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Comparison Between Alkaline and Neutral Variants of Yeast Comet Assay

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  • Institute of Molecular Biology Bulgarian Academy of Sceinces

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Comet assay gain its popularity because it is a fast and relatively easy-to-perform method for assessing damages in DNA. The two persisting variants of the method: alkaline and neutral, lead to some doubts which one would be suitable for a certain purpose. Here we present a comparison between the two versions of the developed by us Yeast Comet Assay (YCA). Since DNA damages can be quite diverse, i.e. single- or double- stranded cuts, oxidative damages, photodimers, etc., we warn that the method of YCA has to be applied with attention and a certain level of understanding concerning the exact damage that is attempted to be assessed.
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... The method is renowned with its sensitivity as it allows measurement of DNA damage at the level of single cells [16,17]. We have performed the neutral variant of the method [18,19]. It is recognised for its high sensitivity allowing detection of DNA breaks at much lower concentrations than its alkaline variant [19]. ...
... We have performed the neutral variant of the method [18,19]. It is recognised for its high sensitivity allowing detection of DNA breaks at much lower concentrations than its alkaline variant [19]. MDA cells were treated with compounds 1 to 6 in concentrations of 30, 75 and 150 μg/ml for 4 h at optimal conditions and after that were subjected to SCGE. ...
Article
Background Quinazolines 1 to 6, with an aromatic or aryl-vinyl substituent in position 2 are selected with the aim to compare their structures and biological activity. The selection includes a natural alkaloid, schizocommunin, and the synthetic 2-(2′-quinolyl)-3H-quinazolin-4-one, known to interact with guanine-quadruplex dependent enzymes, respectively telomerase and topoisomerase. Methods Breast cancer cells of the MDA cell line have been used to study the bioactivity of the tested compounds by the method of Comet Assay and FACS analyses. We model observed effects assuming stacking interactions of studied heterocycles with a naked skeleton of G-quadruplex, consisting of guanine quartet layers and potassium ions. Interaction energies are computed using a dispersion corrected density functional theory method, and an electron-correlated molecular orbital theory method. Results Selected compounds do not remarkably delay nor change the dynamics of cellular progression through the cell cycle phases, while changing significantly cell morphology. Our computational models quantify structural effects on heterocyclic G4-complex stabilization energies, which directly correlate with observed biological activity. Conclusion Our computational model of G-quadruplexes is an acceptable tool for the study of interaction energies of G-quadruplexes and heterocyclic ligands, predicting, and allowing design of novel structures. General significance Genotoxicity of quinazoline-4-one analogues on human breast cancer cells is not related to molecular metabolism but rather to their interference with G-quadruplex regulatory mechanisms. Computed stabilization energies of heterocyclic ligand complexes of G-quadruplexes might be useful in the prediction of novel telomerase/helicase, topoisomerase and NA polymerase dependent drugs.
... Single cell gel electrophoresis (SCGE) or the comet assay method is a practical, simple, versatile, sensitive, economical and elaborative technique in genotoxicity testing for detection of DNA damage and repair (Cortés-Gutiérrez et al., 2012;Nandhakumar et al., 2011;Tice, 1995). This method can measure the single/double-strand DNA breaks, alkali labile sites (apurinic/apyrimidinic sites), DNA cross-links, base/base-pair damages and apoptotic nuclei in the cells (Nandhakumar et al., 2011;Peycheva et al., 2009). ...
... The first variant was used for detection of double stranded DNA breaks and the alkaline variant was utilized for simultaneously uncovering double-and single-stranded DNA breaks. Therefore, since the alkaline assay is rather far more sensitive than the neutral method in DNA damage determination (Peycheva et al., 2009), we performed the alkaline comet assay as described by Duthie (Duthie et al., 2002) and Kamla Al-Salmai (Al-Salmani et al., 2011) with minor modifications. A total of 50 µL of suspended leucocytes in PBS were embedded in 50 µL of 0.75% low-melting-point agarose on the glass slide. ...
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Background The relationship between depression and increased oxidative stress is well known. DNA damage by oxidation factors is an important cause of the aging process in psychiatric disorders. Aims Owing to the scarcity of human studies and high inconsistencies in studies of the effects of antidepressants on DNA damage, the current study was undertaken to investigate the effects of depression and its treatment on DNA damage. Methods In a 15-week open-label study of citalopram ( n = 25) and sertraline ( n = 20), levels of DNA damage were measured by comet assay, proinflammatory (Interlukin-6 (IL-6)) and oxidative DNA damage (8-hydroxy-2’-deoxyguanosine (8-OHdG)) markers by ELISA, and gene expression of base excision repair enzymes (8-oxoguanine glycosylase (OGG1) and poly (ADP)-ribose polymerase-1 (PARP1)) by quantitative real-time polymerase chain reaction in healthy control patients ( n = 14), with depression at the baseline and the same patients after week 15. Results DNA damage, 8-OHdG, IL-6 and expression of PARP1 were elevated in patients with depression compared with the healthy controls ( p < 0.001). Selective serotonin reuptake inhibitor (SSRI) therapy could significantly reduce the depression score ( p < 0.01), DNA damage ( p < 0.001), as well as 8-OHdG and IL-6 ( p < 0.0001). Nevertheless, the expression of PARP1 and OGG1 showed no significant changes after treatment. Conclusions This is the first study on the effect of SSRIs on the DNA damage and some of the repair enzymes in depression. Based on the results, depression can cause increased DNA damage. This damage is followed by activation of compensatory mechanisms whereby the expression of DNA damage repair enzymes is elevated. Finally, the treatment of psychiatric disorder by antidepressants can lower the level of oxidative DNA damage.
