The main contribution is the application of Faster R-CNN for the detection and quantification of DNA damage in comet assay images, by fully automatizing the detection/classification DNA damage task. The experimental results extracted in two real use-case datasets demonstrated (i) the higher robustness of the proposed methodology against other state-of-the-art Deep Learning competitors, (ii) the speeding up of the comet analysis procedure and (iii) the minimization of the intra/inter-operator variability.
... Thus, increased levels of endogenous oxygen species have been observed in lymphocytes from individuals with DS as compared with controls [104]. Additionally, higher levels of macromolecular damage are present in DS subjects, especially occurring on DNA molecules in lymphocytes [104][105][106][107][108], and an acceleration of telomere loss has been described in DS lymphocytes as well [109]. Finally, epigenetic patterns are modified, with differential blood DNA methylation signatures in DS individuals [110,111] and age-related changes in the epigenetic machinery [112]. ...
Article
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Down syndrome is the most common chromosomal disorder, associated with moderate to severe intellectual disability. While life expectancy of Down syndrome population has greatly increased over the last decades, mortality rates are still high and subjects are facing prematurely a phenomenon of atypical and accelerated aging. The presence of an immune impairment in Down syndrome subjects is suggested for a long time by the existence of an increased incidence of infections, the incomplete efficacy of vaccinations, and a high prevalence of autoimmunity. Immunologic abnormalities have been described since many years in this population, both from a numerical and a functional points of view, and these abnormalities can mirror the ones observed during normal aging. In this review, we summarize our knowledge on immunologic disturbances commonly observed in subjects with Down syndrome, and in innate and adaptive immunity, as well as regarding chronic inflammation. We then discuss the role of accelerated aging in these observed abnormalities and finally review the potential age-associated molecular and cellular mechanisms involved.
Chapter
The genome is continuously damaged due to both exogenous agents and endogenous processes. Exogenous agents are, for example, radiation or chemical compounds, whereas endogenous DNA damage mainly occurs due to reactive oxygen species (ROS) generated by cellular metabolic processes. In order to ensure DNA integrity cells have developed specialized DNA repair mechanisms. If DNA lesions are not detected or not resolved effectively DNA damage persists. Accumulation of DNA damage and the incapability of the cellular DNA repair machinery to fix DNA lesions seem to be contributors to aging and to age-associated diseases such as cancer. However, appropriated nutrition can protect against oxidative DNA damage and enhance DNA repair; therefore, dietary interventions might slow down aging processes and decrease cancer incidence.
Chapter
During the past decades, life expectancy of subjects with Down syndrome (DS) has greatly improved, but age-specific mortality rates are still important and DS subjects are characterized by an acceleration of the ageing process, which affects particularly the immune and central nervous systems. In this chapter, we will first review the characteristics of the ageing phenomenon in brain and in immune system in DS and we will then discuss the biological hallmarks of ageing in this specific population. Finally, we will also consider in detail the knowledge on epigenetics in DS, particularly DNA methylation.
Chapter
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For a number of years, coenzyme Q (CoQ10 in humans), was known for its key role in mitochondrial bioenergetics; later studies demonstrated its presence in other subcellular fractions and in plasma, and also extensively investigated its antioxidant role. This chapter discusses the relationship between the acknowledged bioenergetic role of CoQ10 and some clinical effects. The antioxidant properties of CoQ10 are then analyzed especially for their consequences on protection of circulating human low-density lipoproteins and prevention of atherogenesis. The relationship between CoQ10 and statins is also discussed in the light of possible involvement of CoQ10 deficiency in the issue of statin side effects. New aspects of the antioxidant involvement of coenzyme Q are also discussed together with their relevance in cardiovascular disease. Data are reported on the efficacy of CoQ10 in ameliorating endothelial dysfunction in patients affected by ischemic heart disease. Many of the effects of CoQ10, which were classically ascribed to its bioenergetic properties, are now considered as the result of its biochemical interaction with nitric oxide (NO), NO synthase and reactive oxygen species capable of inactivating NO. Clinical studies are reported highlighting the effect of CoQ10 on extracellular SOD, which is deeply involved in endothelial dysfunction. Previous studies have shown decreased levels of CoQ10 in the seminal plasma and sperm cells of infertile men with different kinds of asthenospermia. Research has been extended to supplementation with CoQ10 of infertile men affected by idiopathic asthenozoospermia. CoQ10 levels increased significantly in seminal plasma and sperm cells after 6 months of treatment with concomitant improvement of sperm cell motility.
