Endocytosis, oxidative stress and IL-8 expression in human lung epithelial cells upon treatment with fine and ultrafine TiO{sub 2}: Role of the specific surface area and of surface methylation of the particles

Inhaled ultrafine particles show considerably stronger pulmonary inflammatory effects when tested at equal mass dose with their fine counterparts. However, the responsible mechanisms are not yet fully understood. We investigated the role of particle size and surface chemistry in initiating pro-inflammatory effects in vitro in A549 human lung epithelial cells on treatment with different model TiO{sub 2} particles. Two samples of TiO{sub 2}, i.e. fine (40-300 nm) and ultrafine (20-80 nm) were tested in their native forms as well as upon surface methylation, as was confirmed by Fourier transformed infrared spectroscopy. Radical generation during cell treatment was determined by electron paramagnetic resonance with 5,5-dimethyl-1-pyrroline-N-oxide or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl. Interleukin-8 mRNA expression/release was determined by RT-PCR and ELISA, whereas particle uptake was evaluated by transmission electron microscopy. TiO{sub 2} particles were rapidly taken up by the cells, generally as membrane bound aggregates and large intracellular aggregates in vesicles, vacuoles and lamellar bodies. Aggregate size tended to be smaller for the ultrafine samples and was also smaller for methylated fine TiO{sub 2} when compared to non-methylated fine TiO{sub 2}. No particles were observed inside nuclei or any other vital organelle. Both ultrafine TiO{sub 2} samples but not their fine counterparts elicited significantly stronger oxidant generation and IL-8 release, despite their aggregation state and irrespective of their methylation. The present data indicate that ultrafine TiO{sub 2}, even as aggregates/agglomerates, can trigger inflammatory responses that appear to be driven by their large surface area. Furthermore, our results indicate that these effects result from oxidants generated during particle-cell interactions through a yet to be elucidated mechanism(s)