Detection of alkylation damage in human lymphocyte DNA with the comet assay.

Collins, Andrew; Dušinská, Maria; Horská, Alexandra

doi:10.18388/abp.2001_3895

Cited by 102 publications

(45 citation statements)

References 8 publications

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“…A modified version of the assay can also be performed to detect specific base lesions. This is achieved by inclusion of purified lesion-specific enzymes that converts undetectable base lesion into detectable strand breaks (31,(33)(34)(35).…”

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confidence: 99%

Standard fluorescent imaging of live cells is highly genotoxic

Wood

Weingeist

et al. 2013

Cytometry Pt A

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Fluorescence microscopy is commonly used for imaging live mammalian cells. Here, we describe studies aimed at revealing the potential genotoxic effects of standard fluorescence microscopy. To assess DNA damage, a high throughput platform for single cell gel electrophoresis is used (e.g., the CometChip). Light emitted by three standard filters was studied: a) violet light [340–380 nm], used to excite DAPI and other blue fluorophores, b) blue light [460–500 nm] commonly used to image GFP and Calcein AM, and c) green light [528–553], useful for imaging red fluorophores. Results show that exposure of samples to light during imaging is indeed genotoxic even when the selected wavelengths are outside the range known to induce significant levels. Shorter excitation wavelengths and longer irradiation times lead to higher levels of DNA damage. We have also measured DNA damage in cells expressing enhanced green fluorescent protein (GFP) or stained with Calcein AM, a widely used green fluorophore. Data show that Calcein AM leads to a synergistic increase in the levels of DNA damage and that even cells that are not being directly imaged sustain significant DNA damage from exposure to indirect light. The nature of light-induced DNA damage during imaging was assessed using the Fpg glycosylase, an enzyme that enables quantification of oxidative DNA damage. Oxidative damage was evident in cells exposed to violet light. Furthermore the Fpg glycosylase revealed the presence of oxidative DNA damage in blue-light exposed cells for which DNA damage was not detected using standard analysis conditions. Taken together, the results of these studies call attention to the potential confounding effects of DNA damage induced by standard imaging conditions, and identify wavelength, exposure time and fluorophore as parameters that can be modulated to reduce light-induced DNA damage.

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confidence: 99%

Standard fluorescent imaging of live cells is highly genotoxic

Wood

Weingeist

et al. 2013

Cytometry Pt A

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“…A value (damage index, DI) was assigned to each comet according to its class, using the formula: DI = (0 x n 0 ) + (1 x n 1 ) + (2 x n 2 ) + (3 x n 3 ) + (4 x n 4 ), Where n = number of cells in each class analyzed. The damage index ranged from 0 (completely undamaged: 100 cells x 0) to 400 (with maximum damage: 100 cells x 4) (COLLINS et al, 1995;SILVA et al, 2000;COLLINS et al, 2001). Finally, the specimen total score correspond to scores media of their two slides and represent, thus, the frequency of breaks in their DNA.…”

Section: Methodsmentioning

confidence: 99%

GENOTOXIC EFFECTS OF METHYLMERCURY ON THE FISH <i>Colossoma macropomum</i> (CHARACIFORMES: CHARACIDAE) AS EVALUATED USING THE COMET ASSAY

Rocha¹,

Cavalcanti²,

Pessoa³

et al. 2012

Uakari

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Comet assay has been used increasingly to evaluate genotoxicity of many metals and their organic compounds in aquatic ecosystems. The use of endemic aquatic organisms as biological sentinels has proved to be useful to environmental monitoring. In this study, the genetic damages caused by methylmercury (MeHg) in the widely distributed Amazonian fish, Colossoma macropomum (commonly called tambaqui), was assessed using the comet assay. Specimens of C. macropomum were acclimatized in aquariums to laboratory conditions and then exposed to 2mg.L-1 MeHg by hydric way. Peripheral blood samples were obtained from specimens and subjected to the comet assay. Exposure to MeHg significantly increased the frequency of tailed nucleoids in the erythrocytes of fishes exposed (p<0.0005), indicating DNA damage. The results obtained show that the neotropical fish C. macropomum is not only important as eatable species, but also an adequate model for biological studies that evaluate genotoxic effects of mercury in aquatic environments.

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“…DNA alkylation can also be measured using a modified comet assay. This method involves the digestion of alkylated DNA bases with 3-methyladenine DNA glycosylase (Collins et al, 2001;Hasplova et al, 2012) followed by the standard comet assay to detect where alkyl adducts occur. The advantage of this method is that the alkaline version of the comet assay, as a core method, has an in vivo OECD guideline.…”

Section: Introductionmentioning

confidence: 99%