is regarded as an inert and non-toxic substance by many regulatory bodies; e.g., the FDA approved TiO2
as a food color additive in 1996, and the Material Safety Data Sheets (MSDS) indicate that there are no exposure hazards to the health of occupational workers and public health. However, there have been several studies that have shown that TiO2
is a potential carcinogen and photocatalyst in biological tissues (Burnett and Wang, 2011
). The cytotoxicity and/or phototoxicity of nano-TiO2
is still debated. Also, there is uncertainty about whether the properties of nano-TiO2
differ with its particular form and size. Human skin and ocular tissues are most affected by ambient radiation. In vitro studies have shown that when irradiated, the smallest form of TiO2
, anatase (<25 nm), is phototoxic to human retinal cells (Sanders et al., 2011
). UV-induced production of ROS and the resultant oxidative stress exposure plays an important role in photocarcinogenesis caused by UV irradiation. ROS are believed to be involved in many inflammatory skin disorders, skin cancer formation, and skin aging (He et al., 2005
). Thus we report here a study of the photocytotoxicity and phototoxicity of nano-TiO2
towards human HaCaT keratinocytes.
Our results show that all nano-TiO2
tested in this paper caused UVA and nano-TiO2
dose-dependent phototoxicity in HaCaT cells. As expected, smaller particles resulted in higher phototoxicity than larger particles. Rutile nano-TiO2
showed less phototoxicity than anatase nano-TiO2
. Moreover, small mixed anatase/rutile nano-TiO2
(P25) showed the highest phototoxicity, which is consistent with previous reported results (Hurum et al., 2003
). The cellular toxicity of nano-TiO2
mainly depends on its ROS generation under UVA irradiation. Using ESR and spin trapping technique, a “gold standard” and state-of-the-art tool for detecting and quantifying ROS, we confirmed the generation of hydroxyl radicals and singlet oxygen. ROS generation by these four nano-TiO2
particles is consistent with the phototoxicity studies, further confirming our hypothesis that ROS production was probably involved in the phototoxic mechanism.
In addition to measurement of ROS generation with ESR, we explored two other assays to further characterize the phototoxicity of nano-TiO2
. ROS are known to produce a time-dependent peroxidation of the polyunsaturated lipids in plasma membrane (Yin et al., 2008
; Yin et al., 2009
) as well as peroxidation of lipids and proteins inside the cells (Girotti, 1998
). To mimic oxidation reactions with substrates in biological systems, we used polyunsaturated lipids in plasma membranes and human serum albumin as model targets for ROS. Using innovative ESR oximetry and immune-spin trapping techniques, we easily detected ROS-induced peroxidation, which was found to be in agreement with the ESR-detected ROS production. These results indicate that both methods are useful for qualitative screening of the phototoxicity of nano-TiO2
Based on the results reported in this study, we propose the mechanism shown in . UVA irradiation results in electron-hole pairs in nano-TiO2
. The photoexcited nano-TiO2
serves as the initiating species to transfer energy to molecular oxygen and generate singlet oxygen (1
) or to extract electrons from water or hydroxyl ions to generate hydroxyl radicals (•
OH). Our experimental results also indicate that part of the 1
formation proceeds via a superoxide-dependent mechanism, whereas the OH formation is not via superoxide (). It is well established that in the presence of a lipid or protein, the generated ROS can initiate lipid and/or protein peroxidation. Both ROS and lipid peroxidation are associated with aging-related diseases, including cancer (Aust et al., 1993
; Xia et al., 2007
Proposed mechanism of TiO2 nanoparticle-induced free radicals and lipid/protein peroxidation leading to cell damage.
In conclusion, the studies presented here give an overall in vitro assessment of the phototoxicity by various nano-TiO2 particles towards HaCaT cells under UVA irradiation. This phototoxic damage was apparently mediated by production of ROS by photo-activated nano-TiO2. According to our results ROS are generated by UVA irradiation of nano-TiO2 with anatase and/or mixed anatase/rutile forms. Moreover, the phototoxicity of nano-TiO2 was less with larger particle size and surface areas, indicating that coarse particles of nano-TiO2 may consistently produce less ROS.