Oxidative damage to DNA by diesel exhaust particle exposure in co-cultures of human lung epithelial cells and macrophages

Kim Jantzen 1 Martin Roursgaard 1 Claus Desler 0 Steffen Loft 1 Lene Juel Rasmussen 0 Peter Mller 1 0 Center for Healthy Aging, Department of Cellular and Molecular Medicine, University of Copenhagen , Blegdamsvej 3B, DK-2200 Copenhagen N, Denmark 1 Department of Environmental Health, Faculty of Health Science, University of Copenhagen , ster Farimagsgade 5A, DK-1014 Copenhagen K, Denmark , University of Copenhagen, Universitetsparken 2 , DK-2100 Copenhagen , Denmark The Author 2012. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: . - *To whom correspondence should be addressed. Tel: +45 353 27989; Fax: +45 353 27686; Email: Received on December 12, 2011; revised on May 7, 2012; accepted May 11, 2012 Studies in mono-culture of cells have shown that diesel exhaust particles (DEPs) increase the production of reactive oxygen species (ROS) and oxidative stressrelated damage to DNA. However, the level of particle-generated genotoxicity may depend on interplay between different cell types, e.g. lung epithelium and immune cells. Macrophages have important immune defence functions by engulfing insoluble foreign materials, including particles, although they might also promote or enhance inflammation. We investigated the effect of co-culturing type II lung epithelial A549 cells with macrophages upon treatment with standard reference DEPs, SRM2975 and SRM1650b. The exposure to DEPs did not affect the colony-forming ability of A549 cells in co-culture with THP-1a cells. The DEPs generated DNA strand breaks and oxidatively damaged DNA, measured using the alkaline comet assay as formamidopyrimidine-DNA glycosylase or oxoguanine DNA glycosylase (hOGG1) sensitive sites, in mono-cultures of A549 or THP-1a and co-cultures of A549 and THP-1a cells. The strongest genotoxic effects were observed in A549 mono-cultures and SRM2975 was more potent than SRM1650b. The ROS production only increased in cells exposed to SRM2975, with strongest concentration-dependent effect in the THP-1a mono-cultures. The basal respiration level in THP-1a cells increased on exposure to SRM1650b and SRM2975 without indication of mitochondrial dysfunction. This is consistent with activation of the cells and there was no direct relationship between levels of respiration and ROS production. In conclusion, exposure of mono-cultured cells to DEPs generated oxidative stress to DNA, whereas co-cultures with macrophages had lower levels of oxidatively damaged DNA than A549 epithelial cells. Introduction Mono-cultures of cells are widely used as model systems for toxicological effects of particulate matter, despite the fact that animals and humans are multi-cellular organisms and genotoxic effects of particles may depend on interactions between different types of cells. Airway exposure to particles can cause a mixture of effects related to inflammatory reactions, oxidative stress and genotoxicity in target tissue cells (1). The genotoxicity is considered to arise as a direct effect of the particles or indirectly because of activation of cells from the innate immune system (2). Thus, co-cultivating target cells with cells of the innate immune system may elicit a different response compared to mono-cultures and could serve to heighten the quality of in vitrobased toxicology studies. Diesel exhaust particles (DEPs) are relevant compounds for studies on genotoxicity in co-cultures because there is substantial knowledge about their effects in mono-cultures. Pulmonary exposure to DEPs is associated with increased levels of oxidatively damaged DNA in pulmonary tissues (3). These lesions include the pre-mutagenic 8-oxo-7, 8-dihydro-2-deoxyguanosine (8-oxodG) (47), which may arise as a consequence of increased production of reactive oxygen species (ROS) by more or less direct effects of particles or quinone compounds or by activated inflammatory cells, especially macrophages and neutrophils (8,9). It has been shown that DEPs were located within the inner mitochondrial membrane of cultured macrophages (10). This might cause mitochondrial dysfunction because of particle-mediated ROS production that oxidises disulphide bonds on cysteine residues within the electron transfer chain (ETC) (11). In turn, a dysfunctional ETC has impaired capability of electron transfer and may leak electrons to form superoxide anion radicals, thus further contributing to oxidative stress levels. The aim of this study was to investigate the differences in susceptibility to DEP-induced oxidative stressrelated damage between mono-cultures and co-cultures of lung epithelial cells and macrophages. Materials and methods DEP preparations Standard reference material (SRM)2975 and SRM1650b were purchased from the National Institute of Standards and Technology (Gaithersburg, MD, USA). SRM2975 and SRM1650b are DEPs collected from an industrial diesel-powered forklift (light duty engine) and a heavy-duty diesel engine, respectively. The particles are relatively well characterised, especially in regard to the content of extractable organic compounds and concentrations of transition metals. The supplier has reported in the certificate that the total extractable mass, based on Soxhlet extraction using dichloromethane, is 2.7% and 20.0% for SRM2975 and SRM1650b, respectively. The concentrations of polycyclic aromatic hydrocarbons are 35 and 390 mg/kg in the SRM2975 and SRM1650b, respectively. The concentrations of iron and copper are 3.1 and 20 ng/mg in SRM1650b; the SRM2975 contains 0.9 mg/kg of iron, whereas copper is lower than the limit of detection (12). The DEPs were suspended in distilled H2O (1 mg/ml) by sonication for a total of 8 min in 10 s intervals with 10 s breaks to avoid overheating. Sonication was performed with a Branson Sonicator at 450W and 10% amplitude. This suspension was subsequently diluted in RPMI cell culture media (Invitrogen, Taastrup, Denmark) supplemented with 10% foetal bovine serum (FBS, Biological Industries, Israel). We measured the particle size in the suspensions by Nanoparticle Tracking Analysis (NTA); the results showed a mean diameter of 76 nm for SRM2975 and 166 nm for SRM1650b. The percentage of particles with particle size less than 100 nm was 66% and 14% of the SRM2975 and SRM1650b respectively. Graphs of the particle size distribution analysis are shown in the online supplement (supplementary Figure 1, available at Mutagenesis Online). Cell cultures Type II pneumocytes (A549) and monocytic THP-1 cells were obtained from American Type Culture Collection (Manasses, VA, USA). The A549 cells were grown in F-12 media (Invitrogen, Taastrup, Denmark) supplemented with 10% FBS (Biological industries, Israel), l-glutamine (2.4 mM) and PenStrep (mixture of penicillin and streptomycin antibiotics, 1% v/v). The cells were grown at 37C, 5% CO2. The A549 cells were subcultivated every 23 days by trypsination, whereas the THP-1 cells were su (...truncated)