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Increased DNA damage in blood cells of rat treated with lead as assessed by comet assay
Mohammad Arif,Yearul Kabir,Faizule Hassan,T.M. Zaved Waise
Bangladesh Journal of Pharmacology , 2008,
Abstract: A growing body of evidence suggests that oxidative stress is the key player in the pathogenesis of lead-induced toxicity. The present study investigated lead-induced oxidative DNA damage, if any in rat blood cells by alkaline comet assay. Lead was administered intraperitoneally to rats at doses of 25, 50 and 100 mg/kg body weight for 5 days consecutively. Blood collected on day six from sacrificed lead-treated rats was used to assess the extent of DNA damage by comet assay which entailed measurement of comet length, olive tail moment, tail DNA (%) and tail length. The results showed that treatment with lead significantly increased DNA damage in a dose-dependent manner. Therefore, our data suggests that lead treatment is associated with oxidative stress-induced DNA damage in rat blood cells which could be used as an early bio-marker of lead-toxicity.
Minimum Criteria for the acceptance of in vivo alkaline Comet Assay Reports  [PDF]
European Food Safety Authority
EFSA Journal , 2012, DOI: 10.2903/j.efsa.2012.2977
Abstract: The in vivo alkaline Comet assay detects primary DNA damage in various organs and tissues of exposed animals and can be used to assess the genotoxicity of a great variety of chemical compounds, which include food additives, flavourings, food contact materials, foodborne by-products, pesticides, contaminants, etc. Among the various versions of the assay, the alkaline method (pH of DNA unwinding and electrophoresis buffer > 13) identifies the broadest spectrum of DNA damage and is, therefore, recommended for regulatory purposes. It can detect double- and single-strand breaks, alkali-labile lesions that are expressed as single-strand breaks and single-strand breaks arising as DNA repair intermediates. No OECD Test Guideline yet exists for the Comet assay but internationally agreed protocols are available for performing this test. Since establishing of an OECD Test Guideline for this assay will require some further time, there is a need for an agreed approach on the minimum requirements on conduction and reporting of the in vivo Comet assay, which should be fulfilled for the purposes of EFSA during this transition period.
Sprague Dawley response to the cyclophosphamide and bleomycin in the alkaline comet assay of peripheral blood leukocytes
Daniel Francisco Arencibia Arrebola,Alexis Vidal Novoa,Luis Alfredo Rosario Fernández,Yolanda Emilia Suárez Fernández
RETEL : Revista de Toxicología en Línea , 2010,
Abstract: In this article we decide to evaluate genotoxic effect of the cyclophosphamide and bleomycin in the individual cells by means of alkaline comet assay using the Sprague Dawley rats in both sexes as experimental biomodel. Which were formed 5 experimental groups per sex, the first administered with NaCl 0,9 % by intraperitoneal (i.p) route, the second and third groups were administered with cyclophosphamide by i.p route, with designs of different treatments at doses of 50 mg/kg. The fourth and fifth groups were administered with bleomycin by i.p route, equally in two designs of different treatments at doses of 40 mg/kg. At the end of the experience bigger induction of damage was obtained with the use of the cyclophosphamide and bleomycin, both in the design of 48 and 24 hours administration before the sacrifice. This constitutes under our experimental conditions the two better experimental designs to induce the strand breaks (SB) or alkali-labile sites formation on DNA, increasing considerably the frequency spontaneous present in this species of rat, being useful in studies of drugs evaluation that they have not been explored in to the in vivo antigenotoxicity and genotoxicity environment.
