Toxicology in Vitro 29 (2015) 352–362

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Toxicological assessment of multi-walled carbon nanotubes on A549 human lung epithelial cells Giuseppa Visalli a, Maria Paola Bertuccio a, Daniela Iannazzo b, Anna Piperno c, Alessandro Pistone b, Angela Di Pietro a,⇑ a b c

Department of Biomedical Sciences and Morphological and Functional Images, University of Messina, Italy Department of Electronic Engineering, Industrial Chemistry and Engineering, University of Messina, Italy Department of Chemical Sciences, University of Messina, Italy

a r t i c l e

i n f o

Article history: Received 19 May 2014 Accepted 3 December 2014 Available online 10 December 2014 Keywords: Pristine multi-walled carbon nanotubes (pMWCNT) Acid-treated multiwalled carbon nanotubes (MWCNT-COOH) Oxidative damage Genotoxicity Lysosomal and mitochondrial dysfunction

a b s t r a c t An in vitro model resembling the respiratory epithelium was used to investigate the biological response to laboratory-made pristine and functionalised multi-walled carbon nanotubes (pMWCNT and MWCNTCOOH). Cell uptake was analysed by MWCNT-COOH, FITC labelled and the effect of internalisation was evaluated on the endocytic apparatus, mitochondrial compartment and DNA integrity. In the dose range 12.5–100 lg ml1, cytotoxicity and ROS generation were assayed, evaluating the role of iron (the catalyst used in MWCNTs synthesis). We observed a correlation between MWCNTs uptake and lysosomal dysfunction and an inverse relationship between these two parameters and cell viability (P < 0.01). In particular, pristine-MWCNT caused a time- and dose-dependent ROS increase and higher levels of lipid hydroperoxides compared to the controls. Mitochondrial impairment was observed. Conversely to the functionalised MWCNT, higher micronuclei (MNi) frequency was detected in mono- and binucleate pMWCNT-treated cells, underlining an aneugenic effect due to mechanical damage. Based on the physical and chemical features of MWCNTs, several toxicological pathways could be activated in respiratory epithelium upon their inhalation. The biological impacts of nano-needles were imputable to their efficient and very fast uptake and to the resulting mechanical damages in cell compartments. Lysosomal dysfunction was able to trigger further toxic effects. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction The impact of carbon nanotubes (CNTs) on human health has not yet been clarified although their cytotoxicity has been widely

Abbreviations: pMWCNT, pristine multi-walled carbon nanotubes; MWCNTCOOH, acid-treated multiwalled carbon nanotubes; MWCNT-FITC, fluorescein isothiocyanate-functionalized acid-treated multiwalled carbon nanotubes; THF, tetrahydrofuran; ETA, triethylamine; TGA, thermogravimetric analysis; HRTEM, high-resolution transmission electron microscopy; FCS, fetal calf serum; FACS, fluorescence-activated cell sorting; Dwm, mitochondrial transmembrane potential; DCF-DA, 20 ,70 -dichlorofluorescein-diacetate; DPPP, diphenyl-1-pyrenylphosphine; R123, rhodamine 123; AO, acridine orange; FAU, fluorescence arbitrary units; CLSM, confocal laser scanning microscopy; PUFAs, polyunsaturated side chains; FPG, formamidopyrimidine DNA glycosylase; TM, tail moment; CBMN, cytokinesis-block micronucleus; Cyt-B, cytochalasin B; DFX, deferoxamine mesylate; DMSO, dimethyl sulfoxide. ⇑ Corresponding author at: Department of Biomedical Sciences and Morphological and Functional Images, University of Messina, Via C. Valeria, Gazzi 98100, Messina, Italy. Tel.: +39 090 221 3621; fax: +39 090 221 3351. E-mail address: [email protected] (A. Di Pietro). http://dx.doi.org/10.1016/j.tiv.2014.12.004 0887-2333/Ó 2014 Elsevier Ltd. All rights reserved.

studied. Due to their physico-chemical properties, hundreds of tons of CNTs are produced per year (Zhang et al., 2013). Considering the increased number of people accidentally exposed to their inhalation (Castranova et al., 2013), the potential adverse effects of these nanomaterials cannot be ignored. As reviewed by Kayat et al. (2011), in vivo and in vitro studies, performed to analyse the impact on health by CNTs, have led to contradictory results. These may be ascribed to the different methods of production, to the purification processes and to the intrinsic physico-chemical features of CNTs. CNTs, made by almost pure carbon, are extremely hydrophobic and easily aggregate in liquids. This, by varying the bioavailability, makes assessment of risk for human health more difficult. However, the respiratory system is highly susceptible to the inhalation of CNTs that may trigger pulmonary inflammation, cytotoxic effects and apoptosis (Jacobsen et al., 2009). The strong similarities between the pathogenic mechanism induced by multi-walled CNTs MWCNTs and the one caused by asbestos fibres has been highlighted. Both share the needle-like shape, the pro-oxidant capability and the biopersistence (Poland,

