TiO2-nanoparticles shield HPEKs against ZnO-induced genotoxicity

Materials and Design 88 (2015) 41–50

Contents lists available at ScienceDirect

Materials and Design journal homepage: www.elsevier.com/locate/jmad

TiO2-nanoparticles shield HPEKs against ZnO-induced genotoxicity Mustafa Hussain Kathawala a, Zhao Yun a, Justin Jang Hann Chu b, Kee Woei Ng a,⁎, Say Chye Joachim Loo a,c,⁎⁎ a

School of Materials Science and Engineering, Nanyang Technological University, Singapore Laboratory of Molecular RNA Virology and Antiviral Strategies, Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, Singapore c Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore b

a r t i c l e

i n f o

Article history: Received 27 April 2015 Received in revised form 28 July 2015 Accepted 20 August 2015 Available online 1 September 2015 Keywords: DNA damage Dual nanoparticle Nanoparticles Oxidative stress Titanium oxide Zinc oxide

a b s t r a c t Usage of sunscreens has become commonplace amongst outdoor sports. Recently, nanomaterials have gained increasing market share as ingredients in sunscreens (as well as other topically applied products). In particular ZnO and TiO2 nanoparticles (ZNP and TNP) have found their niche in this application. This study investigated the safety aspects of these nanoparticles from a combinatorial exposure point of view. Focus was on investigating generation of oxidative stress and induction of DNA damage which the two nanoparticles caused. It was found that TNPs triggered stronger oxidative stress than ZNPs but ZNPs remained more potent at causing DNA damage. The individual mechanisms of DNA damage were found to be through oxidative stress for TNPs (indirect genotoxicity) and through Zn2+ ion nuclear uptake resulting in DNA damage for ZNP (direct genotoxicity). Interesting, it was found that intracellular TNPs could adsorb Zn2+ ions and lower their nuclear uptake in turn shielding the HPEKs from ZNP-induced genotoxicity. Toxicological assessments of dual nanoparticle systems remain an unstudied area and based on the results obtained deserves further consideration. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Outdoor sports, like cricket, football, beach volleyball and even running, provide an extended exposure of athletes to the sun. This increases UV-exposure to athletes, and carries a risk of sunburns, excessive tanning, skin-peeling and, in not so extreme cases, even skin cancer. According to the American Cancer Society, skin cancer is by far the most common form of cancer responsible for more than half of the total cancer cases in the USA. To counter this risk, the use of sunscreens have been encouraged to those who are exposed for prolonged periods under the sun. Traditionally, sunscreens mostly constitute of organic ingredients categorized as “chemical” filters. These provide protection by absorbing the harmful UV rays. Inorganic ingredients became popular relatively recently as active agents in sunscreens, because they cause less skin irritation. These were categorized as “physical” filters as they deflect or block UV rays. Since then, microparticles of metallic oxides became the most commonly used physical filters. However, microparticles leave a white cast or tint on the applied skin because of their size. More recently, nanoparticles are slowly replacing their micron-sized counterparts because their nanometric size make them invisible to the naked eye, and thus aesthetically more appealing. ⁎ Corresponding author. ⁎⁎ Correspondence to: S.C.J. Loo, School of Materials Science and Engineering, Nanyang Technological University, Singapore. E-mail addresses: [email protected] (K.W. Ng), [email protected] (S.C.J. Loo).

http://dx.doi.org/10.1016/j.matdes.2015.08.108 0264-1275/© 2015 Elsevier Ltd. All rights reserved.

The two nanomaterials which are chiefly found in modern sunscreens are zinc oxide (ZnO) and titanium dioxide (TiO2). While both these ingredients have been considered safe previously, their reduction in size to the nanometer scale has raised some safety concerns. It is therefore imperative to establish rigorous safety validation in order to avoid any unforeseen health and safety concerns, and/or to allay any negative public opinion on the use of these nanomaterials. While numerous studies in recent years have evaluated in vitro cellular responses to ZnO nanoparticles (ZNP) and TiO2 nanoparticles (TNP), none has addressed the combinatorial effects of these two nanomaterials. Addressing the combinatorial toxicity of these nanomaterials is relevant and important, since sunscreens which possess high Sun Protection Factor (SPF) utilize a combination of these nanoparticles. Our recent studies have found that a combination of ZNP and TNP pose a very different cytotoxic profile from individual nanoparticles. We found that intracellular TNP were able to chelate Zn ions, thus protecting human primary epidermal keratinocytes (HPEKs) from Zn ion induced toxicity. Many studies have focused on the role of Zn ions in ZNP-induced toxicity and therefore it is logical that any effect on its bioavailability would impact its toxic potential [34]. While cytotoxicity remains a vital area of toxicity screening, another highly relevant and often neglected avenue is genotoxicity. Genotoxicity is especially relevant in this context because sunscreens are used to ultimately prevent skin cancer. Any carcinogenic risk of its excipients would imply its usage counterproductive. It is, therefore, imperative that investigations on skin toxicity also focus on DNA damage, a precursor of genotoxic events. Both TNP and ZNP have been shown to possess

42

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

genotoxic potencies, albeit of highly contrasting levels [26]. A comprehensive review of TNP's genotoxic potential was published last year [3]. The assimilation of data incorporated in the review suggests that genotoxicity of TNP is a controversial topic with some clear inconsistencies. For example out of 24 studies which employed the comet assay, 17 reported positive on TNP's genotoxic influence while 7 reported otherwise. On closer inspection, many studies using similar cell lines, concentrations and even crystal structures have reported contradictory results. Even so, mechanistically there was more agreement within the studies arguing in favor of TNP's genotoxic potential. Generation of ROS was consistently suggested as the key factor underlying TNP's genotoxicity. To date, however, to the best of our knowledge, there are no reports that focus on human primary epidermal keratinocytes for genotoxic evaluations. Given that keratinocytes form the first layer of protection to external threats at the skin interface, understanding how TNP can influence them would seem vitally important. The closest models which studies, to date, have used are immortalized keratinocytes, HaCaT cells [9,15]. In addition focus has been more towards studying genotoxicity under UV-exposure [7]. It was shown that TNP can cause up-regulation of genes involved in inflammatory response and cell adhesion for HaCaT cells [9]. ROS generation was shown to be the underlying mechanism causing DNA damage [15]. The DNA damage potential of ZNP is far less controversial. Many studies have shown DNA damage induced by ZNP at concentrations less than 10 μg ml−1 in a number of cell types [11,26,28]. Again ROS generation was strongly suggested as the key underlying mechanism. Our group has previously reported the ability of ZNP to induce DNA damage [25], and the role of p53 in inducing apoptosis when cells are exposed to ZNP. The study also showed how pro-apoptotic pathways including phosphorylated p38 and bax/bcl-2 ratio were upregulated, and how caspases, which are the executors of apoptosis, were activated while anti-apoptotic survivin protein was shown to be suppressed. Studies on primary keratinocytes were again scarce. The only one reported so far showed that ZNP induced DNA damage at concentrations of 8 and 14 μg/ml ZnO NPs after 6 and 24 h of exposure [29]. They induced a dose and time dependent effect in both the micronuclei and Comet assay. Therefore, in this study, the genotoxic potential of combinatorial ZNP and TNP on HPEKs was investigated and compared. The underlying mechanisms were investigated by evaluating nanoparticle uptake and oxidative stress induction. Finally, the combinatorial genotoxic potential of ZNP and TNP was studied and the underlying mechanisms which dominate the summative effect were discussed.