Evaluation of DNA Single and Double Strand Breaks in Women with Cervical Neoplasia Based on Alkaline and Neutral Comet Assay Techniques
Elva I. Cortés-Gutiérrez,Fernando Hernández-Garza,Jorge O. García-Pérez,Martha I. Dávila-Rodríguez,Miguel E. Aguado-Barrera,Ricardo M. Cerda-Flores
Journal of Biomedicine and Biotechnology , 2012, DOI: 10.1155/2012/385245
Abstract: A hospital-based unmatched case-control study was performed in order to determine the relation of DNA single (ssb) and double (dsb) strand breaks in women with and without cervical neoplasia. Cervical epithelial cells of 30 women: 10 with low grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 without cervical lesions were evaluated using alkaline and neutral comet assays. A significant increase in global DNA damage (ssb
Assessment of basal and induced DNA damage on lymphocytes from three mouse lines by means of the alkaline comet assay
Arencibia,Daniel F; Rosario,Luis A; Rodríguez,Yanet;
Biotecnolog?-a Aplicada , 2011,
Abstract: the formation of single-strand breaks and alkali-labile sites on dna has been extensively used for genotoxicity testing, given its involvement in degenerative disorders, cancer and oxidative stress. an alkaline variation of the single-cell electrophoresis (comet) assay used for the detection of dna damage was developed during the eighties, providing data on this phenomenon at the level of individual cells for the first time. the present work employs the alkaline comet assay to compare three mouse lines regarding the basal and cyclophosphamide-induced frequency of single-strand breaks and the appearance of alkali-labile sites in dna from peripheral blood lymphocytes. a total of 10 mice/sex/group of the balb/c, of-1 and nmri lines were treated for 14 days, distributed into an untreated negative control group, two groups receiving excipients and a positive control group receiving intraperitoneal cyclophosphamide at 50 mg/kg. after 14 days, single cells from peripheral blood leukocytes were analyzed by alkaline electrophoresis to estimate dna damage. it was concluded that the best choice for this type of studies is represented by the balb/c line, due to its low basal frequency for the analyzed variables. this result indicates that balb/c may be the best biomodel for preclinical testing of drugs, vaccines and other products.
Paracoccidioidomycosis: no genetic damage in human peripheral blood cells of patients assessed by single-cell gel (comet) assay
Ribeiro-Vieira, Renata Aparecida Martinez Antunes;Ribeiro, Daniel Araki;Salvadori, Daisy Maria Favero;Marques, Sílvio Alencar;
Revista da Sociedade Brasileira de Medicina Tropical , 2007, DOI: 10.1590/S0037-86822007000400021
Abstract: paracoccidioidomycosis is a systemic fungal infection caused by paracoccidioides brasiliensis. as infectious diseases can cause dna damage, the authors aimed at analyzing dna breakage in peripheral blood cells of patients with paracoccidioidomycosis by using the comet assay. the results suggested that paracoccidioidomycosis does not cause genotoxicity.
Radiation-induced DNA damage and repair in human γδ and αβ T-lymphocytes analysed by the alkaline comet assay
Halina Lisowska, Marta Deperas-Kaminska, Siamak Haghdoost, Ingela Parmryd, Andrzej Wojcik
Genome Integrity , 2010, DOI: 10.1186/2041-9414-1-8
Abstract: Human peripheral blood mononuclear cells (PBMC) are used as surrogate tissue for the assessment of individual sensitivity to ionising radiation [1]. Using the G2 chromosomal aberration assay it has been shown by a number of authors that the radiosensitivity of PBMC is higher in cancer patients compared to healthy donors [2-9]. A similar result was reported using the micronucleus assay [10] and the comet assay [11,12] and is generally interpreted as a reflection of genomic instability in PBMC of cancer patients [10].PBMC are composed of different cell subpopulations which are differently radiosensitive. It is generally accepted that B lymphocytes show a higher radiosensitivity than T-lymphocytes that are all CD3+ [13-16]. Among the T-lymphocytes cytotoxic CD8+ cells appear somewhat more sensitive than helper CD4+ cells [16,17], although this was not observed in all studies [18]. A lymphocyte subpopulation which recently attracted interest is composed of T-lymphocytes with γδ T-cell receptors (TCR). The reason for this is the discovery that these cells contribute to immunity against cancer [19]. In peripheral blood γδ lymphocytes account for less than 5% of total T-lymphocytes while their proportion in the intestine can be much higher [20].Given their role in immunosurveillance, the level and/or the activity of γδ T-lymphocytes is expected to be low in PBMC of cancer-prone individuals and high in PBMC of individuals who are resistant to cancer. This is supported by findings in patients with lymphoma, myeloma, breast and nasopharyngeal carcinoma [21-23]. Should the sensitivity to radiation of γδ lymphocytes be lower than that of the more common αβ T-lymphocytes, this could, at least partly explain the low radiosensitivity of PBMC from healthy individuals compared to cancer patients. The present investigation was carried out to test if there are differences in the radiation sensitivity between different TCR subtype populations by comparing the level of DNA damage and th