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2008). In a susceptible animal model CNTs cause adverse pulmonary effects including alveolitis, pulmonary fibrosis, granulomas and genotoxicity (Muller et al., 2005). The aim of this in vitro study was to investigate the interaction with cells and the bioreactivity of MWCNTs synthesised in our laboratory by catalytic chemical vapour deposition (CCVD). Post synthetic treatments modify various properties which may have an impact on the safety of MWCNTs. These include the aspects ratio (i.e. length/diameter), reactivity of surface area, hydrophilicity, interaction with cells and purity. In order to better hypothesise the potential health impact, we assessed the cytotoxicity of pristine multi-walled CNTs (pMWCNT) and acid-treated multi-walled CNTs (MWCNT-COOH). Conversely to occupational risk due to workplace exposure to both types of CNTs, the general population is exposed almost exclusively to functionalised CNTs. After examining in A549 cells the uptake by using MWCNTCOOH, FITC labelled (MWCNT-FITC), we analysed the intracellular ROS and lipid hydroperoxides to estimate the oxidative damage. We also investigated the effects of MWCNTs on cellular endocytic and mitochondrial compartments and we evaluated the genotoxicity by Comet assay and micronuclei (MNi) test. Moreover, to determine the role of iron, used as a catalyst in MWCNTs synthesis, we employed the chelating agent deferoxamine mesylate (DFX) and DMSO, a HO-‘‘scavenger’’.

2. Materials and methods 2.1. Biochemicals and reagents All chemicals and reagents were obtained from Sigma–Aldrich (Milano-Italia) unless otherwise specified.

2.2. Samples preparation and characterisation pMWCNT was synthesised by catalytic chemical vapour deposition (CCVD) from isobutane on Fe/Al2O3 catalyst and was successively purified (purity >95%) (Donato et al., 2007). MWCNT-COOH sample was prepared by the oxidation of pMWCNT using a mixture of sulphuric acid and nitric acid (1:1) (Pistone et al., 2012). MWCNT-FITC, used to assess the cell uptake, was prepared by carboxylation, acylation, amine modification and fluorescein conjugation, as illustrated in Fig. 1A. Briefly, 100 mg MWCNT-COOH was heated at reflux in 30 ml of neat oxalyl chloride for 48 h; the volatile reagent excess was removed under reduced pressure to yield MWCNT-C(O)Cl. The resulting MWCNT-C(O)Cl was suspended in 10 ml of dry THF and 0.028 ml (0.20 mmol) of ETA and 47 mg (0.19 mmol) of {2-[2-(2-Amino-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl ester was added (Iannazzo et al., 2012). The mixture was left to mix for 12 h; the precipitate was filtered under vacuum through a 0.1 mm Millipore membrane and washed with THF and methanol. The residue was treated for 5 h with 5 ml of HCl 4 M in dioxane, filtered and washed with dioxane and deionised water. The amount of free amino groups, expressed as NH2 loading, was calculated by Kaiser Test and was found to be 0.90 mmol g1. The solid phase was dispersed in dry THF (20 ml) with 10 eq of FITC, sonicated for 30 min in a batch (Bandeling Sonorex Type RK5H), and left to mix at reflux for two days. After removal of the solvent, the solid was filtered (Durapore Membrane Filter 0.1 lm) and washed with CH3OH and CHCl3. MWCNTs samples were characterised by TGA (TA instrument, mod. SDTQ600), UV spectra (Nicolet Evolution 500), scanning electron microscopy (SEM, JEOL JSM 5600LV) and HRTEM (200 kV JEOL JEM 2010) by using LaB6 electron gun equipped with a Gatan 794 Multi-Scan CCD camera).

