An ECVAG trial on assessment of oxidative damage to DNA measured by the comet assay

Abstract

The increasing use of single cell gel electrophoresis (the comet assay) highlights its popularity as a method for detecting DNA damage, including the use of enzymes for assessment of oxidatively damaged DNA. However, comparison of DNA damage levels between laboratories can be difficult due to differences in assay protocols (e.g. lysis conditions, enzyme treatment, the duration of the alkaline treatment and electrophoresis) and in the end points used for reporting results (e.g. %DNA in tail, arbitrary units, tail moment and tail length). One way to facilitate comparisons is to convert primary comet assay end points to number of lesions/106 bp by calibration with ionizing radiation. The aim of this study was to investigate the inter-laboratory variation in assessment of oxidatively damaged DNA by the comet assay in terms of oxidized purines converted to strand breaks with formamidopyrimidine DNA glycosylase (FPG). Coded samples with DNA oxidation damage induced by treatment with different concentrations of photosensitizer (Ro 19-8022) plus light and calibration samples irradiated with ionizing radiation were distributed to the 10 participating laboratories to measure DNA damage using their own comet assay protocols. Nine of 10 laboratories reported the same ranking of the level of damage in the coded samples. The variation in assessment of oxidatively damaged DNA was largely due to differences in protocols. After conversion of the data to lesions/106 bp using laboratory-specific calibration curves, the variation between the laboratories was reduced. The contribution of the concentration of photosensitizer to the variation in net FPG-sensitive sites increased from 49 to 73%, whereas the inter-laboratory variation decreased. The participating laboratories were successful in finding a dose–response of oxidatively damaged DNA in coded samples, but there remains a need to standardize the protocols to enable direct comparisons between laboratories.

New nanostructural biomaterials based on active silicate systems and hydroxyapatite: characterization and genotoxicity in human peripheral blood lymphocytes

“…, Johansson et al . ). However, although the comparison of results obtained from in vitro and in vivo genotoxicity assays indicates that the comet assay gives relevant information, it is important to emphasize that in vitro assays do not take into consideration the complex homoeostatic situation that occurs in vivo (Ribeiro , Liuyun et al .…”

Exploring the aneugenic and clastogenic potential in the nanosize range: A549 human lung carcinoma cells and amorphous monodisperse silica nanoparticles as models

“…Briefly, after trypsinization, cells were mixed with 300 ml of low melting point agarose (0.8% w/v in PBS) and loaded onto a normal pre-coated microscopic slide (normal melting point agarose 1% w/v in water). After lysis at 4 C (2.5 M NaCl, 100 mM NA 2 EDTA, 10 mM Tris supplemented with 1% Triton-X100 and 10% DMSO just before use), slides were rinsed twice with enzyme buffer (40 mM HEPES, 100 mM KCl, 0.5 mM EDTA, 0.2 mg/ml BSA; pH 8) prior to incubation with 75 ml formadidopyrimidine DNA glycosylase (FPG; New England Biolabs, Leusden, The Netherlands) for 35 min at 37 C. FPG was chosen because of the great efforts that recently have been made by the European Comet Validation Group (ECVAG) to validate this method (Johansson et al 2010. Furthermore 8-oxo-2¢deoxyguanosine (8-oxo-dG) is the most common oxidative lesion (Pluskota-Karwatka 2008).…”

Influence of experimental conditions on data variability in the liver comet assay

“…While it has been demonstrated that several technical parameters, such as slide preparation, electrophoresis, or scoring of comets can have a strong impact on the assay results (Forchhammer et al, ; Johansson et al, ; Azqueta et al, ), little is known on how initial steps affect the comet assay results. Thus, we evaluated how variations during the processing of liver tissue (meaning from necropsy and tissue sampling until single liver cells are embedded in an agarose layer on the glass slide) impact DNA migration in samples from untreated animals.…”