2. Methods and materials 2.1. Nanoparticle characterization P25 TiO2 NPs (TNP) and ZnO NPs (TNP) were purchased from Evonik Degussa (Germany) and Meliorum Technologies (USA). Transmission electron microscopy (TEM; JEOL 2010) at an accelerating voltage of 200 kV with a Lanthanum Boride (LaB6) cathode, was performed to evaluate particle size and shape. Primary size of the nanoparticles was measured using the ImageJ software. Dynamic light scattering (DLS; Malvern Co., UK) was performed to measure hydrodynamic sizes and Zeta potentials. For Brunauer–Emmett–Teller (BET) surface area measurements, nitrogen adsorption/desorption isotherms were measured at 77 K using ASAP2000 adsorption apparatus from Micromeritics. Solubility of nanoparticles was evaluated using inductively coupled plasma-assisted mass spectrometry (ICP-MS; Agilent 7500 Series). To measure ion chelating effect, CaCl2, ZnSO4 and MgCl2 solutions were prepared and the concentration of the bivalent ions in the initial solutions were measured. Separately, TNP was added to the solutions and removed by centrifugation after 10 mins. The concentration of the bivalent ions were then measured in the supernatant. The difference

between the two measurements was used as the amount of bivalent ions chelated onto the TNP surface. TNPs were tagged with fluorescein Isothiocyanate (FITC) to visualize them. FITC conjugation was performed using an amide linker, aminopropyltriethoxysilane (APTES), and the protocol has been mentioned elsewhere [18,35]. 2.2. Cell culture & maintenance Human primary epidermal keratinocytes (HPEKs; ATCC, USA) were cultured and maintained in serum-free EpiGRO™ Human Epidermal Keratinocyte Complete Medium kit (Chemicon, USA). HPEKs were cultured in standard culture conditions (37 °C, 5% CO2) for up to 7 days. At 80% confluence, the HPEKs were passaged. 2.3. Nanoparticle treatment A number of procedures for treating combinations of nanoparticles to HPEK cultures were employed. The idea behind designing each protocol was to prepare HPEKs which contained extracellular and/or intracellular TNP at the time of ZNP exposure. SIM-exposed HPEKs (SIM stands for simultaneously) were prepared by adding both TNP and ZNP to the HPEKs at the same time. Thus, only extra-cellular TNP are present at the time of ZNP exposure. TNP-exposed HPEKs were prepared by treating HPEKs with TNP first to allow some TNP uptake into the HPEKs. Thus, they contained both extra- and intra-cellular TNP before ZNP was introduced. TNP-loaded HPEKs were prepared in a similar way by first treating with TNP to allow cellular uptake. Thereafter the extracellular medium was replaced to remove any unbound TNP. The result was HPEKs which contained only intracellular TNP at the time of ZNP exposure. 2.4. Dosimetry using the ISDD model Dosimetry was performed for TNP using the in vitro sedimentation, deposition and dosimetry (ISDD) model. Effective density of TNP was measured using the protocol suggested by DeLoid et al. [4]. Briefly, known concentration of TNP was volumetrically centrifuged. The volume of the pellet was used to calculate the effective density. This parameter was fed into ISDD model simulations which were kindly provided by Dr. Justin Teeguarden [13]. 2.5. HPEK viability assay Cell viability was measured using the WST-8 metabolic activity assay (CCK-8; Dojindo Molecular Laboratories Inc., Japan). In a typical experiment, HPEKs were seeded in a 96-well plate at a density of 104 HPEKs/ well in culture medium. After allowing HPEKs to equilibrate overnight, they were treated with ZNP or TNP of different concentrations. Pure medium and medium with NPs (without HPEKs) were used as blank controls. After 24 h of treatment, reconstituted WST-8 mixture was added to each well and incubated for up to two hours after which absorbance at 450 nm was measured using a standard microplate reader. 2.6. Immunofluorescence staining (γ-H2AX assay) HPEKs were grown on 15 mm square no. 1 cover slips (thickness ~160 μm) in 6-well plates to confluence and exposed to nanoparticles for 24 h. As a positive control, ultraviolet irradiation of HPEKs for 5–20 mins was performed to induce DNA damage. A filtered VL-115.C UV lamp (Viber Lourmat, Germany) at 254 nm was used. After the exposure, the HPEKs were incubated in fresh medium for 24 h [12,21]. Post-treatment, HPEKs were fixed using cold 4% paraformaldehyde in PBS (Sigma-Aldrich, USA) at room temperature (RTP) for 10 mins. HPEKs were then washed thoroughly with PBS to remove any leftover NPs and permeabilized with 0.5% v/v Triton X-100 (Sigma-Aldrich,

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

USA) in distilled water for 10 mins at RTP. After further washing, unspecific sites were blocked with 5% w/v bovine serum albumin (BSA; Dako, Denmark) in PBS for 30 mins at RTP. Damaged DNA was tagged using the primary antibody, anti-phospho-histone γ-H2AX (Ser139) rabbit monoclonal antibody (9718; Cell Signaling Technology, USA), diluted 400 times in blocking buffer and incubated overnight at 4 °C. On the following day, HPEKs were washed thoroughly with PBS to remove any unbound antibody and incubated with FITC-conjugated secondary antibody, Alexa Fluor® 568 goat anti-rabbit IgG antibody (A-11034; Invitrogen, USA) diluted 200 times in blocking buffer for 30 mins in the dark at RTP. After washing, the nuclei were stained with Hoechst 33342 (Molecular Probes, Catalogue number H3570). Once the HPEKs were stained, the cover slips were flipped and mounted onto glass microscope slides assisted by cold mounting medium and sealed using transparent nail polish. The HPEKs were either viewed instantly or stored in the dark at 4 °C for up to a week before being viewed.