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2.3. Cells and exposure conditions A549 cells, a human epithelial lung cell line (ATCC, Rockville, USA), were cultured in Ham’s F12 K medium with 2 mM L-glutamine, 10% FCS, 100 IU/ml penicillin and 100 lg/ml streptomycin. Cells were grown at 37 °C in a humidified 5% CO2 atmosphere. Due to their hydrophobicity and large surface area, the examined MWCNTs, and in particular pMWCNT, had a strong propensity to agglomerate in liquid. To minimise the heterogeneous intracellular responses, MWCNT suspensions (100 in PBS) were sonicated for 20 min (frequency 40 kHz). Just before monolayers treatment, the work suspensions, prepared in cell medium with 2% FCS, were further sonicated for 3 min. In each experiment, cell monolayers maintained in the same medium with the addition of PBS instead of the CNT suspensions, were included as negative control. Based on data from the literature and on preliminary tests, we used the concentration interval 12.5–100 lg ml1 to assess dose– effect correlations while, to evaluate the exposure time-effects and the time course of uptake, the dose 50 lg ml1 was used. 2.4. Cytofluorimetric analyses FACS technique (argon laser cytofluorimeter Dako-Galaxy, Denmark), was employed to determine: MWCNT-FITC uptake, viability, intracellular ROS production and lipid hydroperoxides. Moreover, we measured the mitochondrial transmembrane potential (Dwm) and the integrity of the lysosomal compartment. After incubation for the time required in the different experiments, cell suspensions (1  105 cells ml1) were loaded separately with the respective probes. Preliminary tests were carried out in order to select the times and doses of MWCCT exposure which allowed to maintain a population of intact cells comparable to the population of control cells. For the time-course of uptake, aliquots of MWCNT-FITC (50 lg ml1)-treated cells were taken from time 0 to 180 min at intervals of 30 min. To check the fluorescence due to free FITC, a MWCNT-FITC suspension, to the higher dose used in the experiments, was centrifuged and cells were treated with the supernatant. The emission signals were collected in the FL-1 channel. To determine the MWCNTs cytotoxicity in the range 12.5– 100 lg ml1, propidium iodine (3 lg ml1) was used and emission signals were measured in the FL-3 channel. To evaluate intracellular ROS production, DCF-DA (1 lM) was used and the fluorescence ROS-activated was detected in the FL1 channel. Lipid hydroperoxides were detected by using DPPP. The loaded cells were incubated at 37 °C for 3 h and the fluorescent phosphine oxide signal was collected in the FL-1 channel (Di Pietro et al., 2009). Dwm was evaluated by measuring in the FL-2 channel the emission of R123. The fluorochrome (0.2 lM) was added to cell suspensions and incubated at 37 °C for 10 min. The emitted fluorescence was collected in the FL-2 channel (Di Pietro et al., 2011). We assessed integrity of the endocytic apparatus (late endosomes and lysosomes) by employing metachromatic fluorophore acridine orange (AO), which is captured by protons and is collected in the acidic vacuolar compartment. The probe highly concentrated will emit red fluorescence while will release green fluorescence in the cytosol and nucleus where it scarcely accumulates. The loss of red signal and the gain of green fluorescence are indicative of the acid compartment damage. By detections in FL-1 and FL-3 channels we measured AO fluorescence switching, in cell suspensions loaded with AO (5 lg ml1). To estimate lysosomal integrity, further experiments were performed by Lyso-TrackerÒ red probe (Invitrogen). This does not emit

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Fig. 1. MWCNTs synthesis and characterisation. (A) Synthesis of the MWCNT-COOH and MWCNT-FITC samples. Reagent and condition: (a) HNO3/H2SO4; (b) (COCl)2, 2d, reflux; (c) {2-[2-Amino-ethoxy)-ethoxy]-ethyl}-carbamic acid tert-butyl ester, ETA, THF, 12 h; (d) HCl 4 M, 5 h; (e) FITC, THF, 2d, reflux. (B) Comparative study of pMWCNT, MWCNT-COOH and MWCNT-FITC as inferred by TGA (Dm%) at 700 °C under inert atmosphere. (C) TGA profiles of MWCNT-FITC under oxidative conditions. (D) Combined UV spectra for FITC, MWCNT-FITC and MWCNT-COOH samples. (E) EDAX analysis on MWCNT-FITC. (F) SEM image of MWCNT-FITC.

in green fluorescence, allowing a more accurate assessment of MWCNTs-induced damage. Since the sensitivity of photomultipliers is greater in the green than in the red spectral region, AO could amplify the detection of the damage. For each fluorophore used, emission values were corrected by subtracting the values of autofluorescence. Briefly, aliquots of cell suspensions both the MWCNT-treated and controls were subjected to cytofluorimetric analysis in the absence of the probes. In FACS analyses, the weighted averages of emission values for 100 cells were calculated and expressed in FAU. 2.5. Qualitative analyses by optical and confocal microscopy observations To evaluate the MWCNTs uptake and cellular morphological changes, semi-confluent monolayers of A549 were observed by CLSM (TCS-SP2, Leica-Microsystems, Germany). The AO-treated cells were observed also by fluorescence microscopy (Leica DMIRE2).

