[23] Comet assay to detect nitric oxide-dependent DNA damage in mammalian cells

doi:10.1016/S0076-6879(96)69026-0

Methods in Enzymology

Volume 269, 1996, Pages 243-266

https://doi.org/10.1016/S0076-6879(96)69026-0 Get rights and content

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This chapter discusses the use of comet assay to detect nitric oxide-dependent DNA damage in mammalian cells. After hydrogen peroxide, nitric oxide (NO) is the most prevalent mutagen to which human DNA is exposed. NO may interact with cellular amines to form N-nitroso compounds. Its oxidation products nitrite and the higher nitrogen oxides also have biological activity. NO produce DNA strand breakage in mammalian cells by the DNA precipitation assay, in situ nick translation, and the comet assay. The comet assay is a sensitive method for the detection of DNA strand breaks in mammalian cell. The comet assay detects release of DNA from a highly supercoiled DNA-protein complex. In comparison with other sensitive methods, the comet assay is relatively robust and economical in its use of material. It has the specific advantage that as a single-cell assay, it can detect nonuniform response within a cell population, and characterize the behavior of different cell types within a mixed population. Only a small proportion of DNA-damaging agents, including ionizing radiation, bleomycin, and hydrogen peroxide, induces direct breakage of the DNA phosphodiester backbone.