2.7. Oxidative stress quantification HPEKs were exposed to nanoparticles for 4, 8 or 24 h in the same manner as above. As a positive control, ultraviolet irradiation of HPEKs for 20 mins was performed to induce oxidative stress. A filtered VL115.C UV lamp (Viber Lourmat, Germany) at 254 nm was used. Before irradiation, the medium in the wells were removed with the lids off. Immediately after, fresh medium was added and the HPEKs were incubated for 2 h [2]. Treated HPEKs were incubated with CellROX® reagent (Life technologies, US; C10444) according to the manufacturer's suggested protocol. Briefly, CellROX® reagent at a final concentration of 5 μM was added to the HPEKs and incubated for 30 mins. HPEKs were then washed thoroughly with PBS and fixed with cold 4% paraformaldehyde in PBS (Sigma-Aldrich, USA) at RTP for 10 mins. The nuclei of HPEKs were stained using Hoechst 33342 (Molecular Probes, Catalogue number H3570). Once the HPEKs were stained, the cover slips were flipped and mounted onto glass microscope slides assisted by cold mounting medium and sealed using transparent nail polish. The HPEKs were viewed within 24 h according to the manufacturer's instruction.

43

2.10. Ion adsorption To test whether Zn ions could be adsorbed onto the surface of the TNPs, the following study was implemented. 50 μg ml-1 of ZNP in medium was left overnight in order to allow enough time for dissolution. Similarly an equimolar solution ZnSO4, CaCl2 and MgSO4 was also prepared (the concentration was chosen to achieve comparable concentrations of positive ions in all solutions). The next day undissolved ZNP were removed by centrifugation and TNP at various concentrations (0–400 μg ml−1) were introduced to all solutions. After 10 min of equilibration time, centrifugation was performed to spin down the TNP and any adsorbed ions. Aliquots of the supernatant were stored in 4 °C for mass spectrometry. 2.11. Transmission electron microscopy TNP internalization and localization into HPEKs was visualized using transmission electron microscopy (TEM). The used protocol has been described earlier [36]. Briefly, treated cells were fixed overnight with 2.5% glutaraldehyde (SPI, USA) diluted in PBS at 4 °C and post-fixed with 1% osmium tetroxide (SPI, USA) for 1 h at RTP. The resulting pellets were dehydrated using ethanol gradient (25%–100%) and pure acetone, for 20 min each at RTP. Resin infiltration was performed overnight in SPI-Pon™-Araldite® (SPI, USA), followed by embedding pure resin at 60 °C for 72 h. Post-polymerization, sectioning was performed using an ultramicrotome (Leica Ultracut UCT) and collected on 200 mesh copper grids. Finally staining with uranyl acetate and lead citrate was performed before the sections were viewed under a Philips EM 208 transmission electron microscope (accelerating voltage: 100 kV). 2.12. Statistical analysis Statistical significances were only assessed when at least three or more sample replicates were performed, using one-way ANOVA with post-hoc multiple variances and Tukey's equal variance assumed (IBM SPSS v20). 3. Results 3.1. Material characterization

2.8. Intra-cellular and nuclear Zn ions Control, TNP-exposed and TNP-loaded HPEKs were treated using nanoparticles on cover slips in 6-well plates. ZNP concentration tested was 5, 10 and 20 μg ml−1. Samples containing only ZNP (5, 10 and 20 μg ml−1) and TNP (100 μg ml−1) were also tested for comparison. After fixation, HPEKs were stained for free and unbound intracellular Zn ions using Newport Green™ DCF Diacetate (Molecular Probes, Catalogue number N-7991) according to the manufacturer's instructions. Cell nucleus was stained using Hoechst 33342 (Molecular Probes, Catalogue number H3570). Imaging was performed within 24 h and all images were taken at the same instrument settings to allow for post image analysis. Semi-quantitative analysis was done using ImageJ 1.47 V. Nuclear Zn ion concentrations was analyzed by selected the Hoechst stained nuclei as the region of interest (ROI). Color threshold was adjusted to suppress background and cells were counted. The green fluorescence was quantified in terms of the number of bright green specks observed in the nuclei.

2.9. Confocal microscopy Microscopy was performed either on Leica TCS SP5 Broadband Confocal microscope using an oil lens at magnifications up to 60× or Nikon A1-Rsi Confocal Microscope using an oil lens up to 100 ×. Post-image processing was performed using ImageJ 1.47v.

Both nanoparticles were found to have primary size of around 20 nm (Fig. 1). They showed heavy aggregation in water, PBS and cell culture medium. Interestingly, both nanoparticles recorded a negative zeta potential. This is important as negatively charged entities gain entry into the eukaryotic cells via endocytotic machinery [33]. Another point of interest was the solubility of ZNP in cell culture medium which increased with time and reached in excess of 20 μg ml−1 over 24 h. The negative surface demonstrated an ability to adsorb the positive Zn ions in cell culture medium [19]. In this study, the electrostatic attraction was confirmed by testing with other bivalent ions like Ca2 + and Mg2+ both of which were also adsorbed onto the TNP surface. In cell culture medium Zn2 + ions were adsorbed the most, followed by Ca2 +, while Mg2 + were adsorbed minimally (Fig. 2a). Interestingly, when all three ions were present, TNP preferentially adsorbed Zn2 + ions (Fig. 2b). 3.2. ZNP, but not TNP, strongly induce γ-H2AX foci formation in HPEKs nuclei After 24 h of incubation with ZNP, there was significant γ-H2AX foci formation in HPEKs nuclei which indicated double-stranded breaks (DSBs) in the DNA (Fig. 3). At 1 μg ml−1, ZNP induced significant γ-H2AX foci formation, which increased by three-folds when the concentration was increased to 10 μg ml−1, and showed a strong concentration dependency. The level of DNA damage was comparable to the

44

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

Fig. 1. TEM images of (A) ZNP and (B) TNP. (C, D) Nanoparticle characterization data. (E) Nanoparticle solubility.

positive control, i.e. 15 min UV irradiation. TNP, however, did not exert a strong genotoxic influence at low concentrations. At 1 μg ml−1, there was no significant increase in DSBs. However, at higher concentrations of 100 μg ml−1, there was some DSBs induced but the intensity of damage was still much lower than ZNP exposure and the positive control. At this point it is important to note that from our previous studies, the LD50 is ~ 10 μg ml−1 for ZNP and N N 100 μg ml−1 for TNP. Taken together with the fact that ZNP induced a strong genotoxic effect at a dosage as low as 1 μg ml−1, it is clear ZNP is much more cyto- and geno-toxic than TNP. The same has been found for other cell types as well [22,27].

