Titanium dioxide nanoparticles impair lung mitochondrial function
Introduction
Nanotechnology industry is expanding at a rapid rate and deep investigation of the health and environmental effects of these materials is necessary. On the basis of current knowledge, there is increasing requirement for the risk assessment of titanium dioxide nanoparticles (TiO2 NPs) due to increased environmental and occupational exposures and it has been estimated that TiO2 NPs annual production is between 5000 and 6400 tons (Mueller and Nowack, 2008). This nanomaterial is widely used to provide whiteness and opacity to products such as paints, plastics, papers, inks, food colorants, toothpastes and skin care products. For this reason, toxicological properties of TiO2 NPs have been studied on several route of exposure, including dermal, oral and pulmonary exposures. Particularly, after TiO2 NPs inhalation, its internalization is mediated by clathrin-mediated endocytosis, caveolin-mediated endocytosis, and macropinocytosis (Thurn et al., in press) in both phagocytic and non-phagocytic cells. The effect of TiO2 NPs is strongly related to lung inflammation (Hussain et al., 2010, Moon et al., 2010, Li et al., 2010) but TiO2 NPs can also reach extrapulmonary tissue including kidney, liver and brain and it has been demonstrated that reactive oxygen species (ROS) generation is responsible, at least in part, for the inflammatory process (Li et al., 2010). The role of ROS in cellular effects induced by TiO2 NPs exposure has been gained special attention because ROS are implicated in the acquirement of tumorigenic phenotype induced by TiO2 NPs (Onuma et al., 2009). The exact mechanism involved in the carcinogenicity induced by TiO2 NPs is not fully described but the ROS generation is implicated in malignant transformation (Ralph et al., 2010) and cancer (Weinberg et al., 2010). In this regard, mitochondria play a pivotal role by producing almost all the cellular energy when coupling the oxidation of high energy substrates by the respiratory chain through respiratory complexes I–IV to an electrochemical H+ gradient across the inner mitochondrial membrane. The free energy formed by this gradient will be used by ATP synthase (complex V) to the phosphorylation that will generate ATP. Mitochondria are a source of ROS since superoxide anion is formed along electron transfer in the respiratory chain. It will be converted into hydrogen peroxide (H2O2) by superoxide dismutase (SOD), and consecutively, H2O2 turns into water by glutathione peroxidase (GPx) and catalase. Intracellular ROS levels depend, partially, on enzymatic antioxidant enzymes in mitochondria, on substrate and oxygen availability, and on mitochondrial integrity and function. However, under pathological conditions, the increase of mitochondrial ROS affects mitochondrial DNA, ATP synthesis, cellular metabolism, signaling pathways, proliferation and differentiation, and programmed cell death. In this regard, it has been demonstrated that TiO2 NPs is able to induce an increase in ROS production and cause damage to DNA in human lung cells (Bhattacharya et al., 2009) and to induce micronucleus formation in human epidermal cells (Shukla et al., 2011). TiO2 NPs reduces the glutathione content (Shukla et al., 2011), increases lipid peroxidation and decrease SOD levels (Xue et al., 2010). Even though TiO2 NPs are not internalized into mitochondria, there is an increasing evidence for the alterations induced in mitochondria, including cytochrome c release from mitochondria to cytosol, changes in mitochondrial membrane permeability (Zhao et al., 2009) and a decrease in mitochondrial membrane potential (ΔΨm) (Xue et al., 2010). However, the effect of TiO2 NPs on mitochondrial function remains unknown. The present study was designed to investigate the effect of TiO2 NPs in lung mitochondrial function. Furthermore, the redox balance was also examined by the activity of antioxidant enzymes and the amount of NADH levels and ROS generation after mitochondrial exposure to TiO2 NPs. Our results showed that TiO2 NPs can induce mitochondrial dysfunction measured by a decrease in respiratory control rate, oxygen consumption and an increase in P/O rate, repolarization and lag phase. TiO2 NPs also induced a decrease in mitochondrial membrane potential, NADH levels and increases in ROS generation.
