Comet assay based detection of SPION induced DNA damage in human lymphocytes
01 Jan 2016-pp 91-94
TL;DR: The results show that SPION induced genotoxicity is completely dependent on its physicochemical properties and Regulation of these properties by using different coatings could decrease toxicity.
Abstract: Superparamagnetic iron oxide nanoparticle (SPION) coated with suitable biocompatible substances have uses in various biomedical fields, particularly in magnetic resonance imaging, tissue engineering, hyperthermia and drug delivery. In this study we have used two newly formulated SPIONs. SPIONs were coated with biodegradable polymer polylactide co glycolide (PLGA) using of the two types of surfactants-didodecyldimethylammoniumbromide (DMAB) and ±-tocopheryl glycol succinate (TPGS) for surface modification, to extend the application potential in the field of nanomedicine. The present study focuses on the evaluation of genotoxicity if any of the two types of formulated SPIONs on human lymphocyte. Human lymphocytes were exposed to SPIONs at 11.2µg/ml concentrations of Fe in each group for 3 h at 37°C. Single-dose toxicity was tested in isolated lymphocytes using MTT assay. Uncoated SPIONs were found highly toxic while the coated ones showed significantly less cell death. In vitro genotoxicity of the formulated SPIONs showed significantly lower %tail DNA than uncoated SPIONs as detected by comet assay in lymphocytes. The results show that SPION induced genotoxicity is completely dependent on its physicochemical properties. Regulation of these properties by using different coatings could decrease toxicity. Type of surface modification primarily governed the amount of DNA damage as detected by Comet assay. The results also indicate that the coatings on the SPION were biocompatible and suitable for in vivo explorations while the free SPION were found completely unsuitable for in vivo administration.
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TL;DR: Evaluating the comet assay as a genotoxicity test in genetic toxicology of environmental agents, encompassing both experimental animal models and biomonitoring, indicates that the Comet assay is a reliable method for detection of DNA damage in tissues of experimental animals.
Abstract: 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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