Article
Full-text available
The Comet assay (CA) is a sensitive/simple measure of genotoxicity. However, many features of CA contribute variability. To minimize these, we have introduced internal standard materials consisting of ‘reference’ cells which have their DNA substituted with BrdU. Using a fluorescent anti-BrdU antibody, plus an additional barrier filter, comets derived from these cells could be readily distinguished from the ‘test’-cell comets, present in the same gel. In experiments to evaluate the reference cell comets as external and internal standards, the reference and test cells were present in separate gels on the same slide or mixed together in the same gel, respectively, before their co-exposure to X-irradiation. Using the reference cell comets as internal standards led to substantial reductions in the coefficient of variation (CoV) for intra- and inter-experimental measures of comet formation and DNA damage repair; only minor reductions in CoV were noted when the reference and test cell comets were in separate gels. These studies indicate that differences between individual gels appreciably contribute to CA variation. Further studies using the reference cells as internal standards allowed greater significance to be obtained between groups of replicate samples. Ultimately, we anticipate that development will deliver robust quality assurance materials for CA.
Article
Objective: To study the DNA damage levels among the cases of Down's syndrome(DS) with that of the age and sex matched controls using single cell gel eleetrophoresis (SCGE) or Comet assay. Methods: Phytohaemagglutinin stimulated lymphocyte cell culture was carried out using RPMI 1640. G banded metaphase spreads obtained following harvesting was screened using automation software "Ikaros metasystem, Carl Zeiss", Germany. Alkaline comet assay was carried out with the lymphocytes isolated using Histopaque. The lymphocytes were layered on the agarose (NMA,LMA) coated slides. They were then subjected to alkaline medium (PH>13) followed by submarine gel electrophoresis (300mA current), neutralisation (Tris buffer PH-7.5), fixation and staining with silver nitrate. Comet metrics values were obtained using the Comet score software. Results: All 40 cases of DS which was subjected for automation karyotyping showed pure trisomy 21 pattern. The results of Comet assay showed statistically significant elevated levels among cases as compared to controls. Comet length in cases was found to be 45.38 ± 12.5 μm as compared to 24.48 ± 3.2 μm in controls. Similar observations were made for comet tail length (18.35 ± 8.9 μm for cases and 2.61 ± 1.1 μm for controls) and head diameter (27.35 ± 5 μm for cases and 23.08 ± 2.6 μm for controls). Conclusion: There is increased DNA damage among the cases of Down's syndrome which can be attributed to oxidative stress. DNA damage may be responsible for the clinical manifestations and long term outcome among the cases of Down's syndrome.
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Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 μM) at 4 °C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.
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The comet assay is a sensitive and rapid method for DNA strand break detection in individual cells. Its use has increased significantly in the past few years. This paper is a review of the studies published to date that have made use of the comet assay. The principles of strand break detection using both the alkaline and neutral versions of the technique are discussed, and a basic methodology with currently used variations is presented. Applications in different fields are reviewed and possible future directions of the technique are briefly explored.
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The in vivo alkaline single cell gel electrophoresis assay, hereafter the Comet assay, can be used to investigate the genotoxicity of industrial chemicals, biocides, agrochemicals and pharmaceuticals. The major advantages of this assay include the relative ease of application to any tissue of interest, the detection of multiple classes of DNA damage and the generation of data at the level of the single cell. These features give the Comet assay potential advantages over other in vivo test methods, which are limited largely to proliferating cells and/or a single tissue. The Comet assay has demonstrated its reliability in many testing circumstances and is, in general, considered to be acceptable for regulatory purposes. However, despite the considerable data published on the in vivo Comet assay and the general agreement within the international scientific community over many protocol-related issues, it was felt that a document giving detailed practical guidance on the protocol required for regulatory acceptance of the assay was required. In a recent meeting held in conjunction with the 4th International Comet Assay Workshop (Ulm, Germany, 22-25 July 2001) an expert panel reviewed existing data and recent developments of the Comet assay with a view to developing such a document. This paper is intended to act as an update to the more general guidelines which were published as a result of the International Workshop on Genotoxicity Test Procedures. The recommendations are also seen as a major step towards gaining more formal regulatory acceptance of the Comet assay.