Section snippets
Materials
TiO2 NPs was purchased from Aldrich (Cat# 637254). Particle size < 25 nm, spec. surface area 200–220 m2/g, mp 1825 °C, density 3.9 g/mL at 25 °C, bulk density 0.04–0.06 g/mL. MitoTracker Green FM was from Molecular Probes, Inc (Cat# M-7514); mannitol (Cat#M9546), ethylenediaminetetraacetic acid disodium salt (EDTA, Cat#M4884), ditriotritol (DTT, Cat#D9163), PVP 40 (polyvinylpirrolidone, Cat#PVP40), bovine seric albumin (BSA, Cat#4503), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Cat# D6883),
Time-course and concentration/response effects of TiO2 NPs on respiratory control index (RCI)
A time course and concentration/response study was done to investigate the effect of TiO2 NPs on RCI. We found that the RCI, an index of mitochondrial function in control samples decreases after 3 h of isolation procedure and after 5 h, respiratory control index has decreased importantly from 2.25 to 1.8 and after 5 h, RCI was 1.4 (Fig. 1). The decrease of RCI after 1 h of incubation with 1, 5, 10 and 25 μg of TiO2 NPs was 30% on average, which means around 1.65, however, after 1 h of incubation with
Discussion
TiO2 NPs has been shown to induce respiratory disorders in animal models, including lung inflammation (Grassian et al., 2007), emphysema-like lung injury (Chen et al., 2006) and lung cell death (Warheit et al., 2007) and tumor formation (Roller, 2009). The underlying mechanisms of these adverse effects, however, have not fully been characterized. The TiO2 NPs diameter used in this study does not exceed 25 nm size. This size allows TiO2 NPs to penetrate the lung tissue after inhalation, where TiO2
Conclusion
In conclusion, our results show an impairment of mitochondrial function in isolated mitochondrial from whole lung tissue after TiO2 NPs exposure, including decreased ΔΨm, inefficient NADH levels, low oxygen consumption and low ADP phosphorylation. These alterations may force cells to anaerobic respiration promoting change in cell phenotype and severe side effects in metabolism after TiO2 NPs deposition in lung tissue. Finally, further analyzes are needed to go deeper in the effects induced by
Conflict of interest statement
None.
Funding
PAPCA 2010–2011 (Project number 27), DGAPA PAPIIT IN201910 and DGAPA PAPIIT IN211208.
Acknowledgments
This work was supported by PAPCA 2010–2011 (Project number 27), DGAPA PAPIIT IN201910 and DGAPA PAPIIT IN211208.
References (39)
Mitochondrial respiratory control and the polarographic measurement of ADP/O ratios
Methods Enzymol.
(1967)- et al.
The use of monochlorobimane to determine hepatic GSH levels and synthesis
Anal. Biochem.
(1990) - et al.
Significance of ROS in oxygen sensing in cell systems with sensitivity to physiological hypoxia
Respir. Physiol. Neurobiol.
(2002) - et al.
Oligomycin-induced bioenergetic adaptation in cancer cells with heterogeneous bioenergetic organization
J. Biol. Chem.
(2010) - et al.
Comparative pulmonary toxicity study of nano-TiO(2) particles of different sizes and agglomerations in rats: different short- and long-term post-instillation results
Toxicology
(2009) - et al.
Age-related changes in mitochondrial function and antioxidative enzyme activity in fischer 344 rats
Mech. Ageing Dev.
(2007) - et al.
Metformin prevents experimental gentamicin-induced nephropathy by a mitochondria-dependent pathway
Kidney Int.
(2010) - et al.
Nano-scaled particles of titanium dioxide convert benign mouse fibrosarcoma cells into aggressive tumor cells
Am. J. Pathol.
(2009) - et al.
The causes of cancer revisited: “mitochondrial malignancy” and ROS-induced oncogenic transformation – why mitochondria are targets for cancer therapy
Mol. Aspects Med.
(2010) - et al.
Titanium dioxide nanoparticles cause apoptosis in BEAS-2B cells through the caspase 8/t-Bid-independent mitochondrial pathway
Toxicol. Lett.
(2010)
Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts
Exp. Biol. Med.
Titanium dioxide nanoparticles induce oxidative stress and DNA adduct formation but not DNA-breakage in human lung cells
Part. Fibre Toxicol.
Titanium dioxide nanoparticles induce emphysema-like lung injury in mice
FASEB J.
Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer-344 rats with age and caloric restriction
Am. J. Physiol. Regul. Integr. Comp. Physiol.
Diabetes and mitochondrial bioenergetics: alterations with age
J. Biochem. Mol. Toxicol.
Reactions of 1-methyl- 2-phenylindole with malondialdehyde and 4-hydroxyalkenals analytical applications to a colorimetric assay of lipid peroxidation
Chem. Res. Toxicol.
Inhalation exposure study of titanium dioxide nanoparticles with a primary particle size of 2 to 5 nm
Environ. Health Perspect.
The identity of glutathione S-transferase B with ligandin, amajor binding protein of liver
Proc. Natl. Acad. Sci. U.S.A.
Effects of prenatal exposure to surface-coated nanosized titanium dioxide (UV-Titan): a study in mice
Part. Fibre Toxicol.
Cited by (100)
Toxicity spectrum and detrimental effects of titanium dioxide nanoparticles as an emerging pollutant: A review
2024, Desalination and Water TreatmentOxidative stress–mediated nanotoxicity: mechanisms, adverse effects, and oxidative potential of engineered nanomaterials
2022, Advanced Nanomaterials and Their Applications in Renewable Energy, Second EditionEffects, uptake, translocation and toxicity of Ti-based nanoparticles in plants
2022, Toxicity of Nanoparticles in Plants: An Evaluation of Cyto/Morpho-physiological, Biochemical and Molecular ResponsesUnderstanding the concept of signal toxicity and its implications on human health
2022, Pharmacokinetics and Toxicokinetic Considerations - Vol II