In Vitro Phototoxicity and Hazard Identification of Nano-scale Titanium Dioxide☆
Highlights
► Nano-TiO2 enters cells within 24 hours ► Nano-TiO2 causes dose-dependent cytotoxicity greatly enhanced by UVA radiation ► Treatment with nano-TiO2 and UVA produces reactive oxygen species ► Phototoxicity is correlated with particle size, surface area, and TBARS reactivity
Introduction
Engineered nanomaterials, as defined by the National Nanotechnology Initiative (National Science and Technology Council, 2007), are rapidly being developed and deployed in a variety of commercial applications. For the safe use and application of titanium dioxide nanomaterials (nano-TiO2), it is important to define the critical parameters that might influence their impact on health and the environment. Among the more commonly considered properties evaluated in nanotoxicity studies are particle size, surface area, composition, reactivity, coatings, stability, and propensity to agglomerate or aggregate in different media (Balbus et al., 2007, Oberdorster et al., 2005). An additional property of concern for nano-TiO2 is photoreactivity.
Titanium dioxide is one of the most frequently used engineered nanomaterials because it can serve two distinct functions: absorbing and scattering ultraviolet (UV) radiation and/or semiconductor photocatalysis activated by UV radiation (Fujishima et al., 2000, Kwon et al., 2008, Mills and Le Hunte, 1997). As a UV reflecting agent, it is found in a variety of applications, including sunscreens, paints, and coatings, where it serves in a protective role (Klaine et al., 2008). The photocatalytic ability of nano-TiO2 is used in photovoltaic devices and self-cleaning or self-sterilizing product coatings (Kwon et al., 2008, Woodrow Wilson International Center for Scholars, 2011). Additionally, as a photocatalytic agent, nano-TiO2 could be useful for degrading environmental contaminants such as organic compounds (Muneer et al., 2002, Yeber et al., 2000, Yeo and Kang, 2006) and pesticides (Muneer and Bahnema, 2001, Zoh et al., 2005, Zoh et al., 2006), decontaminating indoor air (Salthammer and Fuhrmann, 2007), or sterilizing surfaces or pathogen-contaminated drinking water (Amezaga-Madrid et al., 2002, Paul et al., 2007, Prasad et al., 2009). The two common crystalline forms of nano-TiO2, rutile and anatase, are found in commercially available nano-TiO2 and are considered photocatalytic, with anatase being the more photoreactive (Kwon et al., 2008).
However, the same properties that make nano-TiO2 a photocatalytic agent may also lead to phototoxic reactions in humans. Absorption of UVA radiation activates a surface electron of the TiO2 molecule to the conduction band, leaving a valence band hole that extracts electrons from water or hydroxyl ions, generating hydroxyl radicals. The electrons of the hydroxyl radicals then reduce O2 to produce superoxide anion, which leads to a cascade of reactive oxygen species (ROS) (Brunet et al., 2009, Kwon et al., 2008). UVA radiation can also induce the production of highly reactive singlet oxygen (Konaka et al., 2001). The generation of an ROS cascade can damage tissues through oxidation of nearby lipids, proteins, nucleic acids, or other biomolecules (Straight and Spikes, 1985).
Nano-TiO2 has been shown to be phototoxic to isolated DNA (Hirakawa et al., 2004), bacteria (Adams et al., 2006), and several aquatic species (Hund-Rinke and Simon, 2006; Matsuo et al., 2001), as well as mammalian lymphoma (Nakagawa et al., 1997), ovary (Uchino et al., 2002), dermal fibroblast, and pulmonary epithelial carcinoma cell lines (Sayes et al., 2006). Because skin and eye tissues are exposed to light, they are typically of greatest concern for phototoxic damage. In the skin, phototoxicity can lead to sunburn or an increased risk of cancer (Johnson and Ferguson, 1990). In the eye, phototoxic reactions can damage the lens, retina, or retinal pigment epithelium (RPE), leading over time to cataracts in the lens or photoreceptor loss in the retina (Roberts, 2002).
To our knowledge, the potential for nano-TiO2 phototoxicity in ocular cells has not been previously reported. However, models of human lens (Roberts et al., 2008) and RPE cells have been sensitive to phototoxic effects of other nanomaterials, specifically to hydroxylated fullerene (Nohynek et al., 2008, Wielgus et al., 2010). In addition, cultured RPE cells internalize TiO2 nanoparticles and combine them into perinuclear aggregates in a dose-related fashion (Zucker et al., 2010). In the studies reported in this communication, the potential phototoxicity of several types of nano-TiO2 of various particle sizes and crystalline forms was evaluated in a culture of human-derived RPE cells. The hypotheses tested included: 1) that nano-TiO2 toxicity would be enhanced by UVA radiation, 2) that smaller particles would be more phototoxic than larger particles, and 3) that the anatase crystal form would be the most phototoxic, with mixed anatase/rutile being intermediate and rutile samples being least phototoxic. It was found that: 1) nano-TiO2 was more toxic following exposure to UVA radiation, 2) smaller particles tended to be more toxic than larger ones, and 3) the effect of crystal structure on phototoxicity could not be determined in these experiments. In addition, it was observed that the generation of reactive oxygen species with nano-TiO2 under UVA radiation correlated with cytotoxicity.
Section snippets
ARPE-19 cells
A human-derived RPE cell line (ARPE-19) was obtained from ATCC (Manassas, Virginia) and grown in a 1:1 combination of Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F-12; cat # 11039047, Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; cat # 511150, Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin/streptomycin (cat # 17-602E, Lonza, Walkersville, MD). Phenol red was omitted from the media due to its potential interaction with UV radiation. All
Characterization of TiO2 nanoparticles
DLS measurements of aggregate size varied based on dispersant, concentration, and primary particle size. Aggregates had smaller sizes and were more monodispersed in media with serum than in media without serum, Hank's Buffered Salt Solution, or water. The stabilizing effect of serum was likely due to the association of nanoparticles with serum proteins (Deguchi et al., 2007, Ji et al., 2006). Aggregate size also increased with greater concentrations and primary particle sizes. Aggregates were
Discussion
The data reported in this manuscript show that nano-TiO2 can cause phototoxicity in ocular cells in culture. As hypothesized, the dose-dependent toxicity of nano-TiO2 was much greater after UVA radiation than in the dark. Additionally, smaller particles resulted in higher phototoxicity than larger particles, but the effects of crystal structure on phototoxic potency were not clear. UVA-catalyzed ROS production, as measured by two assays (TBARS and Mitosox), correlated with phototoxicity,
Conflict of interest statement
There are no conflicts of interest.
Acknowledgments
K. Sanders was supported by a contract to EPA (EP09D000042). Thanks are extended to Theresa Freudenrich for her excellent advice and guidance with cell culture, to Kaitlin Daniel for her evaluation of the flow cytometry data and preparation of Fig. 4, Fig. 5, and to Sarah Karafas for her help with the Mitosox experiments. The authors are indebted to Amy Wang, Christy Powers, Steve Diamond, Tom Long, and Carl Blackman for editorial comments on a previous version of the manuscript. This
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