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The salutary characteristics of the tomato are normally related to its content of carotenoids, especially lycopene, and other antioxidants. Our purpose was to verify whether the daily intake of a beverage prototype called Lyc-o-Mato((R)) containing a natural tomato extract (Lyc-o-Mato((R)) oleoresin 6 %) was able to modify plasma and lymphocyte carotenoid concentrations, particularly those of lycopene, phytoene, phytofluene and beta-carotene, and to evaluate whether this intake was sufficient to improve protection against DNA damage in lymphocytes. In a double-blind, cross-over study, twenty-six healthy subjects consumed 250 ml of the drink daily, providing about 6 mg lycopene, 4 mg phytoene, 3 mg phytofluene, 1 mg beta-carotene and 1.8 mg alpha-tocopherol, or a placebo drink. Treatments were separated by a wash-out period. Plasma and lymphocyte carotenoid and alpha-tocopherol concentrations were determined by HPLC, and DNA damage by the comet assay. After 26 d of consumption of the drink, plasma carotenoid levels increased significantly: concentrations of lycopene were 1.7-fold higher (P<0.0001); of phytofluene were 1.6-fold higher (P<0.0001); of phytoene were doubled (P<0.0005); of beta-carotene were 1.3-fold higher (P<0.05). Lymphocyte carotenoid concentrations also increased significantly: that of lycopene doubled (P<0.001); that of phytofluene was 1.8-fold higher (P<0.005); that of phytoene was 2.6-fold higher (P<0.005); that of beta-carotene was 1.5-fold higher (P<0.01). In contrast, the alpha-tocopherol concentration remained nearly constant. The intake of the tomato drink significantly reduced (by about 42 %) DNA damage (P<0.0001) in lymphocytes subjected to oxidative stress. In conclusion, the present study supports the fact that a low intake of carotenoids from tomato products improves cell antioxidant protection.
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The in vivo comet assay (single cell gel electrophoresis assay) in its alkaline version (pH >13) is being increasingly used in genotoxicity testing of substances such as industrial chemicals, biocides, agrochemicals, food additives and pharmaceuticals. Recommendations for an appropriate performance of the test using OECD guidelines for other in vivo genotoxicity tests have been published. In this review, we critically discuss the biological significance of comet assay effects in general and the status of the test in current strategies for genotoxicity testing. Examples for practical applications of the in vivo comet assay and potential consequences of positive and negative test results are given. The significance of comet assay results for hazard identification and risk assessment is discussed. In accordance with international guidelines for genotoxicity testing the in vivo comet assay is recommended for follow-up testing of positive in vitro findings. It is particularly useful as a tool for the evaluation of local genotoxicity, especially for organs/cell types which cannot easily be evaluated with other standard tests. A positive result in an appropriately performed in vivo comet assay indicates genotoxicity of the test compound in the tissue tested and gains particular significance when a mutagenic potential of the test compound has already been demonstrated in vitro. Such findings will have practical consequences in the risk assessment processes and further development of substances.
