Original Contribution ESR evidence for in vivo formation of free radicals in tissue of mice exposed to single-walled carbon nanotubes A.A. Shvedova a,b,n , E.R. Kisin a , A.R. Murray a,b , A. Mouithys-Mickalad c , K. Stadler d , R.P. Mason d , M. Kadiiska d a Pathology and Physiology Research Branch, HELD, National Institute for Occupational Safety and Health, Morgantown, WV 26505, USA b Physiology and Pharmacology, West Virginia University, Morgantown, WV 26506, USA c University of Liege, Liège 4000, Belgium d National Institute of Environmental Health Science, Research Triangle Park, NC 27709, USA article info Article history: Received 2 January 2014 Received in revised form 28 April 2014 Accepted 16 May 2014 Available online 23 May 2014 Keywords: Free radicals Carbon nanotubes in vivo Tissues Oxidative stress Inflammation abstract Nanomaterials are being utilized in an increasing variety of manufactured goods. Because of their unique physicochemical, electrical, mechanical, and thermal properties, single-walled carbon nano- tubes (SWCNTs) have found numerous applications in the electronics, aerospace, chemical, polymer, and pharmaceutical industries. Previously, we have reported that pharyngeal exposure of C57BL/6 mice to SWCNTs caused dose-dependent formation of granulomatous bronchial interstitial pneumonia, fibrosis, oxidative stress, acute inflammatory/cytokine responses, and a decrease in pulmonary function. In the current study, we used electron spin resonance (ESR) to directly assess whether exposure to respirable SWCNTs caused formation of free radicals in the lungs and in two distant organs, the heart and liver. Here we report that exposure to partially purified SWCNTs (HiPco technique, Carbon Nanotechnologies, Inc., Houston, TX, USA) resulted in the augmentation of oxidative stress as evidenced by ESR detection of α-(4-pyridyl-1-oxide)-N-tert-butylnitrone spin-trapped carbon-centered lipid-derived radicals recorded shortly after the treatment. This was accompanied by a significant depletion of antioxidants and elevated biomarkers of inflammation presented by recruitment of inflammatory cells and an increase in proinflammatory cytokines in the lungs, as well as development of multifocal granulomatous pneumonia, interstitial fibrosis, and suppressed pulmonary function. Moreover, pulmonary exposure to SWCNTs also caused the formation of carbon-centered lipid-derived radicals in the heart and liver at later time points (day 7 postexposure). Additionally, SWCNTs induced a significant accumulation of oxidatively modified proteins, increase in lipid peroxidation products, depletion of antioxidants, and inflammatory response in both the heart and the liver. Furthermore, the iron chelator deferoxamine noticeably reduced lung inflammation and oxidative stress, indicating an important role for metal-catalyzed species in lung injury caused by SWCNTs. Overall, we provide direct evidence that lipid-derived free radicals are a critical contributor to tissue damage induced by SWCNTs not only in the lungs, but also in distant organs. Published by Elsevier Inc. Single-walled carbon nanotubes (SWCNTs) are an allotrope of carbon formed by a sheet of graphene rolled into a seamless cylinder with a diameter of 1–4 nm and a length ranging from 100 nm to 1.5 mm. SWCNTs exhibit unique electronic and mechan- ical properties that are used in numerous applications such as field-emission displays, nanocomposites, nanosensors, conductive plastics, paints, technical textiles and repelling features for gar- ments, and biomedical applications. These materials are also on the leading edge of electronic fabrication and are expected to play a major role in the next generation of miniaturized electronics and in energy storage, such as hydrogen fuel cells and other efficient renewable energy sources [1–6]. However, knowledge of the potential health and environmental risks that may occur through- out the entire life cycle of products is limited [5,7]. The intrinsic toxicity of SWCNTs has been attributed to their distinctive physicochemical properties, including their small par- ticle size and the large surface area of the carbon tube decorated with catalytic transition metals (iron, cobalt, nickel, etc.) that are known to trigger generation of free radicals and oxidative stress. Oxidative stress is one of the well-known mechanisms of SWCNT- induced toxicity [8,9]. It is believed that free radical formation by Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/freeradbiomed Free Radical Biology and Medicine http://dx.doi.org/10.1016/j.freeradbiomed.2014.05.010 0891-5849/Published by Elsevier Inc. n Corresponding author at: NIOSH, Pathology and Physiology Research Branch, Health Effects Laboratory Division, 1095 Willowdale Road, Morgantown, WV 26505, United States. Fax: þ304 285 5938. E-mail address: ats1@cdc.gov (A.A. Shvedova). Free Radical Biology and Medicine 73 (2014) 154–165