3.3. TNP are more potent at generating free radicals than ZNP at sub-lethal concentrations One aspect of oxidative stress is the generation of free radicals in response to nanoparticle treatment. NP treated cells were examined for being ROS-affected by the CellROX® dye. ROS-positive cells were further categorized into high, medium and low depending on the intensity of dye fluorescence which was indirect indicative of the level of ROS present in the cells. At sub-lethal concentrations, TNP induced a concentration and time dependent response (Fig. 4).

Fig. 2. Ion adsorptive ability of TNP shown through measuring difference in ionic concentrations of Ca2+, Mg2+ and Zn2+ salt solutions when treated with 100 and 400 μg ml−1 of TNP. In (A) individual solutions of the three ions were prepared and tested while in (B) all three ions were mixed together at comparable concentrations and tested with TNP exposure.

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

45

Fig. 3. (Aa–Ff) Confocal images of HPEKs exposed to ZNP for 24 h with nuclei stained using Hoechst dye (blue) and γ-H2AX foci stained green. (G) Semi-quantitative analysis shown below each image was performed using ImageJ. The data labels show the percentage of cells showing DNA damage. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Others have observed a similar trend not only for keratinocytes cell lines [20] but also other cell lines [3]. At concentrations below 50 μg ml−1, no significant increase in ROS affected cells was observed. At concentrations greater than 50 μg ml− 1, the fraction of ROSpositive cells increased with increasing concentrations and exposure time. The trend directly correlated with the time-weighted average dosage (TWAD) of TNP calculated using the in vitro sedimentation, diffusion and dosimetry (ISDD) model [13]. Upon closer inspection, it was evident that the severity of oxidative stress was also dependent on concentration and exposure time. Samples treated with higher dosages contained a higher fraction of ROS-positive cells in the high category.

Mitochondria is one of the major organelles involved in ROS generation. The localization of ROS was also studied using image analysis. TNP treatment also triggered a higher mitochondrial ROS in a concentration and time dependent manner (Fig. 5b). At the highest concentration and incubation time, however, there was a noticeable drop. This could be due to cell death through apoptosis which can be triggered through oxidative stress. Cells release Ca2+ ions stored in endoplasmic reticulum as a response to ROS, which can in turn depolarize the mitochondrial membrane resulting in cytochrome c release, which is a strong pro-apoptotic factor [14]. In contrast, ZNP exerted relatively lower oxidative stress (Fig. 6) although the generation of free radical followed a concentration

Fig. 4. (Aa–Cc) Confocal images of HPEKs with stained nuclei (blue) after exposure to TNP for 4 h and 8 h showing ROS stained by CellROX® dye (green). (D) Quantitative analysis of ROS generated by TNP. NC is the negative control HPEKs which are not treated with NPs. PC is the positive control which is UV-irradiated HPEKs. A–a: TNP 50 μg ml−1 for 4 h. B–b: TNP 50 μg ml−1 for 8 h. C–c: TNP 100 μg ml−1 for 8 h. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

46

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

Fig. 5. (A) Confocal image showing an overlay of the nuclei stained HPEKs displaying ROS delineated by green CellROX® dye. Using ImageJ software, signal from the cytoplasm was isolated and quantified. An example of such an ROI is shown. Mitochondrial ROS induced in HPEKs by (B) TNP and (C) ZNP at different concentrations and exposure times. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

dependent trend. At sub-lethal concentrations of 2.5 and 5 μg ml−1, an increase in ROS-positive cells was observed indicating the beginning of apoptotic machinery in response to the toxic effects of ZNP. At 10 μg ml−1, this increase plateaued off. A possible reason could be an increase in cell death which causes severely ROS-affected cells to undergo apoptotic cell death. This is supported by the fact that the fraction of ROS-positive cells in the high category were reduced. Nevertheless, the overall oxidative potential of ZNP was much lower than TNP. Closer inspection into the different levels of ROS-affected cells also suggested

that lower severity of ROS was induced. Furthermore and rather interestingly, ZNP did not generate significant mitochondrial ROS (Fig. 5c). 3.4. Intracellular TNP shield HPEKs from ZNP-induced DNA damage As described earlier in the methods section, three kinds of HPEKs were prepared to reproduce HPEKs containing intra- and/or extra-cellular TNP. SIM-exposed HPEKs contained only extracellular TNP, TNP-loaded HPEKs contained only intracellular TNP while TNP-exposed cells

Fig. 6. (Aa–Cc) Confocal images of HPEKs with stained nuclei (blue) after exposure to TNP for 4 h and 8 h showing ROS stained by CellROX® dye (green). (D) Quantitative analysis of ROS generated by TNP. NC is the negative control HPEKs which are not treated with NPs. PC is the positive control which is UV-irradiated HPEKs. A–a: ZNP 2.5 μg ml−1 for 4 h. B–b: ZNP 5 μg ml−1 for 4 h. C–c: ZNP 10 μg ml−1 for 8 h. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

contained TNP both inside and outside the cells. The differently prepared HPEKs were treated with ZNP for 24 h to see how the TNP present can affect the DNA damage induced. Being otherwise of low genotoxic potential, TNP could either further enhance the ZNPinduced genotoxicity or counter it. Interestingly, HPEKs which contained intracellular TNP were completely protected against ZNPinduced DNA damage (Fig. 7a). Both TNP-exposed and TNP-loaded HPEKs did not show any significant increase in DSBs with respect to the control. On the other hand, extracellular TNP did not prompt a similar protective effect as SIM-exposed HPEKs recorded the same level of DSBs as control HPEKs after ZNP treatment. Intracellular, but not extracellular, TNP was effective in alleviating ZNP-induced cell death.