... Further on, the higher sensitivity of S. cerevisiae towards geirradiation (Nemavarkar, Chourasia, & Pasupathy, 2004), oxidative damage during replicative aging (Grzelak, Macierzynska & Bartpsz, 2006), and Cr-(III)-organic compounds (Chatterjee & Luo, 2010) revealed by the Comet Assay was confirmed (Azevedo, Marques, Fokt, Oliveira, & Johansson, 2011;Hrenovi c et al., 2010;Lah, Malovrh, Narat, Cepeljnik, & Marinsek-Logar, 2004). Currently we upgraded the technique and developed conditions for its implementation in order to obtain higher sensitivity and reproducibility of the obtained results (Peycheva, Georgieva, & Miloshev, 2009). ...
... Yeast cultures were grown in YPD medium at 30 C. Cells were cultivated to early logarithmic phase and treated with different concentrations of the compounds. The alkaline Yeast Comet Assay was applied as was published elsewhere (Miloshev et al., 2002;Peycheva et al., 2009) with some modifications. Yeast cells were collected by centrifugation, washed and resuspended in S-buffer (1 mol/L Sorbitol, 25 mmol/L NaH 2 PO 4 , pH 6.5). ...
... Next, in order to further investigate how CBL0137 impacted the DDR alone and in combination with irradiation, we performed alkaline comet assays using GBM NU757, GBM 1016, GBM 3691, and GBM 08-387. The alkaline comet assay allows for comet tails to include DNA fragments resulting mainly from single strand breaks but also double strand breaks to some extent [27][28][29][30]. Cells were exposed to a 6 h pre-treatment of vehicle or 300 nM CBL0137 then left unirradiated or exposed to 3 Gy of irradiation and collected 1 h post irradiation to measure comet tail length. ...
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... This unambiguously entails the application of sensitive tests for fast and accurate evaluation of the potential of any substance that is planned to be implicated in food, pharmaceutical and cosmetic practices to induce general cytotoxicity via inhibiting cell proliferation and/or damage in DNA [22][23][24]. The method of Comet Assay is a brilliant technology for fast and sensitive analysis of genotoxicity [25,26]; it requires single cells, is fast and with high precision evaluates all kinds of DNA damages. Data quantification allows precise estimation of genotoxicity. ...
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... The higher sensitivity of S. cerevisiae cells towards γ-irradiation (Nemavarkar et al., 2004), oxidative damage during replicative ageing (Grzelak et al., 2006), and Cr-(III)organic compounds (Chatterjee and Luo, 2010) revealed by the comet Assay was confirmed (Azevedo et al., 2011;Hrenović et al., 2010;Lah et al., 2004). Currently the technique was upgraded in order to obtain higher sensitivity and reproducibility of the results (Peycheva et al., 2009). ...
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... to reveal simultaneously double-and single-stranded DNA breaks, as well as alkali labile sites, which leads to the general assumption that the alkaline assay is much more sensitive in comparison with its neutral variant in DNA damage detection. [12] Since Singh et al. introduced the alkaline comet assay, its uses and applications have been increasing. The research areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. ...
Method
The comet assay (also known as single‑cell gel electrophoresis) is a technique for the detection of DNA damage at the level of the individual cell. It is a versatile, relatively simple to perform and sensitive method. Although most investigations make use of its ability to measure DNA single‑strand breaks, modifications to the method allow detection of cyclobutane pyrimidine dimers (CPDs), crosslinks, base damage, and apoptotic nuclei. Many investigators also interested in examining the DNA damage as a function of time after exposure to a known genotoxic agent. Here, we present a procedure of comet assay for the detection of DNA strand breaks, base damages, and CPDs that can be used to measure DNA damage during toxicity, oxidative stress, and ultraviolet radiation exposure and it can be applied in human toxicological biomonitoring scenarios
... to reveal simultaneously double-and single-stranded DNA breaks, as well as alkali labile sites, which leads to the general assumption that the alkaline assay is much more sensitive in comparison with its neutral variant in DNA damage detection. [12] Since Singh et al. introduced the alkaline comet assay, its uses and applications have been increasing. The research areas of its current employment in the evaluation of genetic toxicity are vast, either in vitro or in vivo, both in the laboratory and in the environment, terrestrial or aquatic. ...