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Oxidative stress has been implicated in the pathogenesis of chronic diseases related to aging such as cancer and cardiovascular disease. Carotenoids could be a part of a protective strategy to minimize oxidative damage in vulnerable populations, such as the elderly. Our aim was to determine the protective effect of carotenoids against DNA damage. A randomized, double-blind, placebo-controlled intervention study was conducted. Thirty-seven healthy, nonsmoking postmenopausal women aged 50-70 y were randomly assigned to 1 of 5 groups and were instructed to consume a daily dose of mixed carotenoids (beta-carotene, lutein, and lycopene; 4 mg each), 12 mg of a single carotenoid (beta-carotene, lutein, or lycopene), or placebo for 56 d. Plasma carotenoid concentrations were analyzed by using HPLC, and lymphocyte DNA damage was measured by using a single-cell gel electrophoresis (comet) assay. At day 57, all carotenoid-supplemented groups showed significantly lower endogenous DNA damage than at baseline (P < 0.01), whereas the placebo group did not show any significant change. Significantly less (P < 0.05) endogenous DNA damage was found as early as day 15 in the mixed carotenoid (P < 0.01) and beta-carotene (P < 0.05) groups. The results indicate that carotenoid supplementation decreases DNA damage and that a combination of carotenoids (4 mg each of lutein, beta-carotene, and lycopene), an intake that can be achieved by diet, or a larger dose (12 mg) of individual carotenoids exerts protection against DNA damage.
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Health concerns about the exposure to genotoxic and carcinogenic agents in the air are particularly significant for outdoor workers in less developed countries. To investigate the association between personal exposure to a group of air pollutants and severity of DNA damage in outdoor workers from two Mexican cities. DNA damage (Comet assay) and personal exposure to volatile organic compounds, PM(2.5), and ozone were investigated in 55 outdoor and indoor workers from México City and Puebla. In México City, outdoor workers had greater DNA damage, reflected by a longer tail length, than indoor workers (median 46.8 v 30.1 mum), and a greater percentage of highly damaged cells (cells with tail length > or =41 microm); in Puebla, outdoor and indoor workers had similar DNA damage. There were more alkali labile sites in outdoor than indoor workers. The DNA damage magnitude was positively correlated with PM(2.5) and ozone exposure. Outdoor and indoor workers with > or =60% of highly damaged cells (highly damaged workers) had significantly higher exposures to PM(2.5), ozone, and some volatile organic compounds. The main factors associated with the highly damaged workers were ozone, PM(2.5), and 1-ethyl-2-methyl benzene exposure. With this approach, the effects of some air pollutants could be correlated with biological endpoints from the Comet assay. It is suggested that the use of personal exposure assessment and biological endpoints evaluation could be an important tool to generate a more precise assessment of the associated potential health risks.
Article
The comet assay (single-cell gel electrophoresis), which measures DNA strand breaks at the level of single cells, is very easily applied to human lymphocytes, and therefore lends itself to human biomonitoring studies. For the examination of DNA base oxidation (a specific marker of oxidative damage), the assay is modified by including a stage at which the DNA is incubated with a suitable lesion-specific endonuclease. Here we report on the reliability and reproducibility of this approach, from the level of comparing results from duplicate gels prepared from the same sample of cells, up to an assessment of the natural intra- and interindividual variability in lymphocyte DNA damage measured in groups of normal, healthy human volunteers. We applied the assay in investigations of human disease and occupational exposure of factory workers. Environ. Mol. Mutagen. 30:139–146, 1997. © 1997 Wiley-Liss, Inc.
Article
Mammalian cells were after irradiation suspended in melted agarose, and casted on microscope slides. The slides were after gelling at 0 degree C immersed in a neutral detergent solution which lysed the cells. A weak electric field (5 V/cm) was then applied over the gel for 5 minutes. The DNA in the gel was stained with the fluorescent dye acridine orange and gives a green emission in a microscope photometer. DNA had migrated towards the anode and this migration was more pronounced in irradiated than in control cells. The differences in migration pattern were quantitatively measured. The lower detection limit was below 0.5 Gy and a plateau in the dose-effect curve was reached at about 3 Gy. In repair experiments residual DNA damage could be observed after postirradiation incubation for 60 minutes. The advantages of the method is: no radioactive labelling and only a few number of cells is required.
Article
In Down syndrome (DS), oxidative DNA-damage may play a role in the pathogenesis of characteristic mental retardation and precocious dementia of Alzheimer type. We measured the oxidized nucleoside, 8-hydroxy-2'-deoxyguanosine (8-OHdG), in nuclear DNA (nDNA) isolated from four different regions of cerebral cortex and cerebellum in 10 adult DS and 10 Alzheimer's disease (AD) patients compared to normal controls. Levels of 8-OHdG in post-mortem brain tissue were investigated by means of high-performance liquid chromatography with electrochemical detection. There was no significant increase in DS and AD compared to controls in any of the brain regions. Highest amounts of 8-OHdG were in temporal cortex in DS (180.0 +/- 9.6 nmol/g wet weight tissue), AD (172.4 +/- 14.6 nmol/g wet weight tissue) and controls (183.4 +/- 12.7 nmol/g). We conclude that the results provide evidence against an increased reactive oxygen species (ROS) induced damage to nDNA in DS and AD.
