Comparison of Micro- and Nanoscale Fe+3–Containing (Hematite) Particles for Their Toxicological Properties in Human Lung Cells In Vitro

Kunal Bhattacharya 0 3 4 Eik Hoffmann 2 3 Roel F. P. Schins 3 6 Jens Boertz 3 5 Eva-Maria Prantl 3 4 Gerrit M. Alink 3 jj Hugh James Byrne 0 3 Thomas A. J. Kuhlbusch 3 jjj 3 jjjj Qamar Rahman 1 3 Hartmut Wiggers 3 jjjj 3 4 Christof Schulz 3 jjjj 3 4 Elke Dopp 3 4 jjjj 3 0 Nanolab Research Centre, FOCAS Institute, Dublin Institute of Technology , Dublin 6 , Ireland 1 Integral University , Lucknow , India 2 Institute of Biological Sciences, Cell Biology and Biosystems Technology, University of Rostock , 18051 Rostock , Germany 3 Essen , Germany. Fax: 4 Institute of Hygiene and Occupational Medicine, University of Duisburg-Essen , 45122 Essen , Germany 5 Institute for Reference Materials and Measurements (IRMM), European Commission-Joint Research Centre , Geel , Belgium 6 Institut fu r Umweltmedizinische Forschung (IUF) an der Heinrich-Heine University Du sseldorf , Du sseldorf , Germany The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: - The specific properties of nanoscale particles, large surface-tomass ratios and highly reactive surfaces, have increased their commercial application in many fields. However, the same properties are also important for the interaction and bioaccumulation of the nonbiodegradable nanoscale particles in a biological system and are a cause for concern. Hematite (a-Fe2O3), being a mineral form of Fe(III) oxide, is one of the most used iron oxides besides magnetite. The aim of our study was the characterization and comparison of biophysical reactivity and toxicological effects of a-Fe2O3 nano- (d < 100 nm) and microscale (d < 5 mm) particles in human lung cells. Our study demonstrates that the surface reactivity of nanoscale a-Fe2O3 differs from that of microscale particles with respect to the state of agglomeration, radical formation potential, and cellular toxicity. The presence of proteins in culture medium and agglomeration were found to affect the catalytic properties of the hematite nano- and microscale particles. Both the nano- and microscale a-Fe2O3 particles were actively taken up by human lung cells in vitro, although they were not found in the nuclei and mitochondria. Significant genotoxic effects were only found at very high particle concentrations (> 50 mg/ml). The nanoscale particles were slightly more potent in causing cyto- and genotoxicity as compared with their microscale counterparts. Both types of particles induced intracellular generation of reactive oxygen species. This study underlines that a-Fe2O3 nanoscale particles trigger different toxicological reaction pathways than microscale particles. However, the immediate environment of the particles (biomolecules, physiological properties of medium) modulates their toxicity on the basis of agglomeration rather than their actual size. Key Words: nanoscale particles; microscale particles; genotoxicity; cytotoxicity; radical formation. The large surface-to-mass ratios and the reactive surfaces of nanoparticles are important for the interaction and bioaccumulation of the nonbiodegradable nanoscale particles in biological systems and in organisms. This raises concern of possible risks, now also seen and evaluated by international regulatory committees. In order to explore the possible health effects and regulate occupational and nonoccupational exposure scenarios, the Organization for Economic Cooperation and Development (OECD) has generated a list of 14 commercially important nanoparticulate materials, which include iron oxide (OECD, 2010). In this context, it becomes important to determine the bio-nano interaction of the iron oxide nanoscale particles following respiratory exposure. Hematite, being a mineral form of Fe(III) oxide, exists in several polymorphous subtypes (a-, c-Fe2O3), has about 70% iron content, and, due to its utilization as a pigment, is one of the most industrially used forms of iron oxide besides magnetite. The use of Fe2O3 nanoscale particles also includes drug targeting of cancer cells, tracking target cells using labeling, and imaging techniques like magnetic resonance tomography (Kumar et al., 2007). In vivo studies with Fe2O3 nanoscale particles have demonstrated severe inflammatory and toxicity responses in rats exposed to nanoscale particles through inhalation (Wang et al., 2010; Zhu et al., 2008). Fe2O3 particles (diameter < 100 nm) have been found to translocate and interact with the olfactory nerve and trigeminus of brain stem 14 days postinhalation in mice models (Wang et al., 2007). Other studies have demonstrated that Fe2O3 nanoscale particles cause oxidative stress to human bronchoalveolar epithelial and murine neuronal cells leading to loss of cell viability, genotoxicity, and causing a change in electrical activity (Bhattacharya et al., 2009; Gramowski et al., 2010). In contrast to these findings, other studies performed with microscale and nanoparticulate Fe2O3 described them both to be nontoxic under in vitro test conditions in human small airway epithelial and mouse fibroblast cells (exposure concentration up to 400 lg/cm2) (Mahmoudi et al., 2009). Therefore, it is of interest to observe the difference in the bio-nano interaction between Fe2O3 nano- and microscale particles in human lung cells in vitro and to correlate the effect with their physicochemical properties. Most studies under in vitro conditions consider the basic physicochemical characteristics of the nanoscale particles, such as shape, size, and surface coating. However, recent studies have shown that the surface of the nanoscale particles changes after interaction with the surrounding environment through opsonization, solvation, protein corona formation, and agglomeration (Lynch et al., 2007; Nel et al., 2009). The present study characterizes the biophysical reactivity of Fe2O3 nano- (diameter < 100 nm) and microscale (diameter < 5 lm) particles under in vitro test conditions in human lung epithelial and fibroblast cells. Physicochemical properties of Fe2O3 particles were determined through morphological investigations with SEM, specific surface analysis using BrunauerEmmett-Teller (BET) technique, determination of zeta potential and particle size distribution via dynamic light scattering (DLS) and by the determination of total and leachable iron content, and surface reactivity through electron paramagnetic resonance (EPR). The biological responses to the Fe2O3 nano- and microscale particles were studied by transmission electron microscopy (TEM), cyto- and genotoxicity analyses, measurement of intracellular reactive oxygen species (ROS) generation, and mitochondrial membrane potential detection. This study was designed to investigate the influence of particle size on biological effects in human lung cells after exposure. Additionally, the study explores the modification of the metal oxide (hematite) nanoscale particles in their immediate environment (media) in correlation (...truncated)