3.5. Intracellular TNP curtail the nuclear accumulation of Zn ions Zn ions have been identified by many studies as the main toxic species arising from ZNP [17,34]. While oxidative stress is an indirect way of inducing DNA damage and other undesirable cellular effects, one direct way of inducing DNA damage is through nuclear uptake and subsequent oxidation of the DNA. Previously published reports have suggested that TNP do not cross the nuclear barrier in adenocarcinomic human alveolar basal epithelial cells (A-549) and human-derived retinal pigment epithelial cells (ARPE-19) [16,37]. In HPEKs, a similar trend was observed. While TNP was taken up readily through endocytosis and packetized into vesicles, they never entered the nucleus. However, TNP did show a tendency to localize in the peri-nuclear region (Fig. 7). This has been shown in the studies cited as well. One the other hand, ZNP-originated Zn ion may enter the nucleoplasm via the zinc transporting machinery inside the cell [24]. The nuclear localization of Zn ions was measured using a zinc-chelating dye, Newport DCF™. Treatment with ZNP caused a rise in the amount of nuclear Zn ions in a concentration dependent manner (Fig. 7b). Interestingly, however, the nuclear uptake of Zn ions was reduced significantly in the presence of TNP. HPEKs which contained intracellular TNP reduced the nuclear Zn ion concentration to the same level as the negative control (Fig. 7c). On the other hand, SIM-exposed HPEKs also reduced the nuclear Zn ion concentration but by a lesser extent. Although SIM-exposed cells do not contain any intracellular TNP at the time of ZNP exposure, over time the extracellular TNP would be taken up. This could explain why there was only a stifled effect. Nevertheless, the difference between TNP-exposed/TNP-loaded HPEKs and SIM-exposed HPEKs clearly demonstrated that intracellular TNP were chiefly responsible for the drop in nuclear accumulation of Zn ions.

47

3.6. Oxidative stress induced by dual nanoparticles is compounded In contrast to DNA damage, ROS generation in dual nanoparticle did not exhibit antagonism. Instead when both ZNP and TNP were exposed to the HPEKs, ROS generation seemed to be a sum of the individual nanoparticle ROS generation. TNP-exposed HPEKs showed little difference in ROS potential with or without subsequent ZNP treatment (Fig. 9a). This coupled to the low oxidative potential of ZNP at sublethal concentrations indicated a minimal interference in TNP-induced ROS generation. ZNP-exposed HPEKs (these are counterparts of TNP-exposed HPEKs; treated with ZNP before TNP) showed an increase in oxidative potential when treated with TNP (Fig. 9b). This increase corresponded closely to the oxidative potential of TNP alone. Taken together, this observation also pointed to an additive rather than antagonistic effect in dual nanoparticle treatments. 4. Discussion The key finding of this study was that intracellular TNP could shield HPEKs from ZNP-induced DNA damage. We first performed a detailed characterization of the nanoparticles which reveal that both ZNP (935 ± 150 nm) and TNP (1964 ± 80 nm) exhibited a high state aggregation in medium and possessed a negative surface charge in both water and medium (ZNP: −8.0 ± 1.0 mV in water; −9.8 ± 0.2 mV in medium). Negatively charged nanoparticles have been shown to penetrate the plasma membrane via endocytosis which by its nature encapsulates them into endolysosomal vesicles [33]. Our own uptake studies also showed compartmentalization of TNP into vesicles (Fig. 8). TNP uptake into the cell can be thought of as a two-step process. First, TNP diffuses through the cell medium and attaches onto the cell membrane. Our group has observed membrane-bound TNP in confocal images [19] while other groups have also observed similar [15]. The next step is a wrapping of the plasma membrane around TNP in a process known as endocytosis. Although many studies, including our own, have demonstrated the endocytotic ability of TNP, nuclear penetration has not yet been shown [16,30]. However, in all those studies TNP was observed to accumulate around the perinuclear region (Fig. 8). The ability of nanoparticles to induce DNA damage can arise from a variety of scenarios. One of them is direct interaction of nanoparticles with the DNA; however, this requires nuclear penetration. In the case of TNP, only remote and indirect effects would be possible. ROS generation can facilitate a remote effect on DNA damage. This effect is called primary indirect genotoxicity [26]. The surface of TNP is chiefly

Fig. 7. (A) Double stranded breaks (DSBs) occurrences indicating γ-H2AX foci in HPEKs after combinatorial treatment of ZNP and TNP. Quantitative analysis was performed using ImageJ showing the percentage of cells showing DNA damage with respect to NC. (B) Nuclear uptake of Zn2+ ions after ZNP exposure. Inserts show the Newport DCF dye indicating nuclear Zn2+ ions. (C) Nuclear ZI after combinatorial treatment of ZNP and TNP. Values are normalized ZNP 10 and NC is subtracted out. For both, * indicates p b 0.05 w.r.t. NC. Ѱ indicates p b 0.05 w.r.t. ZNP 10.

48

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

Fig. 8. Confocal and TEM images showing TNP internalization, compartmentalization (A–E) and peri-nuclear localization (F). (A–C) HPEKs were treated with FITC-tagged TNP (green) and the nuclei and membranes of HPEKs were stained using Hoechst (blue) and wheat germ agglutinin (red) respectively showing peri-nuclear localization (arrows). (D) TEM image of HPEKs treated with TNP showing TNP internalization, compartmentalization into vesicles and peri-nuclear localization where “N” indicates the nucleus. (E) Magnification of the square region in image D. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

composed of the strongly electronegative oxygen atom which can become hydroxyl groups upon mixing with water [32]. This gives the negative surface charge to the TNP, which can facilitate generation of oxygenated free radicals by reacting to water and hydroxide [6]. In fact, they can even cleave C–H bonds of organic molecules making carbon-centered radicals. Our current study is the first to evaluate DNA damage in HPEKs cells and supported this mechanism as TNP treatment generated substantial amounts of ROS without any exposure

to UV light. This ROS was seen to localize in both nuclei and mitochondria. The nuclear ROS can contribute to oxidation of DNA, which was also observed at similar concentrations. However, the amount of DNA damage induced by TNP was much lower than ZNP even though TNP concentrations were greater by one order of magnitude. This is in line with previous studies on various cell lines which have clearly shown ZNP to be more toxic. Interestingly ZNP were shown to be soluble up to 20 μg ml−1 in the cell culture

Fig. 9. (A) ROS generation of ZNP-exposed cells after 0 and 200 μg ml-1 TNP treatment. The insert shows ROS generation after 200 μg ml-1 (TWAD 46 μg cm-2) TNP treatment of control (non ZNP-exposed) HPEKs. (B) ROS generation of TNP-exposed cells after 0 and 10 μg ml-1 ZNP treatment. The insert shows ROS generation after 10 μg ml-1 ZNP treatment of control (non TNP-exposed) HPEKs.

