Evaluation of cytotoxic, genotoxic and inflammatory response in human alveolar and bronchial epithelial cells exposed to titanium dioxide nanoparticles

Abstract

The toxicity of titanium dioxide nanoparticles (TiO2 -NPs), used in several applications, seems to be influenced by their specific physicochemical characteristics. Cyto-genotoxic and inflammatory effects induced by a mixture of 79% anatase/21% rutile TiO2 -NPs were investigated in human alveolar (A549) and bronchial (BEAS-2B) cells exposed to 1-40 µg ml(-1) 30 min, 2 and 24 h to assess potential pulmonary toxicity. The specific physicochemical properties such as crystallinity, NP size and shape, agglomerate size, surface charge and specific surface area (SSA) were analysed. Cytotoxic effects were studied by evaluating cell viability using the WST1 assay and membrane damage using LDH analysis. Direct/oxidative DNA damage was assessed by the Fpg-comet assay and the inflammatory potential was evaluated as interleukin (IL)-6, IL-8 and tumour necrosis factor (TNF)-α release by enzyme-linked immunosorbant assay (ELISA). In A549 cells no significant viability reduction and moderate membrane damage, only at the highest concentration, were detected, whereas BEAS-2B cells showed a significant viability reduction and early membrane damage starting from 10 µg ml(-1) . Direct/oxidative DNA damage at 40 µg ml(-1) and increased IL-6 release at 5 µg ml(-1) were found only in A549 cells after 2 h. The secretion of pro-inflammatory cytokine IL-6, involved in the early acute inflammatory response, and oxidative DNA damage indicate the promotion of early and transient oxidative-inflammatory effects of tested TiO2 -NPs on human alveolar cells. The findings show a higher susceptibility of normal bronchial cells to cytotoxic effects and higher responsiveness of transformed alveolar cells to genotoxic, oxidative and early inflammatory effects induced by tested TiO2 -NPs. This different cell behaviour after TiO2 -NPs exposure suggests the use of both cell lines and multiple end-points to elucidate NP toxicity on the respiratory system.

Evaluation of uptake, cytotoxicity and inflammatory effects in respiratory cells exposed to pristine and ‐OH and ‐COOH functionalized multi‐wall carbon nanotubes

“…Regarding the similarity of MTT and WST1 results of BEAS‐2B exposed to MWCNTs‐OH, we hypothesize that such nanomaterials, which have in the specific cell medium smaller agglomerate/aggregate size and the highest charge and which do not induce any membrane damage, do not interfere with the intracellular reduction of formazan. The findings confirm the higher susceptibility of BEAS‐2B than A549 cells to different toxicants including NMs as demonstrated in previous studies (Cavallo et al , , ; Ursini et al , ).…”

“…The experimental model used in the present study was also tested to evaluate the toxicity of commercial metal oxide NPs (Ursini et al ., ; Cavallo et al ., ) and highlighted the need to take into account all the physicochemical characteristics of tested NMs and the need to perform a battery of toxicity tests on different cell types, before defining a nanomaterial as toxic. Such a sensitive and reliable in vitro experimental model could represent an useful tool for screening the potential adverse effects of inhaled NMs, reducing in vivo studies, in agreement with the 3R (Replacement, Reduction and Refinement) principles, required by currently available guidelines and good practices for animal experimentation.…”

“…The experimental model used in the present study was also tested to evaluate the toxicity of commercial metal oxide NPs (Ursini et al, 2014b;Cavallo et al, 2015) and highlighted the need to take into account all the physicochemical characteristics of tested NMs and the need to perform a battery of toxicity tests on Figure 8. Cytokine release after 24-h exposure to 20 and 40 μg ml À1 pristine and -OH functionalized multi-wall carbon nanotubes (MWCNTs) and to 1, 5, 10, 20 and 40 μg ml À1 MWCNTs-COOH in A549 cells (left panels) and to 5, 20 and 40 μg ml À1 pristine and MWCNTs-OH and to 20 and 40 μg ml À1 MWCNT-COOH in BEAS-2B cells (right panels).…”

Markers of oxidative damage of nucleic acids and proteins among workers exposed to TiO2(nano) particles

“…The toxic/genotoxic effects induced by reactive oxygen species (ROS) and reactive nitration species (RNS) can contribute to tumourigenesis in multiple ways, including through the formation of DNA adducts, nucleic acids and proteins’ oxidative products 4 5 6 7. The exact site of the DNA damage is unknown and further research is needed to elucidate the mechanisms for ROS formation and carcinogenesis 8.…”

Cyto‐genotoxic and inflammatory effects of commercial Linde Type A (LTA) nanozeolites on human alveolar epithelial cells

“…An in vitro model already applied in the study of cyto‐genotoxicity of other nanomaterials such as multi‐walled carbon nanotubes, titanium dioxide and cobalt oxide (Cavallo et al, ; Ursini et al, ; Ursini et al, ) was used with small modifications. The human lung epithelial (A549) cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in RPMI 1640 (EuroClone Ltd, Paignton, Devon, UK) supplemented with 10% FBS at 37 °C in 5% CO 2 .…”