Article
There is convincing epidemiological and in vitro evidence of chronic oxidative stress in individuals with Down syndrome (DS). These individuals develop Alzheimer like changes in the brain in their 30s and 40s. The incidence of autoimmune diseases and cataracts is significantly increased, and the overall ageing process is accelerated. In vitro studies show that impaired viability of DS neurons may be amended by simple chemical antioxidants, such as vitamin E, BHT and propyl gallate, clearly indicative of oxyl radical involvement. However, because of the lack of in vivo experiments, the role of oxidative stress in DS remains controversial. We report here on the results of the chemical analyses of urine samples of 166 individuals, where DS subjects were matched by their siblings. The levels of 8-hydroxy-2'-deoxyguanosine (2.35 +/- 1.69 in DS vs. 1.35 +/- 1.04 in controls, P = 0.00011), a biomarker of oxidative damage to DNA, and malondialdehyde (0.255 +/- 0.158 in DS vs. 0.204 +/- 0.128 in controls, P = 0.033), a biomarker of lipid peroxidation, are significantly elevated in individuals with DS. Dietary influences failed to show any significant correlation with the oxidative stress biomarkers. These results provide direct evidence for increased oxidative stress in individuals with DS.
Article
Total or partial trisomy of chromosome 21 occurs with relatively high frequency and is responsible for the occurrence of Down syndrome. Phenotypically, individuals with Down syndrome display characteristic morphological features and a variety of clinical disorders. One of the challenges for researchers in this field has been to ascertain and understand the relationship between the Down syndrome phenotype with the gene dosage effect resulting from trisomy of chromosome 21. Much attention therefore, has been given towards investigating the consequences of overexpressing chromosome 21-linked genes. In particular, an extensive analysis of SOD1 and APP have provided important insights as to how perturbations in the expression of their respective genes may contribute to the Down syndrome phenotype. In this review we will highlight studies which support a key role for SOD1 and APP in the pathogenesis of neural abnormalities observed in individuals with Down syndrome. Central to this relationship is how the redox state of the cell is affected and its consequences to neural function and integrity.
Article
Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.
Article
The most widely used system for peripheral blood progenitor cell (PBPC) cryopreservation is controlled-rate freezing (CRF). Uncontrolled-rate freezing (URF) at -80 degrees C has also been used, but its clinical impact has not been studied sufficiently yet. Two groups of patients were compared: Group A consisted of 69 patients autotransplanted with PBPCs cryopreserved with CRF; Group B consisted of 192 patients autotransplanted with PBPCs cryopreserved with URF at -80 degrees C. The same cryoprotectant solution and storage system were used. A significant delay of hematologic reconstitution (HR) in the URF group was observed for neutrophils greater than 0.5 x 10(9) per L and for platelets greater than 20 x 10(9) per L and greater than 50 x 10(9) per L; we did not observe any differences in the clinical course. The long-term HR was comparable in the two groups, all patients showed stable engraftment, and no late graft failures were observed. Our study confirms that URF is safe and allows sustained long-term engraftment without increasing the risks of transplantation, even though the early engraftment after URF is slower.
Article
The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells. Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. The assay has applications in testing novel chemicals for genotoxicity, monitoring environmental contamination with genotoxins, human biomonitoring and molecular epidemiology, and fundamental research in DNA damage and repair. The sensitivity and specificity of the assay are greatly enhanced if the nucleoids are incubated with bacterial repair endonucleases that recognize specific kinds of damage in the DNA and convert lesions to DNA breaks, increasing the amount of DNA in the comet tail. DNA repair can be monitored by incubating cells after treatment with damaging agent and measuring the damage remaining at intervals. Alternatively, the repair activity in a cell extract can be measured by incubating it with nucleoids containing specific damage.