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An evaluation of the differences in DNA damage in lymphocytes and repair efficiencies in patients with schizophrenia and schizoaffective disorder
2018, Schizophrenia Research
Citation Excerpt :
Broken DNA molecules migrate at different rates in electrical fields, as they have different molecular weights and electric charges. The damaged pieces of DNA moving towards an anode present a comet-like image, but undamaged DNA cannot come out of the helical structure (Green et al., 1996; McKelvey-Martin et al., 1993). The parameters used to evaluate damage were the HL (head length, μm), TL (tail length, μm) HD (head density: percentage of DNA in the head), TD (tail density: percentage of DNA in the tail), and TMo (tail moment: expressed in μm, the value obtained by dividing the product of TL and TD by 100).
Schizophrenia and schizoaffective disorder are chronic and debilitating psychiatric disorders. The present study was designed to determine DNA damage in patients with schizophrenia and schizoaffective disorder to assess the roles of oxidative metabolism and DNA repair mechanisms in this process, to assess the contribution of drugs, and thus to demonstrate the differences between schizophrenia and schizoaffective disorder. Thirty schizophrenia and 30 schizoaffective disorder patients, each having at least five years of disease history, aged between 18 and 60 years with no physical or neurological diseases, and 30 healthy volunteers participated in the study. Psychometric scales were applied, and 5 ml of blood was taken from all participants. The DNA damage was measured in lymphocytes by the comet assay method; the total oxidative parameters by ELISA; OGG1 and NEIL1 gene expressions by real-time PCR; and the role of drugs by in vitro assays. The most important finding in this study was that patients with schizophrenia had significantly greater DNA damage than schizoaffective disorder patients and the controls. This study also provides evidence of high oxidative stress statuses and inadequate DNA repair capacities in patients with schizophrenia. Moreover, psychotropic drugs did not induce any DNA damage to the lymphocytes according to in vitro analyses. The use of clozapine and adequate repair processes of the patients were the decisive factors in the prevention of DNA damage. The results of this study provide a reexamination of schizoaffective disorder within the schizophrenia spectrum and indicate that schizoaffective disorder may be considered a different diagnostic category.
3,3′-Dihydroxyisorenieratene and isorenieratene prevent UV-induced DNA damage in human skin fibroblasts
2012, Free Radical Biology and Medicine
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After electrophoresis the slides were washed three times with neutralizing buffer. For final fixation, the slides were kept in ethanol (80%) for 5 min and then dried overnight [19,20]. Directly before fluorescence microscopy, cells were stained with Midori green (Nippon Genetics Europe, Dueren, Germany).
Skin cancer is among the most frequent neoplastic malignancies and exposure to UV irradiation is a major risk factor. In addition to topical sunscreens, photoprotection by dietary antioxidants such as carotenoids or polyphenols has been suggested as a means of prevention. Isorenieratene (IR) and dihydroxyisorenieratene (DHIR) are aromatic carotenoids with particular antioxidant properties produced by Brevibacterium linens. The aim of this study was to investigate the photoprotective and antioxidant activities of DHIR and IR in comparison to the nonaromatic carotenoid lutein in human dermal fibroblasts. Incubation of the cells with DHIR and IR significantly decreased the UV-induced formation of cyclobutane pyrimidine dimers and formation of DNA strand breaks. Lipid oxidation was lowered as determined by the formation of malondialdehyde as a biomarker. Both aromatic carotenoids also prevented oxidatively generated damage to DNA as demonstrated by a decrease in DNA strand breaks associated with the formation of oxidized DNA bases. These data highlight the multifunctional photoprotective properties of aromatic carotenoids, which may be suitable natural compounds for the prevention of skin cancer.
Could a strong alkali deproteinization replace the standard lysis step in alkaline single cell gel electrophoresis (comet) assay (pH > 13)?
2009, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
The alkaline version of single cell gel electrophoresis (comet) assay is widely used for evaluating DNA damage at the individual cell level. The standard alkaline method of the comet assay involves deproteinization of cells embedded in agarose gel using a high salt–detergent lysis buffer, followed by denaturation of DNA and electrophoresis using a strong alkali at pH > 13 [N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175 (1988) 184–191]. However, a recent report showed that a strong alkali treatment results in simultaneous deproteinization of cells and denaturation of genomic DNA [P. Sestili, C. Martinelli, V. Stocchi, The fast halo assay: an improved method to quantify genomic DNA strand breakage at the single cell-level, Mutat. Res. 607 (2006) 205–214]. This study was carried out to test whether the strong alkali deproteinization of cells could replace the high salt–detergent lysis step used in the standard method of the alkaline comet assay. Peripheral blood lymphocytes from 3 healthy individuals were irradiated with gamma rays at doses varying between 0 and 10 Gy. Following irradiation, the comet assay was performed according to the standard alkaline method (pH > 13) and a modified method. In the modified method, agarose embedded cells were treated with a strong alkali (0.3 M NaOH, 0.02 M Trizma and 1 mM EDTA, pH > 13) for 20 min to allow deproteinization of cells and denaturation of DNA. This was followed by electrophoresis using the same alkali solution to obtain comets. DNA damage expressed in terms of comet tail length, percentage of DNA in comet tail and tail moment obtained by the standard alkaline method and the modified method were compared. In both methods, DNA damage showed a good correlation with the dose of gamma ray. The results indicate a satisfactory sensitivity of the modified method in detecting radiation-induced DNA damage in human peripheral blood lymphocytes.