2.6. Analysis of DNA damage by Comet assay To test DNA integrity, the alkaline version of the Comet-assay was performed (Tice et al., 2000). Moreover, to detect the oxidised purines, we used FPG (Collins et al., 2008). To assess FPG-sensitive sites, after cell lysis, the slides were immersed in fresh enzyme buffer (0.1 M KCl, 0.5 mM EDTA, 40 mM HEPES, and 0.2 mg ml1 bovine serum albumin at pH 8) three times, for 5 min each. Next, a commercial Escherichia coli FPG enzyme (1 lg ml1) or the buffer only, was added (80 ll) and gel spots were incubated in a humidity chamber at 37 °C for 1 h. After DNA unwinding, all slides were electrophoresed for 30 min (0.86 V cm1). Analysis of the slides, stained by ethidium bromide (2 lg ml1) was performed at a 400. The acquired images were submitted to the automated image analysis system CASP. Tail moment values (TM) obtained by the alkaline version indicated the direct DNA damage while oxidative DNA damage was obtained subtracting to TM values of FPG treated cells those of buffer treated cells.

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2.7. Cytogenetic analysis The micronuclei (MNi) frequency was assessed by using CBMN cytome assay. MNi are small, round, DNA-containing bodies observed in interphase cells upon treatment with aneugenic (causing chromosomal malsegregation) or clastogenic (producing chromosome breakage) compounds. Sub-confluent monolayers were exposed to MWCNTs (12.5 lg ml1) for 180 min at 37 °C to allow the MWCNT uptake. Further experiments were performed prolonging the MWCNT treatment for 6 h. Next, medium was removed and monolayers, repeatedly washed with PBS, were re-incubated for 24 h in growth medium containing 3 lg ml1 Cyt-B. The incubation time with Cyt-B allowed the expression of damages induced by intracellular MWCNTs. Cells were harvested and spotted (4  104 cells) onto slides which were fixed in methanol, stained by Giemsa, blindly coded and observed at 400. Unlike the clastogenic mechanism, which can be monitored only after cell division (polynucleate cells), the aneugenic effect is also observed in mononucleate cells (Rosefort et al., 2004). By detecting MNi in both cells, we could obtain further information about the mechanism of genotoxicity without requiring fluorescence in situ hybridisation technique (FISH). Morphological markers of cell death (pyknosis, karyorrhexis, karyolysis) were also recorded. 2.8. Experiments with chelating agent DFX and HO scavenger DMSO We employed the chelating agent DFX to determine the bioavailability of iron, used as catalyst in the MWCNT synthesis. FACS analyses (to assess ROS and endocytic apparatus), and Comet-assay were carried out. The experiments were performed by cell exposure to MWCNTs suspensions alone and to the MWCNTs-DFX mixture. This was obtained by mixing double-concentrated MWCNTs suspensions with 0.3 mM DFX (1:1 ratio). The mixture was stirred for 30 min at room temperature before being added to the monolayers. To assess the role of Fenton-like reactions in the MWCNT genotoxicity, FPG-Comet assay was performed by using the HO-scavenger DMSO that was added (0.1%) to the monolayers just before the MWCNTs exposure. 2.9. Statistical analyses All data are presented as mean ± SEM, based on at least three independent experiments. Significance was accepted at p < 0.05. Comparisons and correlations were performed by Kruskal–Wallis, Mann–Whitney and Spearman test, respectively. 3. Results 3.1. MWCNTs characterisation Comparative data of TGA analysis (Fig. 1B) were calculated at 700 °C by thermal decomposition of the surface groups under inert atmosphere. These revealed a weight loss of 19% for MWCNT-FITC samples and 10% for MWCNT-COOH samples, ascribable to the organic groups. Conversely, pMWCNT did not show any significant weight reduction, indicating the absence of functional groups or other thermally-degradable substances on its surface. By oxidative TGA analysis, we instead estimated the amount of iron. In MWCNTs, after total combustion of the organic fraction, a weight residue equal to 4% remained, due to the inorganic fraction (Fig. 1C). Atomic Absorption Spectroscopy analysis of the residues allowed the quantification of an amount of iron close to 3.5–4.0%.

