Impaired Clearance and Enhanced Pulmonary Inflammatory/Fibrotic Response to Carbon Nanotubes in Myeloperoxidase-Deficient Mice Anna A. Shvedova3, Alexandr A. Kapralov1,2, Wei Hong Feng1,2, Elena R. Kisin3, Ashley R. Murray3, Robert R Mercer3, Claudette M. St. Croix2, Megan A. Lang4, Simon C. Watkins4, Nagarjun V. Konduru1,2, Brett L. Allen8, Jennifer Conroy5, Gregg P. Kotchey8, Bashir M. Mohamed 5 , Aidan D. Meade7, Yuri Volkov5,6, Alexander Star8, Bengt Fadeel9, Valerian E. Kagan1,2* 1 Center for Free Radical and Antioxidant Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 2 Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 3 Pathology and Physiology Research Branch, Health Effects Lab Division, National Institute for Occupational Safety and Health, Morgantown, West Virginia, United States of America, 4 Departments of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 5 Department of Clinical Medicine, Trinity College, Dublin, Ireland, 6 Center for Research on Adaptive Nanostructures and Nanodevices, Trinity College, Dublin, Ireland, 7 School of Physics, College of Science and Health, Dublin Institute of Technology, Dublin, Ireland, 8 Department of Chemistry, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America, 9 Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Abstract Advancement of biomedical applications of carbonaceous nanomaterials is hampered by their biopersistence and pro- inflammatory action in vivo. Here, we used myeloperoxidase knockout B6.129X1-MPO (MPO k/o) mice and showed that oxidation and clearance of single walled carbon nanotubes (SWCNT) from the lungs of these animals after pharyngeal aspiration was markedly less effective whereas the inflammatory response was more robust than in wild-type C57Bl/6 mice. Our results provide direct evidence for the participation of MPO – one of the key-orchestrators of inflammatory response – in the in vivo pulmonary oxidative biodegradation of SWCNT and suggest new ways to control the biopersistence of nanomaterials through genetic or pharmacological manipulations. Citation: Shvedova AA, Kapralov AA, Feng WH, Kisin ER, Murray AR, et al. (2012) Impaired Clearance and Enhanced Pulmonary Inflammatory/Fibrotic Response to Carbon Nanotubes in Myeloperoxidase-Deficient Mice. PLoS ONE 7(3): e30923. doi:10.1371/journal.pone.0030923 Editor: Partha Mukhopadhyay, National Institutes of Health, United States of America Received November 1, 2011; Accepted December 24, 2011; Published March 30, 2012 This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Funding: National Institute for Occupational Safety and Health OH008282, National Institutes of Health HL70755, HL094488, U19AI068021, National Institute of Environmental Health Sciences R01ES019304, National Library of Medicine LM007994-05, National Occupational Research Agenda 927000Y, 927Z1LU, Nanotechnology Research Center 927ZJHF, National Science Foundation CAREER 0449117, Air Force Office of Scientific Research FA9550-09-1-0478, 7th Framework Program of the European Commission (EC-FP7-NANOMMUNE-214281),Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning and by the Science Foundation of Ireland, Strategic Research Cluster BioNanointeract and Centre for Research on Adaptive Nanostructures and Nanodevices, Higher Education Authority (Ireland) and Programme for Research in Third-Level Institutions (PRTLI) (Ireland). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kagan@pitt.edu Introduction Biopersistence of carbon nanotubes (CNT) - resulting from their inherent durability [1,2] is one the major stumbling blocks on the way of their broad biomedical applications. This is because engineered CNT represent a possible health risk due to their ability to cause pulmonary inflammation, severe oxidative stress and early onset fibrosis in animals [3,4,5,6]. They also exert genotoxic effects [7] possibly associated with carcinogenesis (e.g., induction of mesotheliomas) [8,9]. These health concerns have been associated – to a large extent – with the reported long ‘‘life- span’’ of SWCNT in the lung thus necessitating exploration of possible metabolic pathways leading to their biodegradation. Although different types of chemical oxidative cutting of CNT - lengthwise and shortening – have been described, they require harsh oxidants (e.g., sulphuric acid plus H2O2 or KMnO4) [10]. Recently, we reported that reactive intermediates of horseradish peroxidase are effective in enzymatic oxidative biodegradation of CNT and graphene oxide [11,12,13]. Moreover, myeloperoxi- dase (MPO), an abundant enzyme of inflammatory cells (neutrophils), - involved in the principal defense mechanisms of innate immunity -was also effective in oxidative biodegradation of CNT in biochemical models and in cells yielding the products that did not cause pulmonary inflammation in mice [14]. However, the relevance of this mechanism for CNT biodegra- dation in vivo was lacking. Here, we employed MPO knockout B6.129X1-MPO (MPO k/o) vs wild-type C57Bl/6 mice (w/t) and demonstrated that clearance and oxidation of single wall carbon nanotubes (SWCNT) in the lungs after their pharyngeal aspiration was markedly less effective whereas inflammatory response was more robust in the former than in the latter. Our results provide direct evidence for the participation of MPO in pulmonary biodegradation of SWCNT in vivo. Based on these data, new genetic and pharmacological approaches can be developed to regulate the biopersistence of nanomaterials in tissues. PLoS ONE | www.plosone.org 1 March 2012 | Volume 7 | Issue 3 | e30923