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The comet assay (also known as single-cell gel electrophoresis) is a technique for the detection of DNA damage at the level of the individual cell. It is a versatile, relatively simple to perform and sensitive method. Although most investigations make use of its ability to measure DNA single-strand breaks, modifications to the method allow detection of cyclobutane pyrimidine dimers (CPDs), crosslinks, base damage, and apoptotic nuclei. Many investigators also interested in examining the DNA damage as a function of time after exposure to a known genotoxic agent. Here, we present a procedure of comet assay for the detection of DNA strand breaks, base damages, and CPDs that can be used to measure DNA damage during toxicity, oxidative stress, and ultraviolet radiation exposure and it can be applied in human toxicological biomonitoring scenarios.
... The method of ChCA performed in the current study is in neutral conditions. Neutral conditions allow a more delicately "sensing" of chromatin loop organization (Peycheva et al., 2009). A representative scheme of the two variants of the method is shown in Fig. 6. ...
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Chromatin structure promotes important epigenetic mechanisms that regulate cellular fate by organizing, preserving and controlling the way by which the genetic information works. Our understanding of chromatin and its functions is sparse and not yet well defined. The uncertainty comes from the complexity of chromatin and is induced by the existence of a large number of nuclear proteins that influence it. The intricate interaction among all these structural and functional nuclear proteins has been under extensive study in the recent years. Here, we show that Saccharomyces cerevisiae linker histone physically interacts with Arp4p (actin-related protein 4) which is a key subunit of three chromatin modifying complexes - INO80, SWR1 and NuA4. A single - point mutation in the actin - fold domain of Arp4p together with the knock-out of the gene for the linker histone in S. cerevisiae severely abrogates cellular and nuclear morphology and leads to complete disorganizing of the higher levels of chromatin organization. Copyright © 2014. Published by Elsevier Ltd.
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The comet assay is a versatile and sensitive method for measuring single- and double-strand breaks in DNA. The mechanism of formation of comets (under neutral or alkaline conditions) is best understood by analogy with nucleoids, in which relaxation of DNA supercoiling in a structural loop of DNA by a single DNA break releases that loop to extend into a halo—or, in the case of the comet assay, to be pulled towards the anode under the electrophoretic field. A consideration of the simple physics underlying electrophoresis leads to a better understanding of the assay. The sensitivity of the assay is only fully appreciated when it is calibrated: between one hundred and several thousand breaks per cell can be determined. By including lesion-specific enzymes in the assay, its range and sensitivity are greatly increased, but it is important to bear in mind that their specificity is not absolute. Different approaches to quantitation of the comet assay are discussed. Arguments are presented against trying to apply the comet assay to the study of apoptosis. Finally, some of the advantages and disadvantages of using the comet assay on lymphocyte samples collected in human studies are rehearsed.
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Generation of DNA damage is considered to be an important initial event in carcinogenesis. The single cell gel electrophoresis (comet) assay is a technically simple and fast method that detects genotoxicity in virtually any mammalian cell type without requirement for cell culture. This review discusses the strength of the comet assay in biomonitoring at its present state of validation. The simple version of the alkaline comet assay detects DNA migration caused by strand breaks, alkaline labile sites, and transient repair sites. By incubation with bacterial glycosylase/endonuclease enzymes, broad classes of oxidative DNA damage, alkylations, and ultraviolet light-induced photoproducts are detected as additional DNA migration. The most widely measured enzyme sensitive sites have been those detected by formamidopyrimidine DNA glycosylase (FPG) and endonuclease III (ENDOIII). Reports from biomonitoring studies show that the basal level of DNA damage in leukocytes is influenced be a variety of lifestyle and environmental exposures, including exercise, air pollution, sunlight, and diet. Although not all types of carcinogenic exposures should be expected to damage DNA in leukocytes, the comet assay is a valuable method for detection of genotoxic exposure in humans. However, the predictive value of the comet assay is unknown because it has not been investigated in prospective cohort studies. Also, it is important that the performance of the assay is investigated in multi-laboratory validation trials. As a tool in risk assessment the comet assay can be used in characterization of hazards.
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Single-cell gel electrophoresis, or the comet assay, is widely used to measure DNA damage and repair. Upon electrophoresis, the DNA of lysed, agarose-embedded cells known as nucleoids, extends towards the anode in a structure resembling a comet, the relative intensity of the tail reflecting the frequency of DNA breaks. The structural organization of the DNA within comet preparations is not fully understood. We have used fluorescent in situ hybridization with large-insert genomic probes and human Cot-I DNA to investigate whether the production of the comet tail is simply explained by the relaxation of supercoiled DNA loops. We find that, under neutral electrophoresis conditions, when the tail and head DNA are double-stranded, the probed sequence of DNA is seen as a linear array, consistent with extension from a fixed point on the nuclear core or matrix. After alkaline electrophoresis, the appearance of the fluorescent probes suggests that linear DNA has coalesced into a granular form.
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