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Toxic effects were reported for pristine-multi-wall carbon nanotubes (p-MWCNTs) while the role of the functionalization on MWCNT-induced toxicity is not yet well defined. We evaluated on human alveolar (A549) epithelial cells and normal bronchial (BEAS-2B) cells exposed to p-MWCNTs, MWCNTs-OH and MWCNTs-COOH: uptake by TEM, cell viability by different assays, membrane damage by the LDH assay and cytokine release by ELISA. The aims of the present study were to: (i) confirm MWCNT cytotoxicity mechanisms hypothesized in our previous studies; (ii) identify the most reliable viability assay to screen MWCNT toxicity; and (iii) to test our model to clarify the role of functionalization on MWCNT-induced toxicity. In A549 cells, p-MWCNTs and MWCNTs-OH were localized free in the cytoplasm and inside vacuoles whereas MWCNTs-COOH were confined inside filled cytoplasmic vesicles. WST-1 and Trypan blue assays showed in A549 cells a similar slight viability reduction for all MWCNTs whereas in BEAS-2B cells WST1 showed a high viability reduction at the highest concentrations, particularly for MWCNTs-COOH. The MTT assay showed a false cytotoxicity as a result of MWCNTs-interference. Pristine and MWCNTs-COOH induced membrane damage, particularly in BEAS-2B cells. MWCNTs-COOH induced interleukin-6 (IL-6) and IL-8 release in A549 cells whereas p-MWCNTs induced IL-8 release in BEAS-2B cells. MWCNTs intracellular localization in A549 cells confirms the toxicity mechanisms previously hypothesized, with p-MWCNTs disrupting the membrane and vesicle-confined MWCNTs-COOH inducing inflammation. WST-1 was more reliable than MTT to test MWCNT-toxicity. BEAS-2B cells were more susceptible then A549 cells, particularly to MWCNT-COOH cytotoxicity. Our results confirm the toxicity of p-MWCNTs and demonstrate, also for the two kinds of tested functionalized MWCNTs toxic effects with a different mechanism of action.

The increasing use of nanomaterials in consumer products highlights the importance of understanding their potential toxic effects. We evaluated cytotoxic and genotoxic/oxidative effects induced by commercial multi-walled carbon nanotubes (MWCNTs) on human lung epithelial (A549) cells treated with 5, 10, 40 and 100 µg ml⁻¹ for different exposure times. Scanning electron microscopy (SEM) analysis, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays were performed to evaluate cytotoxicity. Fpg-modified comet assay was used to evaluate direct-oxidative DNA damage. LDH leakage was detected after 2, 4 and 24 h of exposure and viability reduction was revealed after 24 h. SEM analysis, performed after 4 and 24 h exposure, showed cell surface changes such as lower microvilli density, microvilli structure modifications and the presence of holes in plasma membrane. We found an induction of direct DNA damage after each exposure time and at all concentrations, statistically significant at 10 and 40 µg ml⁻¹ after 2 h, at 5, 10, 100 µg ml⁻¹ after 4 h and at 10 µg ml⁻¹ after 24 h exposure. However, oxidative DNA damage was not found. The results showed an induction of early cytotoxic effects such as loss of membrane integrity, surface morphological changes and MWCNT agglomerate entrance at all concentrations. We also demonstrated the ability of MWCNTs to induce early genotoxicity. This study emphasizes the suitability of our approach to evaluating simultaneously the early response of the cell membrane and DNA to different MWCNT concentrations and exposure times in cells of target organ. The findings contribute to elucidation of the mechanism by which MWCNTs cause toxic effects in an in vitro experimental model.

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.

A radio frequency (RF) (280 MHz) electron paramagnetic resonance (EPR) spectroscopy and imaging apparatus has been used to localize a pyrrolidine nitroxide free radical in the rat abdomen and thorax. The nitroxide 2,2.5.5,-tetramethylpyrrolidine-1-oxyl-3- carboxylic acid (PCA) had a whole body monoexponential decay with half-life of 13.3 +/- 0.7 (n = 4), 19.4 +/- 0.2 (n = 3), and 23 +/- 2 (n = 6) min for 1, 2, and 3 mmol/kg PCA, respectively. Up to seven one-dimensional longitudinal projections were collected on six rats in the presence of a 8 mT/m field gradient. With an injection dose of 3 mmol/kg, PCA half-lives were 19 +/- 1, 17 +/- 2, and 22 +/- 2 min (n = 6) in the lower abdomen, in the liver, and in the thorax, respectively. Thorax half-life was significantly longer than liver half-life. Sequential two-dimensional images of PCA distribution in a plane longitudinal to the rat body were obtained from eight spectra in the presence of a gradient of 12 mT/m (acquisition time 5 min; spatial resolution 8 mm). After 7 min, the nitroxide was detectable in the left side of the thorax area, but it was mostly localized in the liver. PCA was more uniformly distributed in the image collected after 17 min.

The use of pH-sensitive nitroxides, in conjunction with low-frequency EPR, offers a unique opportunity for non-invasive assessment of pH values (in the range 0 to 14) in living animals. In the present study, we have investigated the potential use of pH-sensitive nitroxide free radicals in conjunction with EPR imaging techniques at low and very low frequencies (280 MHz-2.1 GHz). In particular, we have measured the hyperfine splitting (hfs) of a pH-sensitive probe at three different EPR frequencies: 280 MHz, 1.1 GHz and 2.1 GHz. We have also developed EPR imaging experiments with phantoms simulating in vivo conditions, using pH-sensitive probes at 280 MHz (spatial-spatial) and 1.1 GHz (spectral-spatial). Finally, we discuss the actual sensitivity/resolution limits of the EPR imaging techniques at low frequencies. Practical applications of this method in the biomedical field are suggested for the continuous and non-invasive localization of pH in vivo.