Article
Down syndrome (DS) is the congenital birth defect responsible for the greatest number of individuals with mental retardation. It arises due to trisomy of human chromosome 21 (HSA21) or part thereof. To date there have been limited studies of HSA21 gene expression in trisomy 21 conceptuses. In this study we investigate the expression of the HSA21 antioxidant gene, Cu/Zn-superoxide dismutase-1 (SOD1) in various organs of control and DS aborted conceptuses. We show that SOD1 mRNA levels are elevated in DS brain, lung, heart and thymus. DS livers show decreased SOD1 mRNA expression compared with controls. Since non-HSA21 antioxidant genes are reported to be concomitantly upregulated in certain DS tissues, we examined the expression of glutathione peroxidase-1 (GPX1) in control and DS fetal organs. Interestingly, GPX1 expression was unchanged in the majority of DS organs and decreased in DS livers. We examined the SOD1 to GPX1 mRNA ratio in individual organs, as both enzymes form part of the body's defense against oxidative stress, and because a disproportionate increase of SOD1 to GPX1 results in noxious hydroxyl radical damage. All organs investigated show an approximately 2-fold increase in the SOD1 to GPX1 mRNA ratio. We propose that it is the altered antioxidant ratio that contributes to certain aspects of the DS phenotype.
Article
We reviewed the data of 45 alkaline comet assay studies with lymphocytes published during the last three years with the objective of monitoring human exposure to genotoxic agents as a result of occupation, drug treatment, diseases or environmental pollution. The strengths of the studies were that: (i) a lot of data could be obtained within a relatively short period of time in a cost-effective manner, (ii) lymphocytes could be easily collected in a non-invasive way and proved to be good surrogate cells in that they picked up effects caused by agents with different cancer target organs and (iii) a remarkable concordance between comet assay and cytogenetic assay data was proved. However, our analysis revealed some shortcomings of the studies such as: (i) the inclusion of low number of study participants and bias in the number and gender of subjects between control and exposed groups, (ii) lack of qualitative and quantitative exposure data, (iii) lack of consideration of differences in physical activity and diet between control and exposed groups, (iv) difficulty in comparison of the studies due to lack of uniformity in the comet assay procedures such as duration of alkali unwinding and electrophoresis, slide scoring method and the metrics used to assess the extent of DNA damage and (v) controversy in the sensitivity of comet assay since it picked up DNA damage caused by agents such as wood dust, pesticides and hormone preparations which were found to be weak genotoxins or non-genotoxins in other tests, but gave inconsistent results with known mutagens/carcinogens such as tobacco smoke. We feel that for the alkaline comet assay to be an important tool in human biomonitoring studies, serious consideration should be given to the flaws in the design and performance of the assay.
Article
The Comet assay is an often used approach for the assessment of genetic damage in primary cells of exposed populations. In the majority of these studies lymphocytes are used. Therefore, we reviewed human biomonitoring studies of occupational exposure using the Comet assay with lymphocytes. We also tried to elucidate the strengths of the studies, which were that (i) data could be obtained in a fast and cost-effective manner, (ii) the ease at which these cells can be collected and (iii) a remarkable concordance between Comet assay and cytogenetic assays. However, the analysis also revealed some shortcomings: (i) the low number of study participants, (ii) the bias in the distribution of gender, (iii) lack of qualitative and quantitative exposure data, (iv) omission to consider differences in physical activity and diet between control and exposed groups, (v) lack of uniformity in the Comet assay procedures, and (vi) controversy in the sensitivity of Comet assay since it picked up DNA damage caused by agents which were found to be weak genotoxicants or non-genotoxicants in other tests, but gave inconsistent results with known mutagens/carcinogens such as cigarette smoke.
