DNA damage induced by micro- and nanoparticles—interaction with FPG influences the detection of DNA oxidation in the comet assay

J. Kain 1 y H. L. Karlsson 0 1 L. Moller 1 0 Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet , SE-171 77 Stockholm , Sweden 1 Unit for Analytical Toxicology, Department of Biosciences and Nutrition at Novum, Karolinska Institutet , SE-141 83 Huddinge , Sweden TThheeAAuutthhoorr22001122..PPuubblilsishheeddbbyyOOxxfofordrdUUninvievresristiytyPrPersesssononbebheahlafloffotfhethUeKUKEnEvnirvoinromnemnetanltaMl uMtaugtaegneSnoScioectiye.ty. AAllll rriigghhttss rreesseerrvveedd.. FFoorr ppeerrmmiissssiioonnss,,pplleeaasseeee--mmaaiill::jjoouurrnnaallss..ppeerrmmiissssiioonnss@@oouupp..ccoomm.. - *To whom correspondence should be addressed: Tel: 46 8 524 810 75; Fax: 46 8 774 68 33; Email: Received on May 16, 2011; revised on February 10, 2012; accepted on February 14, 2012 Reliable methods for evaluation of toxicity from particles, such as manufactured nanoparticles, are needed. One promising tool is the comet assay, often used to measure DNA breaks (strand breaks and alkali-labile sites) as well as oxidatively damaged DNA, the latter by addition of specific DNA repair enzymes such as formamidopyrimidine DNA glycosylase (FPG). The aim of this study was to investigate the use of the comet assay for analysis of DNA oxidation by a range of micro- and nanoparticles in the lung cell lines A549 and BEAS-2B and to test the hypothesis that nanoparticles present in the cells during the assay performance may interact with FPG. This was done by investigating the ability of micro- and nanoparticles (stainless steel, subway particles, MnO2, Ag, CeO2, Co3O4, Fe3O4, NiO and SiO2) to induce DNA breaks, oxidatively damaged DNA (FPG sites, dominantly 8oxoguanine), intracellular production of reactive oxygen species (ROS) and non-cellular oxidation of the DNA base guanine, as well as by studying interactions of the particles and their released ions with FPG. Several particles caused DNA breaks, but low levels of FPG sites. The ability of FPG to detect DNA oxidation induced by a photosensitiser was however shown. An oxidative capacity of the particles was indicated by increased levels of intracellular ROS, and especially Ag and subway particles caused non-cellular oxidation of guanine. Incubation of FPG with the particles led to less FPG activity, particularly with nanoparticles of Ag but also with CeO2, Co3O4 and SiO2. Further investigations of these particles revealed that for Ag, the decreased activity was mainly due to released Ag ions, whereas for CeO2 and Co3O4, FPG interactions were due to the particles. We conclude that measurement of oxidatively damaged DNA in cells exposed to nanoparticles may be underestimated in the comet assay due to interactions with FPG. Introduction The link between exposure to ambient particles in air pollution and health effects, such as cancer, cardiovascular and respiratory diseases, is well known and inflammation and oxidative stress are believed to be the main routes from the particle exposure to diseases (1,2). Particles from other sources, such as metal-based nanoparticles in consumer products, from industrial production sites and other environments such as the subway system are less studied and imply new challenges when it comes to human health risk assessment. Nanoparticles, often classified as particles with at least one dimension ,100 nm, are due to their many interesting properties more extensively used today in medical applications as well as in different industrial and consumer products. The unique properties of nanoparticles include a large surface area per unit mass, which can display a large amount of reactive surface molecules leading to high reactivity, as well as unique material properties affecting, e.g. electrical conductivity, magnetic characteristics and hardness. Such properties may not only be beneficial in different contexts but can also induce toxicity (3). Due to the expanding use of nanoparticles and increased exposure risks, there is an urgent need for risk assessment and legislation. This may also concern particles from occupational settings or other milieus, e.g. the subway system, where little is known about the toxicity of the emitted particles. Many in vitro toxicity assays designed for testing of chemicals are also used for particles. Particles do, however, display several unique physicochemical properties due to their solidity, such as size, shape, agglomeration properties, elemental purity and surface area, which make them different when compared to chemicals. Some toxicity assays may therefore not be appropriate to use for particles. The unique particle properties may lead to interaction in assays and give misleading information about toxicity (4). The interference can, e.g. be due to the fact that nanoparticles have optical absorbance at the same wavelength as a coloured product in an assay or adsorb dyes or cytokines that are supposed to be measured (5). Reaction between the particle surface and the dye, endotoxin contamination of the particles and magnetic properties may also cause problems (5). In the present need for screening methods for evaluation of particle toxicity, it is therefore important to evaluate the reliability of the applied assays. One promising tool in toxicity testing of particles is the comet assay (single-cell gel electrophoresis), where DNA damage is measured in single cells. In this method, cells are lysed, the DNA is denaturated and electrophoresis is performed. During the electrophoresis, strand breaks in the DNA allow DNA to migrate out of the nucleoid, which appears as comets when evaluated microscopically. The amount of DNA in the comet tail represents DNA breaks in the form of strand breaks (SB) and alkali-labile sites (ALS). By using DNA repair enzymes, often formamidopyrimidine DNA glycosylase (FPG), more specific damage can also be measured. FPG predominantly detects oxidatively damaged DNA (FPG sites), which can be a product of oxidative stress, in the form of oxidised purines, primarily 8oxoguanine and ring-opened formamidopyrimidine bases (e.g. FapyGua) (6). Formation of 8-oxoguanine, which is mutagenic and in general assumed to be related to carcinogenesis, is considered to be a marker for oxidative stress (7). Oxidative stress and 8-oxoguanine have been shown to be increased in cancer tissue (8,9) and studies have, by using the comet assay, linked human ambient particle exposure to oxidatively damaged DNA in lymphocytes, as a measure of systemic oxidative stress (10,11). There is no standardised protocol for the comet assay, but the method has been evaluated in inter-laboratory trials conducted by the European Standards Committee on Oxidative DNA Damage (ESCODD) (12) and by the European Comet Assay Validation Group (ECVAG) (13,14). The importance of the protocol used has also been highlighted in two recently conducted studies (15,16). The comet assay seems to be a good method to use for evaluation of DNA damage induced by chemi (...truncated)