Environmental and occupational biomonitoring using the Comet assay
2009, Mutation Research - Reviews in Mutation Research
Biomonitoring of human populations exposed to potential mutagens or carcinogens can provide an early detection system for the initiation of cell disregulation in the development of cancer. In recent years, the Comet assay, also known as a “single cell gel” (SCG) electrophoresis assay, has become an important tool for assessing DNA damage in exposed populations. This is the method of choice for population-based studies of environmental and occupational exposure to air pollutants, metals, pesticides, radiation, and other xenobiotics as we show in this review. To appreciate the role of the Comet assay in the field of biomonitoring, we review data from 122 studies that employed the assay. These studies evaluated environmental versus occupational exposures and the levels of DNA damage in cells of individuals exposed in each case. Our review of the literature reveals the importance of the need to establish standard methodological conditions that affect unwinding and electrophoresis times and tail values (tail length, tail DNA, tail moment), with the goal of being able to compare data collected in different laboratories throughout the world. The Comet assay is susceptible to subtle artifacts of manipulation depending on the type and timing of sampling performed. Therefore, in the reporting of DNA damage detected by the Comet assay, the context of how the DNA damage was created also needs to be reported and considered in the interpretation of Comet assay results. The success of the Comet assay is reflected by its use over the past 20 years in the field of biomonitoring, and by the increasing number of studies that continue to report its use. As the shortcomings of the assay are identified and considered in the interpretation of DNA damage detection, the Comet assay will continue to provide improved reliability as a biomarker in human biomonitoring studies.
Photodynamic therapy with zinc-tetra(p-sulfophenyl)porphyrin bound to cyclodextrin induces single strand breaks of cellular DNA in G361 melanoma cells
2005, Toxicology in Vitro
The basis of photodynamic therapy (PDT) is the phototoxicity resulting from co-action of light, sensitizer and oxygen. In this study we demonstrate in vitro phototoxicity measurement on G361 cell lines using ZnTPPS₄ sensitizer bound to cyclodextrin hpβCD. We have proved its photodamage effect on cancer cell lines in the visible region of spectrum. We used the halogen lamp (24 V/250 W) as a source of radiation. After 24 h incubation of cell cultures with 10 μM ZnTPPS₄ and 1 mM cyclodextrine hpβCD, the cells were irradiated for 7.5 min at the total irradiation dose of 12.5 J cm⁻². Analysis of DNA damage in the cell line after PDT was proved by comet assay and using inversion fluorescent microscope with image analysis. This treatment method gave rise to DNA damage. The used radiation dose of visible light in the absence of sensitizers does not induce DNA breaks in tumour cells. In conclusion, binding of ZnTPPS₄ sensitizer to cyclodextrin hpβCD may improve the efficacy of PDT for the treatment of malign melanoma.
Analysis of DNA damage in sea lamprey (Petromyzon marinus) spermatozoa by UV, hydrogen peroxide, and the toxicant bisazir
2005, Aquatic Toxicology
In this study we sought to demonstrate that Comet assay can be applied to sea lamprey sperm DNA fragmentation and used to describe the relationship between sperm DNA damage and sperm fertilizing ability. We show that the assay can be used reliably and accurately, and unlike in the case of mammals, there is no need for additional steps related to improvement of efficacy of lysis and DNA decondensation. This agrees with the presence of histone proteins in lamprey sperm. An increase in DNA fragmentation was noted during short-term storage of milt on ice (0–4 days). We demonstrated genotoxic effects of UV radiation and oxidative stress (exposure to hydrogen peroxide) and found that oxidative damage to sperm DNA was likely repaired after fertilization in the embryo. Repairing capacity of the oocyte toward sperm DNA lesions caused by UV was restricted. Toxic effect of p,p-bis-(1-aziridinyl)-N-methylphosphinothioic acid (p,p-bis(1-aziridinyl)-N-methylphosphinothioic amide), a sea lamprey chemosterilant, could not be linked to DNA fragmentation in the in vitro tests. Its genotoxicity in vivo may possibly be associated with other mechanisms of DNA degradation (oxidation or DNA–protein and DNA–DNA cross-linking). In conclusion, this study demonstrates that Comet assay can be successfully applied to monitor effects of environmental disturbances and imposed injuries in sea lamprey spermatozoa and possibly other species of ancient fish with acrosomal sperm.

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[23] Comet assay to detect nitric oxide-dependent DNA damage in mammalian cells

Publisher Summary

Mutat. Res.

FEBS Lett.

Mutat. Res.

Mutat. Res.

FEBS Lett.

Biochem. Biophys. Res. Commun.

Exp. Cell Res.

Mutat. Res.

Exp. Cell Res.

Mutat. Res.

Mutat. Res.

Anal. Biochem.

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Nutr. Cancer

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Biochim. Biophys. Acta

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Toxicol. In Vitro

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Science