Cytotoxic, genotoxic and the hemolytic effect of titanium dioxide (TiO 2 ) nanoparticles on human erythrocyte and lymphocyte cells in vitro Manosij Ghosh a , Anirban Chakraborty b and Anita Mukherjee a * ABSTRACT: With the increasing clinical use of titanium dioxide (TiO 2 ) nanoparticles, a better understanding of their safety in the blood stream is required. The present study evaluates the toxic effect of commercially available TiO 2 nanoparticles (~100 nm) using a battery of cytotoxic, genotoxic, hemolytic and morphological parameters. The cytotoxic effects of TiO 2 nanoparticles in human lymphocyte cells were studied with respect to membrane damage, mitochondrial function, metabolic activity and lysosomal membrane stability. Genotoxicity in lymphocyte cells was quantitated using a comet assay. The mode of cell death (apoptosis/necrosis) was evaluated using PI/Annexin V staining. TiO 2 nanoparticles were also evaluated for their hemolytic prop- erties, osmotic fragility and interaction with hemoglobin. Human erythrocyte cells were studied for morphological alterations using atomic force microscopy (AFM). Results suggest that the particles could induce a significant reduction in mitochondrial dehydrogenase activity in human lymphocyte cells. Membrane integrity remained unaffected by nanoparticle treatment. DNA damage and apoptosis were induced by TiO 2 nanoparticles in a dose-dependent manner. A study on human erythrocyte cells revealed a hemolytic property of TiO 2 nanoparticles characterized by spherocytosis and echinocytosis. Spectral analysis revealed a hemoglobin TiO 2 nanoparticle interaction. Our in vitro study results suggest that commercially available blood contacting nano- particles (TiO 2 nanoparticle) should be carefully evaluated for their toxic potential. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: hemolysis; genotoxicity; cytotoxicity; reactive oxygen species; atomic force microscopy; echinocyte Introduction Coarse and fine (> 100 nm in diameter) particles of titanium dioxide (TiO 2 ) were classified as biologically inert in both human and animals (Chen and Fayerweather, 1988; Bernard et al., 1990; Hart and Hesterberg, 1998). According to the US National Nanotechnology Initiative TiO 2 nanoparticles are amongst the most manufactured, and are widely used in a broad range of products such as food colorants (Jin et al., 2008; Vamanu et al., 2008), sunscreen and cosmetics. However, the environmental fate and behaviour of TiO 2 nanoparticles is debatable and a rapidly expanding area of research. The small size of nanoparticles facilitates their uptake into cells as well as transcytosis across epithelial cells into blood and lymph circulation (Gheshlaghi et al., 2008). As aggregates or single particles, TiO 2 nanoparti- cles can not only enter cells, but also enter mitochondria and nuclei (Gheshlaghi et al., 2008). TiO 2 nanoparticles can interact with cellular proteins such as microtubules (Gheshlaghi et al., 2008) and also with DNA (Ghosh et al., 2010). In spite of being classified as a carcinogen by the International Agency for Re- search on Cancer (http://monographs.iarc.fr/ENG/Publications/ techrep42/TR42-summary.pdf) several contradictory reports exist regarding the toxicity of TiO 2 nanoparticles. Several previ- ous studies, in vitro and in vivo have shown TiO 2 nanoparticles to induce DNA damage and cause genetic instability and an inflammatory response (Wang et al., 2006; Reeves et al., 2007; Kang et al., 2008; Vevers and Jha, 2008; Trouiller et al., 2009; Wu et al., 2009; Xu et al., 2009; Bernardeschi et al., 2010; Ghosh et al., 2010) in animal cells. However, some previous studies sug- gest that TiO 2 nanoparticles/ultrafine particles are not genotoxic to certain cell lines (Driscoll et al., 1997; Linnainmaa et al., 1997; Bhattacharya et al., 2009). The toxicological report of TiO 2 nano- particles in vitro has been elegantly reviewed by Iavicoli et al. (2011). The lack of homogeneity has been highlighted. Thus addi- tional research in the field of in vitro TiO 2 nanoparticle toxicity, with proper characterization of the material, effect of low dose exposure on genotoxicity and carcinogenicity is priority (Iavicoli et al., 2011; Arora et al., 2012). Moreover with advances in nanotechnology, no clear regulatory guideline(s) on the testing/ evaluation of nanoparticle exists. Hence it would be important to understand that with an ever increasing number of nanomater- ials in the market, an efficient and cost effective study should be designed so as to cover the major aspects of toxicity and obtain reliable results in less time. Hence the in vitro toxicological studies have become extremely relevant and important. Our previous study (Ghosh et al., 2010) reported that TiO 2 nanoparticles could interact with genetic material in plants *Correspondence to: Anita Mukherjee, Cell Biology and Genetic Toxicology Labora- tory, Centre of Advanced Study, Department of Botany, University of Calcutta, 35 Ballygunge Circular Road, Kolkata, India. Email: anitamukherjee28@gmail.com a Cell Biology and Genetic Toxicology Laboratory, Centre of Advanced Study, Department of Botany, University of Calcutta, Kolkata, India b Department of Chemical Engineering, University of Calcutta, Kolkata, India J. Appl. Toxicol. 2013 Copyright © 2013 John Wiley & Sons, Ltd. Research Article Received: 14 October 2012, Revised: 26 December 2012, Accepted: 13 January 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/jat.2863