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

medium used. This was close to the concentrations of ZNP treated to cells. Unsurprisingly the HPEKs experienced a sharp increase in intracellular Zn ion concentration. However, unlike TNP, the Zn ions did penetrate into the nuclei. Zn ions are a critical aspect of cellular homeostasis because of the central role Zn ions play in the folding of various proteins amongst many other functions. The Zn ion concentration is maintained within a tight range. In response to increase in the Zn ion concentration the expression of the apo-metallothioneins (apo-MT) is upregulated via metal-regulatory transcription factor (MTF-1) [24]. The apo-MT can bind 7 and transport the ions into the nucleus via the nuclear pores. Over here Zn ions are released into the nucleoplasm where they are released into the nucleoplasm. Once released the Zn ions can directly interact with the cellular DNA. They are known to attach (damage) to purines and other sites. Such DNA damage is categorized as primary direct genotoxicity, and is generally very potent due to its direct approach. Our results are in line with this mechanism as ZNP treatment increases intracellular Zn ion concentration, nuclear Zn ion concentration and DSBs in the DNA of HPEKs. Numerous studies have suggested that ZNP can induce ROS-mediated DNA damage [26]. However, in our study we did not see substantial ROS-generation when HPEKs were exposed to sub-lethal ZNP concentrations. The same concentrations did, however, induce high occurrence of DSBs which suggests ROS does not play a central role in ZNP induced genotoxicity. The adsorption of positive Zn ions onto the negative surface of TNP was chiefly observed. In another study, this effect was seen to facilitate scavenging of intracellular Zn ions and bring down free Zn ion concentration in the HPEKs alleviating the Zn ion induced cytotoxicity [19]. In this study, we focused on Zn ion concentration inside the nuclei and observed that intracellular TNP effectively neutralized the nuclear uptake of Zn ions. This in turn also effectively shielded the HPEKs from ZNPinduced DNA damage. The machinery responsible for trafficking cytoplasmic Zn ions into the nucleus was pointed out as the metallothionein (MT) Zn binders [24]. TNP could possibly be competing with apo-MT for Zn ions. Previous studies have shown that MT also plays an important antioxidant role in cells [31]. In response to oxidative stress, MTs release Zn ions and induce an antioxidant effect [31]. It is possible that TNPinduced oxidative stress can trigger the release of Zn ions by MT. This coupled with the ion scavenging effect of TNP could be working together to block off entry of Zn ions into the nuclei. The same effect was not created, at least not as effectively, by extracellular TNP which reaffirmed the need for an intracellular Zn ion scavenger. Previously, it had been reported that Zn ion chelator can have a similar protective effect by reducing the Zn ion availability [1]. Interestingly, the intracellular and toxic Zn ion chelator, N,N,N',N'tetrakis(2-pyridylmethyl)ethylene diamine (TPEN), when treated along with lethal concentrations of ZNP did not cause any toxicity. It is also important to note that the reduction in DNA damage is perfectly correlated to the level of nuclear Zn ions. This is another confirmation of the role nuclear uptake of Zn ions plays in inducing primary direct genotoxicity. While the ion scavenging ability of TNP is clear, other mechanisms may also be at play contributing to the reduction of intracellular Zn ion concentration. One possibility is that TNP uptake has saturated the endocytic machinery of HPEKs, rendering them unable to take up more ZNP. However, this is possibly not a major contributing factor because most of the ZNP are presented to the HPEKs not in particulate form but as Zn ions. Another possibility is that TNP uptake could influence the ion uptake machinery of HPEKs. An examination of a report on the effects of ultrafine TiO2 particles on gene expression profile in human keratinocytes revealed that key genes involved in Zn ion uptake and regulation were affected [9]. Specifically members of the SLC30 family, SCL39 family and metal response-element transcriptional factor (MTF-1) were all affected. It had been shown that modulation of these genes can modulate intra-cellular Zn ion concentrations [5,8,10,23] making such an indirect effect a possibility.

