Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Sensitive detection of DNA oxidation damage induced by nanomaterials Andrew Collins a,b, ⁎ , Naouale El Yamani b,c , Maria Dusinska c a University of Oslo, Department of Nutrition, Oslo, Norway b NorGenotech AS, Skreia, Norway c Norwegian Institute for Air Research, Department of Environmental Chemistry, Kjeller, Norway ARTICLE INFO Keywords: Nanomaterials Comet assay DNA oxidation Lesion-specific enzymes ABSTRACT From a toxicological point of view, nanomaterials are of interest; because – on account of their great surface area relative to mass - they tend to be more reactive than the bulk chemicals from which they are derived. They might in some cases have the potential to damage DNA directly, or could act via the induction of oxidative stress. The comet assay (single cell gel electrophoresis) is widely used to measure DNA strand breaks and also oxidised bases, by including in the procedure digestion with lesion-specific enzymes such as formamidopyrimidine DNA glycosylase (which converts oxidised purines to breaks) or endonuclease III (recognising oxidised pyrimidines). We summarise reports in which these enzymes have been used to study a variety of nanomaterials in diverse cell types. We also stress that it is important to carry out tests of cell viability alongside the genotoxicity assay, since cytotoxicity can lead to adventitious DNA damage. Different concentrations of nanomaterials should be investigated, concentrating on a non-cytotoxic range; and incubating for short and longer periods can give valuable information about the mode of damage induction. The use of lesion-specific enzymes can substantially enhance the sensitivity of the comet assay in detecting genotoxic effects. 1. Introduction Hazard assessment of nanomaterials (NMs) presents particular challenges. Physicochemical properties of NMs differ significantly from those of corresponding bulk chemicals, mainly because of their high surface area relative to mass, which tends to increase reactivity. They are also better able to penetrate cellular and even, in certain cases, nuclear membranes (e.g. [1]), and so potentially have access to chromatin at all stages of the cell cycle. In addition to the possibility of direct, damaging interactions with DNA, NMs might cause oxidative damage – either as part of the cellular response to their presence, or as a secondary effect of inflammation induced by the particles. The comet assay (single cell gel electrophoresis) [2] is a commonly used method for assessing DNA damage in many different cell types. Essentially, a single cell suspension is mixed with agarose and set on a glass slide or Gelbond film. Lysis in Triton X-100 removes membranes and soluble cell components, and high molarity NaCl strips histones from the DNA, leaving it as a series of supercoiled loops attached to a matrix – a structure known as a nucleoid. DNA breaks are detected by their ability to relax supercoiling, so that loops containing a single (or double) strand break (SB) are able to extend towards the anode during electrophoresis, forming a comet-like image as viewed by fluorescence microscopy. The % of DNA in the tail is proportional to the break frequency. Normally the mean or median % tail DNA from 50 or 100 comets is taken as the measure of DNA damage, and if required it can be converted to a break frequency using a calibration curve based on X- or γ-irradiation, which has a known breakage rate per Gray [3]. Other lesions than strand breaks can be detected by including incubation with a lesion-specific endonuclease in the assay, after lysis; net enzyme-sensitive sites are estimated by subtracting the % tail DNA after lysis alone (or with buffer incubation) from the % tail DNA after enzyme incubation. The first enzyme so used was EndoIII (endonu- clease III, Nth) [4], which converts oxidised pyrimidines to breaks. Formamidopyrimidine DNA glycosylase (Fpg) recognises 8-Oxo-7,8- dihydroguanine (8-oxoG) and formamidopyrimidines (ring-opened purines) and is widely used to estimate DNA oxidation damage, for instance in white blood cells collected during human biomonitoring studies [5], as well as in genotoxicity testing. 8-Oxoguanine DNA glycosylase (OGG1), the mammalian analogue of Fpg, is occasionally used; it has a high specificity for oxidised purines, whereas Fpg also recognises some alkylated bases. 2. Recent investigations of toxic effects of nanomaterials, using Fpg to detect DNA oxidation There is good reason to examine NM effects with Fpg in the comet http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.001 Received 28 September 2016; Received in revised form 2 January 2017; Accepted 1 February 2017 ⁎ Correspondence to: University of Oslo, Department of Nutrition, PB 1046 Blindern, 0316 Oslo, Norway. E-mail address: a.r.collins@medisin.uio.no (A. Collins). Free Radical Biology and Medicine (xxxx) xxxx–xxxx 0891-5849/ © 2017 Elsevier Inc. All rights reserved. Please cite this article as: Collins, A., Free Radical Biology and Medicine (2017), http://dx.doi.org/10.1016/j.freeradbiomed.2017.02.001