Toxicological evaluation of the TiO2 anatase and rutile crystalline phases in H9c2 cardiac cells

Abstract

Although TiO2 particles constitute a highly used material in consumer products, including food and pharmaceutical industries, considerable experimental evidence suggests that TiO2 particle exposure could be harmful and cause adverse health effects. Generally, the most studied factor for toxicity is size as nano, and fine particles are considered more toxic than bulk forms. The second structural factor most studied is the crystalline phase. The TiO2 rutile phase is considered a more inert phase than the highly active, high-refractive-index anatase phase. The cytotoxicity of TiO2-anatase has been related to that these particles can induce higher production of reactive oxygen species (ROS), which is a trigger of apoptosis pathway and alteration of mitochondrial membrane potential in cells. However, such a toxicological susceptibility to the TiO2-anatase phase may differ from the one initiated by the TiO2-rutile phase, suggesting a different cell death mechanism, which is not known at the detail. In this thesis, a series of experimental measurements were carried out to analyze TiO2-anatase and TiO2-rutile submicron-sized particles' physical properties. The TiO2 particles in anatase phase were transformed to rutile phase through a heating process, and then both were analyzed by Raman spectroscopy, powder X-ray diffraction (XRD), scanning electron microscopy (SEM), dynamic light scattering (DLS), and zeta potential. The evaluation of the toxicity of TiO2 particles in H9c2 cardiac cells to identify the role of the crystalline phase that may pose a health risk in a dose-dependent manner is the main objective of this study. The TiO2-anatase and TiO2-rutile particles' toxicity assessment was conducted in vitro, evaluating the metabolic activity, the plasma membrane integrity, mitochondrial superoxide production, and intracellular redox state. The particles' characterization by XRD and Raman spectroscopy confirmed the successful transformation of anatase particles to the rutile phase through a heating process. By DLS, it was confirmed that the hydrodynamic particle diameter was 166 nm and 468 nm for anatase and rutile, respectively. At the same time, further analysis by XLPA methods: Williamson-Hall and Warren Averbach showed that the apparent crystallite size of anatase is larger than for rutile. SEM microscopy identified that anatase particles had a spheric-like shape while for rutile were slightly more elongated. H9c2 cells show metabolic activity inhibition of 50% at an approximate value of 30 μg/mL when exposed to either anatase or rutile particles for 24 h. However, the dose-dependent inhibition at lower or higher values of the IC50 is dependent on the crystalline structure. Neither anatase phase nor rutile phase reduces the number of viable cells through necrosis; however, cell death has been categorized as early or late apoptosis for both particles. No significant alteration of the intracellular redox state at any particle exposure concentration between 0.3 μg/mL – 30 μg/mL was observed. On the other hand, for anatase, a 3-fold increase in mitochondrial superoxide production at 30 μg/mL was found, indicating that the intrinsic mitochondrial apoptotic pathway might mediate the apoptosis. However, for rutile, there is no increase in mitochondrial ROS production, suggesting that the cell death mechanism is dependent on a different metabolic pathway independent of the mitochondria.