49

Lastly, the interaction between Zn ions and TNP did not inhibit the oxidative stress exhibited by both the species. Out of the two, TNP was the more potent in creating free radicals. It might be possible that Zn ions which adsorb onto the TNP can quench the active negative surface of TNP which generates ROS. While this mechanism cannot be ignored, the effect can only be minimal at the concentrations used in this study. A simple calculation, considering hydrodynamic diameter of TNP aggregate of approximately ≈ 2 μm, effective aggregate density measured using volumetric centrifugation of 1.255 g ml−1 [4] and assuming a circular non-porous aggregate, would reveal that TNP possesses 2.4 × 106 cm2 μg−1 of surface. On the other hand, even at the highest dosage of ZNP tested (i.e. 20 μg), the maximum surface that the ZI can cover considering they are packed next to each other is at best 0.8 cm2. The numbers are so disproportionately tilted towards the TNP surface that any effect would be negligible. It is therefore not surprising that ZNP has no effect on the oxidative stress induced by TNP. 5. Conclusion This study was aimed to understand genotoxic effects of TNP and ZNP on HPEKs, both individually and in combination. The results discussed here suggest that intracellular TNP are effective in shielding HPEKs from the primary direct genotoxicity induced by ZNP nanoparticles. The mechanism behind this protection was ion adsorption onto the TNP surface which immobilized the Zn ions and mitigating them from entering the nucleoplasm to interact with DNA. Funding information This work was supported by the Singapore Centre on Environmental Life Sciences Engineering (SCELSE) (M4330001.C70.703012), the School of Materials Science and Engineering (M020070110), National Medical Research Council, Ministry of Health (NMRC/CIRG/1342/2012, MOH), and the NTU-National Healthcare Group (NTU-NHG) grant (ARG/14012). Acknowledgments The authors would like to thank Assistant Professor David Leong (National University of Singapore) for his support on ICP-MS analysis. In addition, the authors would like to acknowledge Nikon Imaging Centre, Singapore for the usage of Nikon A1R + si Confocal Microscope. Special thanks to Jian Chow Soo, Application Engineer, for his kind technical assistance. References [1] T. Buerki-Thurnherr, L. Xiao, L. Diener, O. Arslan, C. Hirsch, X. Maeder-Althaus, K. Grieder, B. Wampfler, S. Mathur, P. Wick, H.F. Krug, In vitro mechanistic study towards a better understanding of ZnO nanoparticle toxicity, Nanotoxicology 7 (2013) 402–416, http://dx.doi.org/10.3109/17435390.2012.666575. [2] W.-H. Chan, J.-S. Yu, Inhibition of UV irradiation-induced oxidative stress and apoptotic biochemical changes in human epidermal carcinoma A431 cells by genistein, J. Cell. Biochem. 78 (2000) 73–84, http://dx.doi.org/10.1002/(SICI)1097-4644(20000701)78: 1b73::AID-JCB7N3.0.CO;2-P. [3] T. Chen, J. Yan, Y. Li, Genotoxicity of titanium dioxide nanoparticles, J. Food Drug Anal. 22 (2014) 95–104, http://dx.doi.org/10.1016/j.jfda.2014.01.008. [4] G. DeLoid, J.M. Cohen, T. Darrah, R. Derk, L. Rojanasakul, G. Pyrgiotakis, W. Wohlleben, P. Demokritou, Estimating the effective density of engineered nanomaterials for in vitro dosimetry, Nat. Commun. 5 (2014), http://dx.doi.org/10. 1038/ncomms4514. [5] S. Devergnas, F. Chimienti, N. Naud, A. Pennequin, Y. Coquerel, J. Chantegrel, A. Favier, M. Seve, Differential regulation of zinc efflux transporters ZnT-1, ZnT-5 and ZnT-7 gene expression by zinc levels: a real-time RT-PCR study, Biochem. Pharmacol. 68 (2004) 699–709, http://dx.doi.org/10.1016/j.bcp.2004.05.024. [6] I. Fenoglio, G. Greco, S. Livraghi, B. Fubini, Non-UV-induced radical reactions at the surface of TiO2 nanoparticles that may trigger toxic responses, Chemistry 15 (2009) 4614–4621, http://dx.doi.org/10.1002/chem.200802542. [7] I. Fenoglio, J. Ponti, E. Alloa, M. Ghiazza, I. Corazzari, R. Capomaccio, D. Rembges, S. Oliaro-Bosso, F. Rossi, Singlet oxygen plays a key role in the toxicity and DNA damage caused by nanometric TiO2 in human keratinocytes, Nanoscale 5 (2013) 6567–6576, http://dx.doi.org/10.1039/c3nr01191g.

50

M.H. Kathawala et al. / Materials and Design 88 (2015) 41–50

[8] R.B. Franklin, J. Ma, J. Zou, Z. Guan, B.I. Kukoyi, P. Feng, L.C. Costello, Human ZIP1 is a major zinc uptake transporter for the accumulation of zinc in prostate cells, J. Inorg. Biochem. 96 (2003) 435–442, http://dx.doi.org/10.1016/S0162-0134(03)00249-6. [9] K. Fujita, M. Horie, H. Kato, S. Endoh, M. Suzuki, A. Nakamura, A. Miyauchi, K. Yamamoto, S. Kinugasa, K. Nishio, Y. Yoshida, H. Iwahashi, J. Nakanishi, Effects of ultrafine TiO2 particles on gene expression profile in human keratinocytes without illumination: involvement of extracellular matrix and cell adhesion, Toxicol. Lett. 191 (2009) 109–117, http://dx.doi.org/10.1016/j.toxlet.2009.08.011. [10] L.A. Gaither, D.J. Eide, The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells, J. Biol. Chem. 276 (2001) 22258–22264, http://dx.doi. org/10.1074/jbc.M101772200. [11] S. Hackenberg, F.-Z. Zimmermann, A. Scherzed, G. Friehs, K. Froelich, C. Ginzkey, C. Koehler, M. Burghartz, R. Hagen, N. Kleinsasser, Repetitive exposure to zinc oxide nanoparticles induces dna damage in human nasal mucosa mini organ cultures, Environ. Mol. Mutagen. 52 (2011) 582–589, http://dx.doi.org/10.1002/em.20661. [12] S. Hanasoge, M. Ljungman, H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase, Carcinogenesis 28 (2007) 2298–2304, http://dx.doi.org/10.1093/carcin/bgm157. [13] P. Hinderliter, K. Minard, G. Orr, W. Chrisler, B. Thrall, J. Pounds, J. Teeguarden, ISDD: a computational model of particle sedimentation, diffusion and target cell dosimetry for in vitro toxicity studies, Part. Fibre Toxicol. 7 (2010) 36, http://dx.doi.org/10. 1186/1743-8977-7-36. [14] N. Jacobsen, P. Møller, K. Jensen, U. Vogel, O. Ladefoged, S. Loft, H. Wallin, Lung inflammation and genotoxicity following pulmonary exposure to nanoparticles in ApoE−/− mice, Part. Fibre Toxicol. 6 (2009) 2, http://dx.doi.org/10.1186/17438977-6-2. [15] A. Jaeger, D.G. Weiss, L. Jonas, R. Kriehuber, Oxidative stress-induced cytotoxic and genotoxic effects of nano-sized titanium dioxide particles in human HaCaT keratinocytes, Toxicology 296 (2012) 27–36, http://dx.doi.org/10.1016/j.tox.2012. 02.016. [16] G. Janer, E. Mas del Molino, E. Fernández-Rosas, A. Fernández, S. Vázquez-Campos, Cell uptake and oral absorption of titanium dioxide nanoparticles, Toxicol. Lett. 228 (2014) 103–110, http://dx.doi.org/10.1016/j.toxlet.2014.04.014. [17] Y.Y. Kao, Y.C. Chen, T.J. Cheng, Y.M. Chiung, P.S. Liu, Zinc oxide nanoparticles interfere with zinc ion homeostasis to cause cytotoxicity, Toxicol. Sci. 125 (2011) 462–472, http://dx.doi.org/10.1093/toxsci/kfr319. [18] M.H. Kathawala, S.P. Khoo, T. Sudhaharan, X. Zhao, J.S.C. Loo, S. Ahmed, K.W. Ng, Fluorescence techniques used to measure interactions between hydroxyapatite nanoparticles and epidermal growth factor receptors, Biotechnol. J. (2014), http:// dx.doi.org/10.1002/biot.201400404. [19] M.H. Kathawala, K.W. Ng, S.C.J. Loo, TiO2 nanoparticles alleviate toxicity by reducing free Zn2+ ion in human primary epidermal keratinocytes exposed to ZnO nanoparticles, J. Nanoparticle Res. 17 (2015) 1–17, http://dx.doi.org/10.1007/s11051-0153068-4. [20] P. Kocbek, K. Teskač, M.E. Kreft, J. Kristl, Toxicological aspects of long-term treatment of keratinocytes with ZnO and TiO2 nanoparticles, Small 6 (2010) 1908–1917, http://dx.doi.org/10.1002/smll.201000032. [21] X. Lu, D.P. Lane, Differential induction of transcriptionally active p53 following UV or ionizing radiation: defects in chromosome instability syndromes? Cell 75 (1993) 765–778, http://dx.doi.org/10.1016/0092-8674(93)90496-D. [22] N.A. Monteiro-Riviere, K. Wiench, R. Landsiedel, S. Schulte, A.O. Inman, J.E. Riviere, Safety evaluation of sunscreen formulations containing titanium dioxide and zinc oxide nanoparticles in UVB sunburned skin: an in vitro and in vivo study, Toxicol. Sci. 123 (2011) 264–280, http://dx.doi.org/10.1093/toxsci/kfr148.

