ORIGINAL ARTICLE Length-dependent effect of single-walled carbon nanotube exposure in a dynamic cell growth environment of human alveolar epithelial cells Hemang J. Patel 1 and Soonjo Kwon 1 Despite the great use of nanomaterials for engineering and medical applications, nanomaterials may have adverse consequences by accidental exposure, because of their nanoscale size, composition and shape. Like many nanomaterials, carbon nanotubes (CNTs) have been used for many proven applications, but the size of the CNTs makes them more readily become airborne and can therefore create the risk of being inhaled by a worker. In this study, we evaluated single-walled CNT (SWCNT)-induced effects on cellular responses such as cell proliferation, inflammatory response and oxidative stress in dynamic cell growth condition. A dynamic cell growth environment was established to mimic the dynamic changes in the amount of circumferential and longitudinal expansion and contraction occurred during normal breathing movement in the lung. Two different length (short: outer diameter (OD) 1--2nm, length 0.5--2 mm; long: OD 1--2nm, length 5--30 mm) of SWCNTs were used at different exposure concentrations (5, 10 and 20 mg/ml) during the different exposure duration (24, 48 and 72 h). Dynamic environment facilitated altered interaction between SWCNTs and A549 monolayer. Cellular responses in dynamic condition were significantly different from those in static condition. Moreover, cellular responses were dependent on the length of SWCNTs both in static and dynamic cell growth conditions. Journal of Exposure Science and Environmental Epidemiology (2013) 23, 101--108; doi:10.1038/jes.2012.75; published online 1 August 2012 Keywords: A549; short-SWCNT; dynamic environment; IL-8; oxidative stress; long-SWCNT INTRODUCTION The respiratory system is susceptible to be insulted by various airborne environmental factors including airborne toxic materials. Particles that can enter the respiratory system are categorized into three major regions: (1) dust reaching the gas exchange or alveolar region called respirable dust; (2) dust reaching the tracheobronchial region and alveolar region called thoracic dust; and (3) dust entering the nose and mouth called an inhalable dust. 1 Respirable dust is smaller than about 4 mm aerodynamic equivalent diameters, thoracic dust is smaller than about 10 mm and inhalable dust is smaller than about 100 mm. Depending on their size, particles can travel to various regions of the lung. Larger airborne particles tend to deposit in the upper respiratory system. 1 Like many nanomaterials, carbon nanotubes (CNTs) have been used for many proven applications. Carbon is generally recognized as having low toxicity in its pure form. However, high aspect ratio (diameter vs length) of CNTs may lead to enhanced toxicity through mechanisms, such as reactive oxygen species (ROS) generation in the lungs. In a manufacturing environment, CNTs are handled in much larger quantities, subjecting the workers to a higher risk of exposure to these potentially hazardous nanopar- ticles. The nanotechnology community in the United States, led by NIOSH (the National Institute for Occupational Health and Safety) and OSHA (Occupational Safety and Health Administration) has been devoting efforts to issue ‘‘best practices’’ guide for safely working with nanomaterials. However, the development is still in its infant stage, and there is a strong need for science- based methodologies to predict the health and toxicological effects of CNTs. Intensive studies on the toxicity of CNTs have shown that exposure to CNTs results in pulmonary inflammation. 2--8 The inflammatory lung reactions (alveolitis), from CNT exposure, are a source of genetic lesions, which could eventually lead to the development of lung cancer. 2 In vivo studies performed, to evaluate CNT toxicity, using guinea pigs and rats showed the appearance of multifocal granulomas, resulting in inflammatory reactions of the terminal and respiratory bronchioles. Mild fibrosis in the alveolar septa was also observed. 9 Donaldson et al. 10 described three properties of CNTs associated with pathogenicity in particles. They are (1) nanoparticles showing more toxicity than larger sized particles, (2) fiber-shaped particles behaving like asbestos and other pathogenic fibers, which have toxicity associated with their needle-like shape and (3) biologically persistent. They also pointed out that CNTs are possibly one of the least biodegradable man-made materials ever devised. Concerns over the increased emissions of CNTs into the environmental compartments (air, water and soil) mainly due to improper disposal of CNTs were also raised. 9 Recent studies for nanomaterials indicate: (1) CNTs and fullerenes have produced toxic effects on biological systems; 9,11--14 (2) evidence that nanoparticles can translocate to bloodstream; 15,16 and (3) evidence that nanoparticles can cross blood--brain barrier. 17 Many recent studies have demonstrated the effect of CNT dimension on cytotoxicity in different cell types such 3T3 fibroblasts, RAW 264.7, Received 8 January 2012; accepted 11 June 2012; published online 1 August 2012 1 Department of Biological Engineering, Utah State University, Logan, Utah 84322, USA. Correspondence to: Dr. Soonjo Kwon, Department of Biological Engineering, Utah State University, 4105 Old Main Hill, Logan, UT 84322-4105, USA. Tel.: þ 1 435 797 7481. Fax: þ 1 435 797 1248. E-mail: soonjo.kwon@usu.edu Journal of Exposure Science and Environmental Epidemiology (2013) 23, 101 -- 108 & 2013 Nature America, Inc. All rights reserved 1559-0631/13 www.nature.com/jes