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The UV spectra of unconjugated-FITC, MWCNT-COOH and MWCNT-FITC and were determined (Fig. 1D). Unconjugated FITC showed absorbance at 460 and 485 nm, MWCNT-FITC samples had an evident peak at 493 nm, whereas MWCNT-COOH samples, lacking the fluorochrome, did not present any absorbance between 400 and 600 nm. Sulphur atoms due to fluorescein was detected by the EDAX spectrum of MWCNT-FITC (Fig. 1E). The SEM image of rough MWCNT-FITC samples (Fig. 1F) indicates bundles of nanotubes. HRTEM images of MWCNTs (Fig. 2) show that pMWCNT was entangled and randomly oriented with the outer surface smooth and well graphitised. A multi-walled tube structure of 15–20 layers is shown and the graphite layers did not always stay continuous along the growth orientation, showing point defects. Moreover, pMWCNT had an average length of 10–20 lm and a diameter close to 15–30 nm. MWCNT-COOH samples appeared dispersed and considerably shortened compared to pMWCNT (average length between 200 and 1000 nm). MWCNT-COOH showed an erosion of external layer in many points as a result of the oxidative insertion of functional groups. MWCNT-FITC exhibited amorphous material often covering MWCNTs, due to organic residue bound on the COOH groups.

3.2. Kinetic of MWCNTs uptake by A549 cells and cell viability MWCNTs uptake or adsorption on cellular membranes was examined by CLSM (Fig. 3A–C). In contrast to PBS-treated cells (A), A549 monolayers exposed for 180 min either to oxidised (B) or pMWCNT (C) (50 lg ml1), showed intracellular aggregates. These appeared bulkier and more numerous in pMWCNT- than in MWCNT-COOH-treated cells. To quantify MWCNTs uptake and cell viability, A549 monolayers were treated with 50 lg ml1 of MWCNT-FITC. Samples were collected at 0, 30, 60, 90, 120, 150, and 180 min and examined by FACS analysis. The emission values increased with the exposure time and were mainly due to FITC labelled CNTs since in the cells exposed to MWCNT-FITC supernatant the fluorescence was tenfold lower (Fig. 3D). In these cells the emission values did not increase over time, which was in contrast to the results from MWCNT-treated cells. These, already after 30 min, showed significantly higher values compared to t 0 values (P < 0.05). The emitted fluorescence continued to increase with time and the maximum value was recorded at 90–120 min of exposure (1100 FAU/100 cells). At 180 min, despite the curve still shifting towards higher values, the weighted average of FAU (1000) decreased. This was probably due to the break-up of cells with the highest uptake. The kinetic of MWCNTs uptake indicated the high speed of the cells-nanotubes interaction that was observable by CLSM analysis (Fig. 3E). Compared to negative control and supernatant-treated cells, only FITC-conjugated MWCNTs treated cells were strongly fluorescent. Furthermore the comparison of the images (Fig. 3E and B) highlights the strong similarity of the treated cells with MWCNT-COOH and MWCNT-FITC respectively. Conversely to the trend of the MWCNT-FITC internalisation, the cell viability was of 100% at t 0 and dropped to less than 30% at 180 min (Fig. 3F). The inverse relationship between MWCNT-FITC uptake and viability (r = 0.983; P < 0.01) strongly suggested the harmful effect of the dose tested, already after a short exposure. Fig. 3G shows the dose–response curves of viability obtained after exposure for 60 min to CNTs (range 12.5–100 lg ml1). On the basis of dose–effect curves, we used 50 lg ml1 for the experiments with a single exposure dose to highlight a more complete range of effects MWCNTs-induced.

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Fig. 2. HRTEM images of pMWCNT, MWCNT-COOH and MWCNT-FITC. Low and high magnification images are displayed on the left and right side. The bar is equal to 20 nm.

3.3. MWCNTs-mediated cytotoxicity: Membrane destabilization of acidic vacuolar compartment in exposed cells The effects on the endocytic compartment of exposed cells for 120 and 180 min were studied by AO. Conversely to PBS-treated cells (Fig. 4A), which showed a rich set of intact lysosomes, the cells exposed both to pristine or oxidised MWCNTs (50 lg ml1) clearly revealed lysosomal damage. After 180 min of exposure, we highlighted a diffuse green cytosolic fluorescence (Fig. 4B and C) that was associated with the morphological changes of cell death, as pyknosis and karyorrhexis. Damage was more pronounced in pMWCNT-treated cells, several of which emitted (Fig. 4C) only green fluorescence and showed blebs surrounding the cells. AO emission was also determined by quantitative cytofluorimetric analyses. Calculating the ratios FL3/FL1 emission values, we observed a progressive significant decrease (Fig. 4-Table). Even if pMWCNT-treated cells showed a more pronounced lysosomal membrane permeabilisation, no significant differences were recorded in comparison with MWCNT-COOH.

