Titanium dioxide nanoparticles enhance production of superoxide anion and alter the antioxidant system in human osteoblast cells

Figures & data

Figure 1 Histogram of titanium oxide-nanoparticle (TiO₂NP) size distribution. The predominant size for TiO₂NPs ranged from 10 to 15 nm. The size distribution was obtained from six independent dynamic light scattering measurements.

Figure 2 Distribution of titanium oxide nanoparticles (TiO₂NPs) in hFOB 1.19 cells.

Notes: (A) Arrows indicate TiO₂NP (50 μg/mL) uptake by cells following 24 hours’ incubation. (B) A representative image of the osteoblasts showing normal cell ultrastructure after incubation with nanoparticles (NPs) at 50 μg/mL for 24 hours. (C) Altered mitochondrial morphology after 48 hours’ exposure to TiO₂NPs (50 μg/mL). Arrows indicate mitochondrial profiles with condensed matrix. (D) Ultrastructural evidence of the presence of NPs within the cell after 24 hours’ exposure to TiO₂NPs. Note aggregates of TiO₂NPs inside the cell vacuoles. Arrow indicates internalization of nanoparticles.

Figure 3 Ultrastructural features of osteoblasts incubated with 100 μg/mL titanium oxide nanoparticles (TiO₂NPs) for 48 hours.

Notes: (A) Intensive cytotoxic vacuolization of mitochondria. (B) Autophagolysosomes with remnants of organelles. Arrow indicates well-developed Golgi apparatus. (C) Vacuolization of cytoplasm. (D) Cell undergoing necrosis showing disintegration of cytoplasmic membrane and electron-lucent nuclear chromatin.

Figure 4 Increased generation of superoxide anion (O₂^•−) in hFOB 1.19 cells following exposure to titanium oxide nanoparticles (TiO₂NPs).

Notes: Data shown are means ± standard deviations of triplicate determinations from four independent experiments. *P<0.05; ***P<0.001; treatments vs control.

Figure 5 Reduction of superoxide dismutase (SOD) activity by titanium oxide nanoparticles (TiO₂NPs) in hFOB 1.19 cells. The exposure of cells to TiO₂NPs decreased SOD activity.

Notes: Data shown are means ± standard deviations of duplicate determinations from four independent experiments. **P<0.01 compared with control.

Figure 6 Reduction of superoxide dismutase (SOD) protein levels by titanium oxide nanoparticles (TiO₂NPs) in hFOB 1.19 cells. The exposure of cells to TiO₂NPs decreased SOD1 (A) and SOD2 (B) protein levels. Insets show representative immunoblots.

Notes: Data shown are means ± standard deviations of triplicate determinations from three independent experiments. *P<0.05; **P<0.01; compared with control.

Figure 7 Reduction of sirtuin 3 (SIR3) protein levels by titanium oxide nanoparticles (TiO₂NPs) in hFOB 1.19 cells. The treatment of cells with TiO₂NPs decreased SIR3 protein levels. Inset shows a representative immunoblot.

Notes: Data shown are means ± standard deviations of triplicate determinations from three independent experiments. **P<0.01; compared with control.

Table 1 Pearson’s correlation coefficient between sirtuin 3 (SIR3) and manganese superoxide dismutase (MnSOD)/superoxide anion (O₂^•−)

Parameter	na	rb	P-value
SIR3/MnSOD protein level	15	0.766	<0.001
SIR3 protein level/O2^•− level	15	−0.789	<0.001

Note:

a n, number of observations per parameter;

b r, correlation coefficient.

Figure 8 Reduction of total antioxidant capacity (TAC) by titanium oxide nanoparticles (TiO₂NPs) in hFOB 1.19 cells.

Notes: Data shown are means ± standard deviations of triplicate determinations from three independent experiments. *P<0.05; ***P<0.01; compared with control.

Figure 9 Increased levels of malondialdehyde (MDA) following exposure to titanium oxide nanoparticles (TiO₂NPs) in hFOB 1.19 cells.

Notes: Data shown are means ± standard deviations of triplicate determinations from three independent experiments. *P<0.05; ***P<0.001; treatments vs control.

Figure 10 Reversal of titanium oxide-nanoparticle (TiO₂NP)-induced cytotoxicity by superoxide dismutase (SOD) in hFOB 1.19 cells. SOD attenuates TiO₂NP-induced decrease in cell viability (A), alkaline phosphatase (ALP) activity (B), and increase in malondialdehyde (MDA) production (C).

Notes: Data shown are means ± standard deviations of triplicate determinations from three independent experiments. *P<0.05; **P<0.01; ***P<0.001; TiO₂NPs-treated cells vs TiO₂NPs-treated cells in the presence of SOD.

Figure S1 A preliminary concentration–response study. Cell viability after 24 hours’ treatment with titanium oxide nanoparticles (TiO₂NPs) (5, 50, 250, and 500 μg/mL). The exposure of cells to TiO₂NPs for 24 hours decreased hFOB 1.19 cell viability.

Notes: Data shown are means of duplicate determinations from two independent experiments.

Figure S2 Cell viability after 24 and 48 hours of treatment with titanium oxide nanoparticles (TiO₂NPs). The exposure of cells to TiO₂NPs for 48 hours decreased hFOB 1.19 cell viability.

Notes: Data shown are means ± standard deviations of triplicate determinations from three independent experiments. *P<0.05; ***P<0.001 compared with control.

Figure S3 Titanium oxide nanoparticles (TiO₂NPs) result in lactate dehydrogenase (LDH) release following 48 hours’ incubation with hFOB 1.19 cells.

Notes: Data shown are means ± standard deviations of triplicate determinations from three independent experiments. LDH release in control (untreated cells) was 9.5%±2% and this was significantly increased following treatment with TiO₂NPs. *P<0.05; ***P<0.001 compared with control.

Figure S4 Reduction of alkaline phosphatase (ALP) activity by titanium oxide nanoparticles (TiO₂NPs). The 48-hour treatment of cells with TiO₂NPs decreased hFOB 1.19 ALP activity.

Notes: Data shown are means ± standard deviations of triplicate determinations from independent experiments done in triplicate. *P<0.05; ***P<0.001 compared with control.