Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase

Abstract

Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.

Fig 1. TEM images of (A) 0.5 mg/mL TiO₂ NPs alone; (B): enlarged view of the cluster; (C) 0.5 mg/mL TiO₂ NPs with membrane fraction (MF) (25μg/mL cyt b558) and 50 μg/mL aggregated trimera;. (The black bar gives the scale: 250 nm for Fig 1A and 1C and 30 nm for Fig 1B).

The samples are dried (see materials and methods).

Fig 2. Fluorescence emission spectra of the trimera-TiO₂ NPs suspensions.

The solution contains 5 μg/mL (60 nM) trimera and TiO₂ NPs at the concentrations of 0, 10, 40, 80 and 100 μg/mL in a final volume of 3 mL of buffer (PBS supplemented with 10 mM MgSO₄). The emission spectra were measured using an excitation wavelength of 290 nm as described in the Materials and Methods section. Results are representative of at least three independent experiments. In inset: enlargement of the fluorescence spectrum in the region 360-500 nm for three solutions. Fluorescence spectra of 5 μg/mL trimera alone (green), 100 μg/ mL TiO₂ NPs alone (red) 5 μg/mL trimera in the presence of 100 μg/ mL TiO₂ NPs (blue).

Fig 3. SRCD spectra of trimera alone and in the presence of either TiO2 NPs or AA.

SRCD spectra of the trimera (18 μM) alone (Blue) and in the presence of TiO₂ nanoparticles (60 μg/mL) (sky blue) and AA (300μM) (red). The solvent was NaF 100mM /NaPi 10 mM pH 7, 25°C. The points are experimental, the curves are the fits using BeStSel [49].

Fig 4. Kinetics of superoxide anion production in presence of TiO₂ NPs.

Neutrophil membrane fractions (5 nM cyt b558) and trimera 200 nM were incubated together in the presence of 40 μM AA and (0, 20, 30 μg/mL) TiO₂ NPs. The production was initiated by addition of NADPH (250 μM) and the rate of O₂ ^-• was quantified by the reduction of cyt c (50 μM). Control was performed by the addition of 50 μg/mL SOD. (on the fig: 20μg/mL TiO₂ in the presence of SOD). The initial rates of production of superoxide are the following: 92.0±0.3, 134.0±0.5, 119.2±0.4 mol O₂ ^•-/s/Mol Cyt b ₅₅₈ for TiO2 NPs 0, 20, 30 μg/m respectively.

Fig 5. Dependence of NADPH oxidase activity as a function of TiO₂ NPs concentration.

Neutrophil membrane fractions (5 nM cyt b558) and trimera 200 nM (blue dots) or the cytosolic subunits (p67^phox 200 nM, p47^phox 260 nM and Rac 580 nM) (red squares) were incubated together in the presence of 40 μM AA and TiO₂ NPs. Oxidase activities were expressed as the percent of activity measured in the absence of TiO₂ NPs (90 mol O₂ ^.-/s/mol cyt b558), and determined as 100%. Points are an average of 3 independent measurements. The dotted curve is a visual fit for both systems.

Fig 6. Dependence of NADPH oxidase activity as a function of TiO₂ NPs concentrations in the absence of arachidonic acid.

Membrane fractions (4 nM cyt b558) with trimera 200 nM were incubated 4 min in the presence of 0, 20 or 40 μg/mL TiO₂ NPs. Control experiment representing 100% (83 mol O₂ ^•−/s/ mol cyt b558) of the activity was realized in presence of 40 μM AA and in absence of TiO₂ NPs. The rates of superoxide production were measured as described in Materials and Methods. Data are the average of 3 independent measurements.

Fig 7. Effect of TiO₂ NPs on the AA-dependent activation profile.

Neutrophil membrane fractions and trimera were incubated together in the presence of different concentration of AA. The TiO₂ NPs concentration was as follow, blue dots: no TiO₂ NPs; red squares: 20 μg/mL TiO₂ NPs. Oxidase activities were expressed as the percent of activity measured in the presence of 40 μM AA (85 mol O₂ ^.-/s/mol cyt b558) set as 100%. The curves are visual fits of the experimental points and the maxima have been indicated by crosses. The rate of O₂ ^•− production was measured as described in Materials and Methods.

Fig 8. Effect of TiO₂ NPs as a function of its sequence of addition in the cell free system.

Neutrophil membrane fractions (4 nM cyt b558) and 200 nM trimera were incubated together in the presence of 40 μM AA and TiO₂ NPs (10, 20, 40 μg/mL). TiO₂ NPs was added to the solution either after the membrane fractions or after the membrane fractions and trimera or after the membrane fractions, trimera and AA. Oxidase activity was expressed as the percent of activity measured in the absence of TiO₂ NPs (84 mol O₂ ^.-/s/mol cyt b558) set as 100%. Results are presented as the mean±SD of 3 independent experiments.