Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver

Particle and Fibre Toxicology Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver Julie A Bourdon 2 Anne T Saber 0 Nicklas R Jacobsen 0 Keld A Jensen 0 Anne M Madsen 0 Jacob S Lamson 0 Hkan Wallin 0 1 Peter Mller 1 Steffen Loft 1 Carole L Yauk 2 Ulla B Vogel 0 0 National Research Centre for the Working Environment , Copenhagen , Denmark 1 Univesity of Copenhagen, Department of Public Health, Section of Environmental Health , Copenhagen , Denmark 2 Health Canada, Environmental and Radiation Health Sciences Directorate, Mechanistic Studies Division , Tunney's Pasture, Ottawa , Canada Background: Widespread occupational exposure to carbon black nanoparticles (CBNPs) raises concerns over their safety. CBNPs are genotoxic in vitro but less is known about their genotoxicity in various organs in vivo. Methods: We investigated inflammatory and acute phase responses, DNA strand breaks (SB) and oxidatively damaged DNA in C57BL/6 mice 1, 3 and 28 days after a single instillation of 0.018, 0.054 or 0.162 mg Printex 90 CBNPs, alongside sham controls. Bronchoalveolar lavage (BAL) fluid was analyzed for cellular composition. SB in BAL cells, whole lung and liver were assessed using the alkaline comet assay. Formamidopyrimidine DNA glycosylase (FPG) sensitive sites were assessed as an indicator of oxidatively damaged DNA. Pulmonary and hepatic acute phase response was evaluated by Saa3 mRNA real-time quantitative PCR. Results: Inflammation was strongest 1 and 3 days post-exposure, and remained elevated for the two highest doses (i.e., 0.054 and 0.162 mg) 28 days post-exposure (P < 0.001). SB were detected in lung at all doses on postexposure day 1 (P < 0.001) and remained elevated at the two highest doses until day 28 (P < 0.05). BAL cell DNA SB were elevated relative to controls at least at the highest dose on all post-exposure days (P < 0.05). The level of FPG sensitive sites in lung was increased throughout with significant increases occurring on post-exposure days 1 and 3, in comparison to controls (P < 0.001-0.05). SB in liver were detected on post-exposure days 1 (P < 0.001) and 28 (P < 0.001). Polymorphonuclear (PMN) cell counts in BAL correlated strongly with FPG sensitive sites in lung (r = 0.88, P < 0.001), whereas no such correlation was observed with SB (r = 0.52, P = 0.08). CBNP increased the expression of Saa3 mRNA in lung tissue on day 1 (all doses), 3 (all doses) and 28 (0.054 and 0.162 mg), but not in liver. Conclusions: Deposition of CBNPs in lung induces inflammatory and genotoxic effects in mouse lung that persist considerably after the initial exposure. Our results demonstrate that CBNPs may cause genotoxicity both in the primary exposed tissue, lung and BAL cells, and in a secondary tissue, the liver. Oxidative stress; Genotoxicity; DNA strand breaks; Inflammation; Nanoparticles; Carbon Black - Background The use of nanoparticles (NPs) in consumer products and applications continues to rise [1]. In parallel, the potential for NP mediated toxicity is a growing public concern. Many of the unique properties exhibited by NPs increase the likelihood of deleterious biological interactions and subsequently, the risk of adverse health outcomes [2-4]. Understanding the repercussions of inhaling NPs is particularly important because NPs penetrate deeper regions of the lung (e.g., alveoli and pulmonary interstitium) [5,6], are translocated from lung to systemic circulation more readily [7,8], and are cleared from the lungs less effectively [9] than their larger counterparts. As such, there is a great probability of cellular interactions, necessitating investigations of NPmediated toxicity and risk of health consequences. Carbon black (CB) has been widely investigated since its use as a benchmark control for in vivo toxicological evaluation of diesel exhaust particles and as a model of urban air pollution particulate matter almost three decades ago [10,11]. Since then, CB has become the focus of numerous toxicity studies as well as an important reference material (i.e., Printex 90) [12,13]. CBNPs are reactive oxygen species (ROS) generators as shown in cellular [14,15] and acellular systems [16]. Moreover, inhalation or intratracheal instillation exposures to CBNPs result in large pulmonary inflammatory responses in rodents [17-24], which can greatly exacerbate ROS generation via activation of polymorphonuclear (PMN) granulocytes [25]. As such, it is expected that CBNPs can mediate secondary genotoxicity by means of inflammation and oxidative stress. CBNPs are genotoxic in vitro, as shown by increases in DNA base oxidation [26], mutation frequency [26,27], strand breaks [28,29] and micronucleus frequency in lung epithelial cells [30] as well as increases in strand breaks in fibroblasts [31]. However, less is known about the genotoxicity of CBNPs in vivo. A few studies in rats have demonstrated CBNP-induced DNA base oxidation [32] and increased mutation frequency [20]. However, rats may not be the most suitable model for exposure to particulates due to their predisposition to particle overload. Studies in mice have demonstrated DNA strand breaks in BAL cells [21,33] and one study has established CBNP-induced lung DNA strand breaks, but this was found using a high dose (i.e., 0.2 mg) immediately (3 hours) post instillation [30]. The growing demand for CBNPs for diverse commercial applications (e.g., rubber products and pigments) raises health concerns for the increasing number of individuals routinely exposed, especially in occupational settings where relatively high levels of exposure may occur. As such, it is critical to establish whether genotoxicity and oxidative stress arise in vivo at low doses of exposure and in extrapulmonary tissues, and to determine whether these effects are associated with inflammation and/or persist for long periods of time following the initial exposure. Here, we investigate the relationships between inflammation and genotoxic outcomes over time after a single exposure to Printex 90 CBNPs in BAL cells, lung and liver. Mice were exposed via intratracheal instillation using various doses (i.e., 0.018, 0.054 and 0.162 mg) and post-exposure recovery time-points (i.e., 1, 3 and 28 days), alongside sham controls. We report that instillation of CBNPs leads to prolonged generation of DNA damage in BAL cells, lung and liver of exposed mice as well as persistent pulmonary inflammation, acute phase response and oxidatively damaged DNA. Results Particle characterization Printex 90 CBNPs were a gift from Evonik/Degussa (Frankfurt, Germany). The manufacturer reported an average primary particle size of 14 nm and an organic impurity content of less than 1%. The specific surface area was determined to be 295-338 m2/g, corresponding to a theoretical average spherical particle size of 8.1-9.5 nm [34]. The total carbon content measured was greater than 99 wt%, with 0.82 nitrogen and 0.01 hydrogen w (...truncated)