Dose–effect assessment in endocytic compartment was performed also by employing Lyso-TrackerÒ red probe (Fig. 4D). In the range 12.5–100 lg ml1 a significant dose-dependent response was observed (r = 0.955 for pristine and r = 0.899 for oxidised; P < 0.01). At the highest dose, the percentage of intact acid apparatus was 48.5 and 58.7 for pMWCNT and MWCNT-COOH. For the same dose and exposure time, the values detected by this probe were about a half and one third for pMWCNT and MWCNT-COOH in comparison with the AO detected values and were more in agreement with the viability values. 3.4. MWCNTs-mediated cytotoxicity: ROS generation, lipid peroxidation, mitochondrial impairment To assess the relationship among MWCNTs uptake, cell viability and oxidative damage, a time course analysis of ROS in untreatedand treated-cells (50 lg ml1 of MWCNTs) was performed (Fig. 5A and B). Internalisation of both nanotubes induced a time-dependent increase of emission values. Compared to controls, at 30 min (Fig. 5A) the FAU were higher than 2- and 4-fold in oxidised

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Fig. 3. MWCNTs uptake (qualitative and quantitative assessment) and cell viability. (A–C) Images of A549 semiconfluent monolayers by transmission microscopy after exposure for 180 min to MWCNTs (50 lg ml1). (A) Untreated, (B) MWCNT-COOH- and (C) pMWCNT-treated cells. (D). Time-course of MWCNT-FITC uptake (50 lg ml1) assessed by FACS analysis. To check the background values due to free FITC probe, the cells were treated for 30 min with MWCNT-FITC supernatant (the curves are constructed with data from three independent experiments). (E) Fluorescence images under CLSM showing the MWCNT-FITC uptake (50 lg ml1) after 60 min (3) compared to untreated (1) and FTC-treated cells (2). (F) Relationship between MWCNT-FITC uptake and cell viability (r = 0.983; P < 0.01). (G) Dose–response curves to value viability in MWCNTs exposed cells (60 min; range 12.5–100 lg ml1).

and pristine MWCNTs-treated cells (P < 0.01). At 180 min (Fig. 5B), the kinetic analysis showed a progressive increase of ROS production. For all of the examined time points (the data obtained at intermediate times are not shown), pMWCNT showed higher pro-oxidant capability compared to MWCNT-COOH. To evaluate the effects of ROS overproduction on membrane integrity, we measured lipid hydroperoxides that are the major early reaction product of lipid peroxidation. In cells treated with both MWCNT types (50 lg ml1) we detected an increased percentage of lipid hydroperoxides in comparison to the control at each time point (Fig. 5C). The trend closely followed the ROS production curve (r = 0.989 and 0.991 in pristine- and oxidisedMWCNTs). Fig. 5D illustrates the percentage decrease of Dwm in MWCNTsexposed cells for 60 min, compared to control (100). The observed

mitochondrial failure emphasised the metabolic impairment MWCNTs-induced. In the range 12.5–100 lg ml1 we underlined the dose-dependent effect (r = 0.889 and 0.851 in pristineand oxidised-nanotubes; P < 0.05). At the highest dose, Dwm values were about half, making the differences significant in comparison to control (P < 0.05). 3.5. MWCNTs-mediated genotoxicity: assessment by Comet assay and MNi test We examined the DNA damage by Comet-assay, treating the cells to 12.5 lg ml1for 60 min to prevent possible interference due to the electrophoretic DNA mobility of the dead cells. In the alkaline version of the Comet-assay, the sensitivity is mainly ascribed to the detection of abasic (alkali-labile) sites, produced

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Fig. 4. Membrane destabilization of acidic vacuolar compartment in MWCNTs exposed cells. (A–C) Images of AO-loaded cells (400 magnifications) after exposure for 180 min. (A) Untreated cells showing intact lysosomes. (B) and (C) A549 treated with MWCNT-COOH and pMWCNT (50 lg ml1) respectively. Diffuse green cytosolic fluorescence accompanied by morphological changes, typical of apoptosis and/or necrosis, highlight the damage to late endosomes and lysosomes. In C image, intact lysosomes are not apparent while blebs and karyorrhexis is evident. Table – results of quantitative analyses of cell exposed to MWCNTs and AO-loaded. The values indicate the relative membrane integrity (RMI) of endocytic compartment, expressed as percentage of the ratio FL3/FL1 values, in comparison to the control (100). (D) Acidic organelle integrity, in comparison to the control (100), assessed by FACS analysis of cells loaded by LysoTrackerÒ red, after 120 min of MWCNTs exposure. ⁄P < 0.05, and ⁄⁄P < 0.01, as compared with controls. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

