No evidence of the long-term in vitro toxicity of Aeroxide P25 TiO₂ nanoparticles in three mammalian cell lines despite the initial reduction of cellular mitochondrial activity

TiO₂ NPs powder of nominal size 21 nm, anatase (80%)/rutile (20%) polymorphs (Aeroxide P25), factory-made by Degussa-Evonik (Essen, Germany) was kindly delivered by the European Commission Joint Research Center (EC JRS) depository. Nanoparticles stock solution was prepared at 2 mg/mL NPs in distilled water with sonification at 420 J/cm³. Directly after sonication, 100 μL of BSA and 100 μL of a 10× concentrated phosphate-buffered saline (PBS) were given to 800 μL aliquots of suspension (Protocol 1 described in Ref. [8]). The hydrodynamic diameter and zeta potential (ζ) analysis was performed using dynamic light scattering (DLS) with the Zestasizer Nano ZS system (Malvern, UK) at 25°C with a scattering angle of 173°. Stock solutions were diluted at 1:8 in a complete culture media and analysed in triplicate with 14-sub runs. The pH of suspensions was 7.4. Zeta potentials were calculated using the Smoluchowski limit for the Henry equation with a setting calculated for practical use (f(ka) = 1.5).

A full characterization of the TiO₂ NPs is given (ibid.). Briefly, the particle size distribution of TiO₂ NPs was narrow and uniform, with an average hydrodynamic diameter of 132 nm, depending on the culture medium. The ξ-potential was negative, on average about (–33 mV), depending on the culture medium.

The cell lines investigated were human colorectal adenocarcinoma HT29, human epithelial cell line A549 and human hepatic cell line HepG2; all three were obtained from the American Type Tissue Culture Collection (ATCC, Rockville, MD, USA) and maintained according to ATCC protocols.

Suitable numbers of cells were seeded on 5 cm Petri dishes and left to settle down without nanoparticles for 24 h to achieve optimal cell attachment to the plastic surface. Afterwards, the nanoparticle suspension was added to the cell cultures to obtain the final concentrations of 10 μg/mL, 50 μg/mL and 100 μg/mL for 2 h or 24 h. After incubation, the medium containing NPs was removed; for comet assay and γH2AX foci experiments, the plates were washed twice with 5 mL of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄ and 2 mM KH₂PO₄; pH 7.4). After washing, the cells were trypsinized and suspended in a fresh culture medium.

The positive controls in micronuclei and γH2AX foci experiments were NP-untreated cells X-irradiated with the dose of 2 Gy. X-irradiation was performed in an ice water bath, using a Smart200 (Yxlon) defectoscope operating at 200 kV and 4.5 mA, with 3 mm Al filtration, at a dose rate of 1.14 Gy/min.

The comet assay (single-cell gel electrophoresis) was performed as described in Refs. [9, 10]. The Comet Assay IV Image Analysis System (Perceptive Instruments, UK) performed an image analysis of the data. One hundred randomly selected comets per slide were analysed. The percentage of DNA in the comet’s tail was used in this study to measure DNA damage.

An estimation of the γ-H2AX foci number in NP--treated cells was carried out as described in Ref. [11]. In short, cells were fixed in methanol at –20°C for 5 min and rehydrated in PBS for 3 × 10 min. Unspecific binding was blocked overnight in the refrigerator in a blocking buffer (2% BSA, 10% dried milk powder) in KCM+T buffer (120 mM KCl/20 mM NaCl/10 mM Tris·HCl/0.5M EDTA/0.1% Triton X-100, pH 8.0). Samples were treated with antiphospho-histone H2AX (Ser 139) antibody (1:200; Upstate, Millipore) in a blocking buffer for 1 h at room temperature and washed in KCM+T buffer for 3 × 10 min. Thereafter, they were then incubated with Alexa Fluor 488 goat-anti-mouse IgG secondary antibody (1:200; Molecular Probes, Eugene, OR, USA) in a blocking buffer for 1 h at room temperature. The cells were washed in KCM+T buffer (4 × 10 min) and mounted with Vectashield mounting medium for fluorescence with 4’,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Foci were counted under a fluorescence microscope (Zeiss Axioplan-2) by the automated image-analysis system (MetaSystems, Altlussheim, Germany). In a single experiment, at least 2 × 200 cells were analysed per dose point. To test the reproducibility of the analysis, three independent experiments were performed.