[23] S.A. Myers, A. Nield, M. Myers, Zinc transporters, mechanisms of action and therapeutic utility: implications for type 2 diabetes mellitus, J. Nutr. Metab. 2012 (2012) 13, http://dx.doi.org/10.1155/2012/173712. [24] L. Nejdl, B. Ruttkay-Nedecky, J. Kudr, S. Krizkova, K. Smerkova, S. Dostalova, M. Vaculovicova, P. Kopel, J. Zehnalek, L. Trnkova, P. Babula, V. Adam, R. Kizek, DNA interaction with zinc(II) ions, Int. J. Biol. Macromol. 64 (2014) 281–287, http://dx.doi. org/10.1016/j.toxlet.2014.04.014. [25] K.W. Ng, S.P.K. Khoo, B.C. Heng, M.I. Setyawati, E.C. Tan, X.X. Zhao, S.J. Xiong, W.R. Fang, D.T. Leong, J.S.C. Loo, The role of the tumor suppressor p53 pathway in the cellular DNA damage response to zinc oxide nanoparticles, Biomaterials 32 (2011) 8218–8225, http://dx.doi.org/10.1016/j.biomaterials.2011.07.036. [26] K.W. Ng, Y. Zhao, M.H. Kathawala, S. Xiong, C.F. Cheok, S.C.J. Loo, Genotoxicity and carcinogenicity of nanomaterials, Handbook of Safety Assessment of Nanomaterials, Pan Stanford Series on Biomedical NanotechnologyPan Stanford 2014, pp. 341–387, http://dx.doi.org/10.1201/b15668-13. [27] I.F. Osman, A. Baumgartner, E. Cemeli, J.N. Fletcher, D. Anderson, Genotoxicity and cytotoxicity of zinc oxide and titanium dioxide in HEp-2 cells, Nanomedicine 5 (2010) 1193–1203, http://dx.doi.org/10.2217/nnm.10.52. [28] V. Sharma, D. Anderson, A. Dhawan, Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria mediated apoptosis in human liver cells (HepG2), Apoptosis 17 (2012) 852–870, http://dx.doi.org/10.1007/s10495-0120705-6. [29] V. Sharma, S.K. Singh, D. Anderson, D.J. Tobin, A. Dhawan, Zinc oxide nanoparticle induced genotoxicity in primary human epidermal keratinocytes, J. Nanosci. Nanotechnol. 11 (2011) 3782–3788, http://dx.doi.org/10.1166/jnn.2011.4250. [30] H. Shi, R. Magaye, V. Castranova, J. Zhao, Titanium dioxide nanoparticles: a review of current toxicological data, Part. Fibre Toxicol. 10 (2013) 15, http://dx.doi.org/10. 1186/1743-8977-10-15. [31] Z.E. Suntres, E.M. Lui, Antioxidant effect of zinc and zinc-metallothionein in the acute cytotoxicity of hydrogen peroxide in Ehrlich Ascites tumour cells, Chem. Biol. Interact. 162 (2006) 11–23, http://dx.doi.org/10.1016/j.cbi.2006.04.007. [32] R. Tedja, A.H. Soeriyadi, M.R. Whittaker, M. Lim, C. Marquis, C. Boyer, T.P. Davis, R. Amal, Effect of TiO2 nanoparticle surface functionalization on protein adsorption, cellular uptake and cytotoxicity: the attachment of PEG comb polymers using catalytic chain transfer and thiol-ene chemistry, Polym. Chem. 3 (2012) 2743–2751, http://dx.doi.org/10.1039/C2PY20450A. [33] A. Verma, F. Stellacci, Effect of surface properties on nanoparticle-cell interactions, Small 6 (2010) 12–21, http://dx.doi.org/10.1002/smll.200901158. [34] T. Xia, M. Kovochich, M. Liong, L. Mädler, B. Gilbert, H. Shi, J.I. Yeh, J.I. Zink, A.E. Nel, Comparison of the mechanism of toxicity of zinc oxide and cerium oxide nanoparticles based on dissolution and oxidative stress properties, ACS Nano 2 (2008) 2121–2134, http://dx.doi.org/10.1021/nn800511k. [35] X. Zhao, K.J. Ong, J.D. Ede, J.L. Stafford, K.W. Ng, G.G. Goss, S.C.J. Loo, Evaluating the toxicity of hydroxyapatite nanoparticles in catfish cells and zebrafish embryos, Small 9 (2013) 1734–1741, http://dx.doi.org/10.1002/smll.201200639. [36] Y. Zhao, J.L.C. Howe, Z. Yu, D.T. Leong, J.J.H. Chu, J.S.C. Loo, K.W. Ng, Exposure to titanium dioxide nanoparticles induces autophagy in primary human keratinocytes, Small 9 (2013) 387–392, http://dx.doi.org/10.1002/smll.201201363. [37] R.M. Zucker, E.J. Massaro, K.M. Sanders, L.L. Degn, W.K. Boyes, Detection of TiO2 nanoparticles in cells by flow cytometry, Cytometry A 77 (2010) 677–685, http:// dx.doi.org/10.1002/cyto.a.20927.