Article
Nineteen scorers from seven Cuban laboratories participated in this slide exercise designed to test the influence of the scorer on the accuracy, sensitivity and variability of the comet assay when a visual method of DNA damage evaluation is used. The assay was performed using human lymphocytes from a single donor exposed in vitro for 5 min at 0 degrees C to doses of 0, 5, 10, 25, 50, 100 and 200 microM of hydrogen peroxide. Each participant scored the same set of 14 coded slides with silver stained comets. The comets were classified visually into five categories according to the appearance resulting from the relative proportion of DNA in the tail. The extent of DNA damage was expressed in arbitrary units. At zero dose the median values of 12 scorers out of 19 were included between the values of the overall 25 and 75 per thousand. This proportion remains practically the same as the dose increases. The lowest dose detected by this method for the majority of scorers (11) was 10 microM. The coefficient of variation at the control dose was the highest (median value 26%), progressively declined to 20%, and starting from 25 microM, values are around 10%. The results of the exercise show the reliability of the silver staining and visual scoring for the comet method.
Article
Generation of DNA damage is considered to be an important initial event in carcinogenesis. A considerable battery of assays exists for the detection of different genotoxic effects of compounds in experimental systems, or for investigations of exposure to genotoxic agents in environmental or occupational settings. Some of the tests may have limited use because of complicated technical setup or because they only are applicable to a few cell types. The single cell gel electrophoresis (comet) assay is technically simple, relatively fast, cheap, and DNA damage can be investigated in virtually all mammalian cell types without requirement for cell culture. The aim of this thesis was to evaluate the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring. The comet assay detects strand breaks (SB). The cells are embedded in agarose and lysed, generating nucleus-like structures in the gel (referred to as nucleoids). Following alkaline electrophoresis, the DNA strands migrate toward the anode, and the extent of migration depends on the number of SB in the nucleoid. The migration is visualized and scored in a fluorescence microscope after staining. Broad classes of oxidative DNA damage can be detected as additional SB if nucleoids are incubated with bacterial DNA glycosylase/endonuclease enzymes. Oxidized pyrimidines and purines can be detected by incubation with endonuclease III and formamidopyrimidine DNA glycosylase, respectively. The animal experimental studies indicated that the comet assay was able to detect genotoxic effects of diesel exhaust particles in lung tissue, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced DNA damage in colon epithelial cells and liver tissue, and benzene-induced damage in bone marrow and liver cells. The strength of the comet assay was further outlined by application of repair enzymes, indicating no oxidative DNA base damage following IQ treatment. High levels of oxidative DNA lesions were detected after exposure to benzene or X-ray irradiation. The comet assay did not detect DNA damage in colon or liver following ingestion of diets containing of high contents of animal fat or sucrose, although other indices of DNA damage were found. Determined from the results of a large Japanese study, the discrimination between carcinogens and non-carcinogens appears to be similar between the comet assay and alkaline elution, which also detects SB. This suggests that the comet assay is a reliable genotoxicity test in animal experimental systems. In the biomonitoring studies, we investigated the effect of common exposures and lifestyle factors (rather than effects of known carcinogens) on the level of oxidative DNA damage in mononuclear blood cells of humans. In the first study, based on repeated measurements, it was shown that interindividual variation and seasonal variation were major determinants for the basal level of SB, whereas no effect of age, exercise, or antioxidant intake could be detected. The effect of exercise was further investigated under both normoxic and hypoxic circumstances, showing a strong effect of hypoxia, and only effect of exercise in terms of SB in hypoxia. In a placebo-controlled parallel dietary fruit and vegetable (or the corresponding amount of antioxidants) intervention study, no effects of the level of oxidative DNA damage or sensitivity to hydrogen peroxide were observed. Although this may seem in contrast to other antioxidant intervention studies, a critical literature survey of antioxidant intervention studies on oxidative DNA damage suggested that well-controlled studies tended to show no effect of antioxidant supplementation. In summary, the aggregated data from the publications included in this thesis, and other publications encompassing the comet assay, indicate that the comet assay is a reliable method for detection of DNA damage in tissues of experimental animals. Although not all types of genotoxic exposures should be expected to result in DNA damage in mononuclear blood cells, the comet assay seems to be a valuable tool for detection of genotoxic exposure in humans. The comet assay indicates that DNA damage is abundant in mammalian cells and affected by lifestyle and many environmental exposures, including diet, exercise, hypoxia, and sunlight.
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Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing
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