After completing treatment with NP, the cells were washed thrice with a forward scatter (FCS)-free culture medium and left in a full culture medium for 24 h in the incubator. Afterwards, the cells were fixed with cold methanol:glacial acetic acid 3:1 for 15 min, washed with distilled water, air-dried and stained with 4’,6-diamidino-2-phenylindole (DAPI) according to Ref. [12]. To determine the number of cells with micronuclei, at least 2000 cells were analysed under a fluorescence microscope (Zeiss Axioplan-2) at 400× magnification. Objects close to mitotic cells or adhering to nuclei were excluded from the analysis. Since each cell line had characteristic morphologic features, microscopic preparations of X-irradiated (2 Gy) cells served as standards.

HepG2, HT29 and A549 cells were incubated with Aeroxide P25 TiO₂ NP for 2 h and 24 h as described above. Staurosporine (for HepG2 – 1 μM; and for A549 and HT29 – 2 μM) served as the positive control. The frequencies of viable, apoptotic and necrotic cells were detected with the Annexin V-FITC apoptosis detection kit I (BD Pharmingen, USA), according to the manufacturer’s recommendations. The fluorescence was measured by flow cytometry (LSR II flow cytometer [Becton Dickinson]). A computer system BD FACS DiVa (version 6.0, Becton Dickinson) was used for data acquisition and analysis.

After treatment with NPs, cell lysates were prepared as follows. The culture medium was removed and a culture plate (about 5 × 10⁵ cells) was rinsed three times with 5 mL PBS. Cells were detached in 1 mL of lysis buffer containing the following: 20 mM HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) pH 7.4, 250 mM NaCl, 1% Nonidet P-40, 5% glycerol, 3 mM ethylenediaminetetraacetic acid (EDTA), 3 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA), 1 mM sodium ortovanadate, 50 mM NaF, 1 mM PMSF, 20 mM β-glycerophosphate, 30 mM sodium pyrophosphate and protease inhibitor cocktail (Roche Diagnostics Indianapolis, IN, USA), and lysed on the plate for 20 min. The resulting mixture was centrifuged for 20 min, 20,000 ×g. All steps were performed on ice using pre-cooled buffers. The supernatant (cell lysate) was collected, frozen in small aliquots and stored at –80°C. Protein content in the lysate was determined by Bradford’s method. All lysates were standardized for the protein content of 2 mg/mL. The caspase 3 activity was measured by the enzyme-linked immunosorbent assay (ELISA) with the Human Caspase-3 (active) test (Invitrogen Corporation, Camarillo, CA, USA), according to the manufacturer’s guidelines.

Mitochondrial activity (MTT) assay was used to measure the activity of mitochondrial dehydrogenases and was carried out as described in Ref. [13].

The assay was carried out according to Ref. [14].

To assess the effect of NP on cell survival, a clonogenic assay was used. The stock suspension of NP, 2 mg/mL, was freshly prepared as described above. Appropriate volumes of NP were mixed with cell suspensions and the cells were seeded onto 60 mm Petri plates at a density of 700–1000 per plate in three replicates. The cells were growing in the presence of NP until visible clones were formed (usually 8–14 days); they were then fixed with formaldehyde and Giemsa stained. Clones were counted and survival was calculated as a percentage of the clone number in the untreated (control) cell sample. Individual points in the survival curves were shown as means + standard deviation (SD) of three to five independent experiments.

Intracellular ROS formation was studied based on intracellular peroxide-dependent oxidation of 2’,7’-dichlorodihydrofluorescein diacetate (DCFH--DA; Sigma Aldrich) to undergo conversion into the fluorescent compound 2’,7’-dichlorofluorescein (DCF). Cells were plated in 6-well plates at a density of 2.5 × 10⁵ cells per well and cultured for 24 h. Aliquots of NP suspensions increasing in concentration were added to the wells, and they were treated for 2 h. After that time, the cells were detached, resuspended in the fresh medium and DCFH-DA solution was added to the final concentration of 5 μM. After 30 min incubation, FCS, side scatters (SSC) parameters and fluorescence of cells were measured with a BD LSR Fortessa cytometer (BD Biosciences).

Statistical analysis of the obtained data was performed using Statistica 7.1 software (StatSoft Inc., Tulsa, OK, USA). The data were expressed as mean ± SD of at least three independent experiments. To calculate the significance of a particular experimental variable, an analysis of variance (ANOVA) was performed, followed by a post hoc Tukey’s test. The indicated statistical significance was evaluated using pairwise comparison by Student’s t-test. Differences were considered statistically significant when the P-value was less than 0.05.