by DNA glycosylases during the repair mechanisms to restore the double-strand integrity. To further amplify the sensitivity, we used FPG restriction enzymes to catalyse the oxidised purines excision. We evaluated the direct DNA damage by the alkaline version of Comet-assay and in Fig. 6A are reported the interquartile range of TM. The obtained values increased by 3- and 2-fold in cells treated with pristine- and oxidised-nanotubes (P < 0.01), compared to untreated cells. Following the FPG treatment we assessed more specifically oxidative DNA damage. As shown in Fig. 6A that reports the values achieved by subtracting the baseline damage, the significance was maintained for both the nanomaterials (P < 0.01). To estimate the MWCNTs genotoxicity applying a more predictive marker of effect and differentiating between aneugenic and clastogenic effects, we evaluated MNi induction in mononucleate and multinucleate cells. Fig. 6B reports the results of cytogenetic analysis after exposure to 12.5 lg ml1. MNi frequencies in pMWCNT-treated cells was significantly higher than the control (P < 0.01) and showed comparable values between mono- and

binucleate cells. Conversely, no significant differences were observed in MWCNT-COOH-exposed cells compared to the control. Besides the high MNi induction, pMWCNT exposure caused a strong cytotoxicity as highlighted by pyknosis and karyorrhexis values that were always significantly higher than in PBS- and oxidised MWCNT-treated cells. Similar results were obtained prolonging the MWCNT treatment for 6 h. For both CNTs, only pyknosis, karyorrhexis and karyolysis showed no significant further increases while a weak rise of MNi frequency was observed solely in p-MWCNT treated cells (data not shown)’’. 3.6. Experiments with chelating agent DFX and HO scavenger DMSO The oxidative role of iron, used as catalyst during the nanotubes production, was verified by exposing cells to MWCNTs-DFX mixture and by examining them by FACS analysis and Comet assay. The chelating treatment of MWCNTs completely inhibited the ROS overproduction in the exposed cells. After 120 min of exposure to 50 lg ml1 (Fig. 7A), DCF-fluorescence was reduced by 2- and

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Fig. 5. ROS production and oxidative damages MWCNT-induced. (A and B) Time course of ROS production in PBS and MWCNTs (50 lg ml1) treated cells for 30 min. (A), and180 min (B). The curves relative to 60 and 120 min are not shown. The shift of the curves towards higher emission values showed the progressive ROS increase. (C) Percentage increases of lipid hydroperoxides, compared to controls (0%), in cell treated with 50 lg ml1 of MWCNTs at different incubation times. (D) Percentage decreases of Dwm, compared to controls (100%), in cells exposed to MWCNTs (range 12.5–100 lg ml1). ⁄P < 0.05, and ⁄⁄P < 0.01, as compared with controls.

4-times in cells treated with the MWCNTs-DFX mixture of oxidised- and pristine-nanotubes (P < 0.01 in comparison to MWCNTs alone treated cells). These values overlapped with the DCF-fluorescence detected in controls and were consistent with the Comet assay that showed a complete inhibition of DNA damage induced by MWCNTs (P < 0.01 in comparison to MWCNTs alone treated cells) (data not shown). Conversely, the presence of DFX did not decrease the membrane permeabilisation of acid organelles caused by both nanotubes (Fig. 7B and C). The FL1 and FL3 emission values were similar in cells exposed to DFX mixture and to MWNTCs alone, underlining that the damage to the endocytic compartment was not irondependent. The FPG-Comet assay, by using the HO-scavenger DMSO in the MWCNTs-exposed cells, confirmed that the DNA oxidative damage was due to Fenton reactions catalysed by the iron (Fig. 7D). The values obtained in DMSO pre-treated cells exposed for 60 min to the MWCNTs (50 lg ml1) were similar to negative controls, formed by cells treated with DMSO alone. 4. Discussion

Fig. 6. MWCNTs-mediated genotoxicity. (A) DNA damage triggered by MWCNTs assessed by Comet-assay (alkaline version to evaluate basal DNA damage and after FPG-treatment to evaluate DNA oxidative damage) in cells exposed for 60 min (12.5 lg ml1). The graph reports the interquartile range and the median values of TM. DNA oxidative damage was obtained subtracting to TM values of FPG treated cells those of buffer treated cells. (B) MNi frequency in mononucleate and in multinucleated cells and the morphological markers of cell death in control and MWCNTs-treated cells (12.5 lg ml1 for 180 min). ⁄P < 0.05, and ⁄⁄P < 0.01, as compared with controls.