Factors influencing heterogeneity of radiation-induced DNA-damage measured by the alkaline comet assay
Clemens Seidel, Christine Lautenschl?ger, Jürgen Dunst, Arndt-Christian Müller
Radiation Oncology , 2012, DOI: 10.1186/1748-717x-7-61
Abstract: Two-hundred leukocytes per sample of healthy donors were split into four groups. I: Intact chromatin structure; II: Nucleoids of histone-depleted DNA; III: Nucleoids of histone-depleted DNA with 90?mM DMSO as antioxidant. Response to single (I-III) and twice (IV) irradiation with 4?Gy and repair kinetics were evaluated using %Tail-DNA. Heterogeneity of DNA damage was determined by calculation of variance of DNA-damage (V) and mean variance (Mvar), mutual comparisons were done by one-way analysis of variance (ANOVA).Heterogeneity of initial DNA-damage (I, 0?min repair) increased without histones (II). Absence of histones was balanced by addition of antioxidants (III). Repair reduced heterogeneity of all samples (with and without irradiation). However double irradiation plus repair led to a higher level of heterogeneity distinguishable from single irradiation and repair in intact cells. Increase of mean DNA damage was associated with a similarly elevated variance of DNA damage (r?=?+0.88).Heterogeneity of DNA-damage can be modified by histone level, antioxidant concentration, repair and radiation dose and was positively correlated with DNA damage. Experimental conditions might be optimized by reducing scatter of comet assay data by repair and antioxidants, potentially allowing better discrimination of small differences. Amount of heterogeneity measured by variance might be an additional useful parameter to characterize radiosensitivity.
INDIRECT SAFETY ASSESSMENT OF ECTODERMAL MERCURY EXPOSURE BY TRADITIONAL MEDICAL FORMULATIONS ON FRESHWATER CAT FISH CLARIAS BATRACHUS USING MICRONUCLEUS ASSAY AND ALKALINE SINGLE-CELL GEL ELECTROPHORESIS (COMET) ASSAY  [PDF]
Anand Prem Rajan,Nathiya T,Alphonse Maria A
International Journal of Research in Ayurveda and Pharmacy , 2012,
Abstract: Mercury and its salts are the major constituents of Ayurvedic, Chinese and Tibetan traditional formulations. The mercury is extensively reported to accumulate in food chain and cause many neurological disorders in environment. The safety assessment of mercury on the ectodermal application on animal model is rarely reported. The void in the scientific study on external exposure of mercury and its genotoxic inside the body lead to the necessity for the present study. The freshwater cat fish Clarias batrachus was used for broad specificity genotoxic indicators micronucleus assay and alkaline single-cell gel electrophoresis (comet) assays. The fish was exposed to 0.03 ppm of mercuric chloride for a period of 7, 14, 28 and 35 days ectodermally. The blood sample was assayed for the genotoxicity. The results revealed undoubted DNA damage through the micronuclei and alkaline single-cell gel electrophoresis (comet) assays. Hence it is concluded the usage of traditional medicines containing the mercury may be toxic at genetic level in prolonged usage.
Hepatocytes, rather than leukocytes reverse DNA damage in vivo induced by whole body y-irradiation of mice, as shown by the alkaline comet assay
PINCHEIRA,JUANA; CARRERA,PILAR; MARCELAIN,KATHERINE; DE LA TORRE,CONSUELO;
Biological Research , 2008, DOI: 10.4067/S0716-97602008000200011
Abstract: dna damage repair was assessed in quiescent (g0) leukocytes and in hepatocytes of mice, after 1 and 2 hours recovery from a single whole body y-irradiation with 0.5, 1 or 2 gy. evaluation of single-strand breaks (ssb) and alkali-labile sites together were carried out by a single-cell electrophoresis at ph>13.0 (alkaline comet assay). in non-irradiated (control) mice, the constitutive, endogenous dna damage (basal) was around 1.5 times higher in leukocytes than in hepatocytes. irradiation immediately increased ssb frequency in both cell types, in a dose-dependent manner. two sequential phases took place during the in vivo repair of the radio-induced dna lesions. the earliest one, present in both hepatocytes and leukocytes, further increased the ssb frequency, making evident the processing of some primary lesions in dna bases into the ssb repair intermediates. in a second phase, ssb frequency decreased because of their removal. in hepatocytes, such a frequency regressed to the constitutive basal level after 2 hours recovery from either 0.5 orí gy. on the other hand, the ssb repair phase was specifically abrogated in leukocytes, at the doses and recovery times analyzed. thus, the efficiency of in vivo repair of radio-induced dna damage in dormant cells (lymphocytes) is quite different from that in hepatocytes whose low proliferation activity accounts only for cell renewal.
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