Analysis of the size and zeta potential of Aeroxide P25 TiO₂ NPs in different culture media used in this study revealed no major differences (Table 1). The hydrodynamic size of NPs in all media was in the range of 250–300 nm, whereas the zeta potential was about 25–29 mV The values of the polydispersity index below 0.3 indicate acceptable homogeneity of all NPs suspensions.

The formation of DNA single-strand breaks (SSB) and net formamide-pyrimidine glycosylase (FPG) sites in cells treated for 2 h or 24 h with Aeroxide P25 TiO₂ NP at three concentrations is shown in Fig. 1. In A549 cells the TiO₂ NP induced a statistically significant increase in both SSB and base damage induction, except the lowest NPs concentration. After the 24 h treatment, the FPG sites did not differ from the control, whereas a statistically significant difference was found in the extent of SSB in cells treated with 50 mg/mL. No significant difference in the induction of DNA damage was found in HT29 despite the treatment time and NPs concentration. A similar pattern was observed in HepG2 cells. HepG2 cells respond to 2 h TiO₂ NP treatment with the lowest DNA damage level (both SSB and base damage). However, 24 h TiO₂ NP treatment with the highest concentration induced marked, statistically significant base damage.

Fig. 1.

The effect of 2 h and 24 h treatment on micronuclei frequency using the same NP concentrations as those used for comet assay is shown in Table 2. The effect of X-irradiation with 2 Gy is given in the same figure for comparison (a positive control). There were no statistically significant differences in micronuclei frequencies between the control and the treated cells in the three cell lines examined.

The same table shows the mean number of γH2AX foci per nucleus after 2 h and 24 h treatment of the cells with the same NP concentrations as those used for comet assay measurements. The effect of X-irradiation with 2 Gy is given for comparison (a positive control). There were no differences in γH2AX foci numbers between the control and the treated cells in the three cell lines examined.

The results of apoptosis induction in Aeroxide P25 TiO₂ NP-treated cells are shown in Table 3. The staurosporine-induced apoptosis is shown for comparison (a positive control). Though an increase in the number of apoptotic cells was observed in all cell lines for almost all treatment regimes, the increase was very weak, as compared to the effect of staurosporine. For example, treatment of the cells with 100 μg/mL, Aeroxide P25 TiO₂ NP for 24 h resulted in less than 10% of apoptotic cells, whereas treatment of the same cells with staurosporine revealed 50-60% of apoptosis.

These results were further confirmed by analysis of caspase 3 activation in treated cells (Table 4). Following induction of apoptosis, elevated caspase 3 activity was observed only in HepG2 and HT29 cells treated with the highest dose of the nanomaterial. Though statistically significant, the caspase 3 activation by Aeroxide P25 TiO₂ NP was very low, for example, two orders of magnitude lower than that induced by staurosporine in Jurkat cells.

The effect of different concentrations of Aeroxide P25 TiO₂ NPs on mitochondrial homoeostasis was determined by MTT assay. A549 cells appeared to be the most vulnerable to Aeroxide P25 treatment, as a slight decrease in mitochondrial activity was observed even 24 h after treatment. However, a decrease in mitochondrial activity higher than 15% was observed in neither A549 cells nor HT29 cells. No changes in mitochondrial activity were observed in HepG2 cells (Fig. 2).

Fig. 2.

Short-term survival (up to 72 h) after 24 h treatment with Aeroxide P25 TiO₂ NP was measured by neutral red assay (Fig. 3). A slight decrease in cell survival was observed for all cell lines 48 h and 72 h after treatment with the highest doses of Aeroxide P25. However, in line with a decrease in mitochondrial activity, the decrease in cell survival was not higher than 15%.

Fig. 3.

In opposition to the MTT assay and neutral red assay results, the determination of the cloning ability of cells continuously treated with TiO₂ NP revealed no apparent toxicity of Aeroxide P25 (Fig. 4). The most susceptible cell line was HT29 with a survival rate of 80% or higher despite the NPs concentration. Survival rates of the two remaining cell lines (A549 and HepG2) were around 100%, indicating no toxicity of the tested compound.

Fig. 4.

The analysis of Aeroxide P25 TiO₂ NP-induced ROS formation revealed that the highest concentration of nanoparticles significantly elevated ROS in all cell lines (Fig. 5). The highest production of ROS was observed for HepG2 cells.

Fig. 5.