To improve the knowledge on the safety of MWCNTs, we used an in vitro model resembling the respiratory epithelium. We studied the interaction with the cells and the bioreactivity of pristineand COOH functionalised-MWCNTs, synthesised and characterised in our laboratory. Similarly to the airborne ultrafine matter, the effects of these synthetic particles on the cells may be attributed to physical and chemical features whose influence on the MWCNTs toxicity is still rather contradictory. Some authors showed that functional groups increase the cytotoxicity (Magrez et al., 2006; Ursini et al., 2012), whereas others, similarly to us observed that sidewall functionalisation seems to improve CNTs biocompatibility (Bottini et al., 2006; Muller et al., 2008). The different behaviour could be due to the higher surface hydrophobicity of the pMWCNT

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Fig. 7. Effects of chelating agent DFX and HO scavenger DMSO on redox imbalance and lysosomal damages. (A) ROS production after exposure for 180 min in cells treated with MWCNTs alone (50 lg ml1) and with the mixture MWCNTs-DFX. (B and C) Membrane permeabilization of acid organelles in negative control and in cells exposed to MWCNTs and to MWCNTs-DFX mixtures (50 lg ml1). Each bar shows the ratio FL3/FL1 values of AO. (B) after 120 min. (C) after 180 min. (D) FPG-Comet assay by using the  HO-scavenger DMSO in the MWCNTs-exposed cells. Negative control was formed by cells treated with DMSO alone. DNA oxidative damage was obtained subtracting to TM values of FPG treated cells those of buffer treated cells.

that favours their interaction with cell membranes. In addition, carboxylation of MWCNTs, significantly improving their suspension and reducing agglomeration, further reduces the CNT-cell interactions. Moreover, like MWCNTs-induced phlogosis (Poland, 2008) directly related on the aspect ratio, cytotoxicity may also depend on the length/diameter ratio. Acidic oxidative treatment significantly reduced the length of CNTs and our pMWCNT was at least one order of magnitude longer than MWCNT-COOH (length 10– 20 lm vs. 200–1000 nm). We revealed that MWCNTs probably enter the cells by endocytosis. Moreover, we showed that the kinetic of uptake was very fast and that the internalisation was time-dependent. The capability of A549 cells to internalise MWCNT through hole in the plasma membrane was described by Cavallo et al. (2012). Moreover, CNTs cellular uptake may occur through passive diffusion (nanopenetration) or by the mechanisms of endocytosis/phagocytosis (Firme and Bandaru, 2010). However, the efficient uptake of nano-needles depends on their hydrophobicity (Antonelli et al., 2010) and in A549 cell line the endocytosis is caveolae and/or clathrin-induced (Guo et al., 2012). We observed that MWCNTs internalisation caused time-dependent lysosomal intracellular dysfunction, more pronounced in cells treated with pMWCNT. Lysosomal damage was inversely related to the cell viability, thus underlining the role of endocytic dysfunction in the CNTs pathogenicity, previously observed in keratinocytes (Riviere et al., 2005). Changes in lysosomal permeability lead to the release of hydrolytic enzymes in the cytoplasm, triggering apoptosis. We underlined that the fast lysosomal membrane destabilisation was associated with the loss of Dwm, similarly to what was observed by Sohaebuddin et al. (2010). In addition, we characterised the A549 cell response to both types of MWCNTs by examining ROS production, lipid peroxidation and mitochondrial impairment. Oxidative stress is a common

mechanism involved in the cytotoxicity of CNTs (Garza et al., 2008; Ursini et al., 2012). Our MWCNTs caused a rapid timedependent ROS increase, which was more evident in pMWCNT exposed cells. Consistently with the ROS overproduction, we detected significant time- as well as dose-dependent increases in lipid hydroperoxides, early markers of lipid peroxidation. This strongly highlighted the pathogenic pathway of redox imbalance on pulmonary epithelium. Mitochondrial dysfunction may be due to the lipid peroxidation MWCNTs-induced. PUFAs, present in the mitochondrial membrane, are particularly susceptible to free radical-initiated oxidation which determines Dwm decrease and metabolic impairment because of the shift from oxidative phosphorylation to anaerobic glycolysis (Wei et al., 2009). Due to the bioenergetic phenotype of A549 (Wu et al., 2007) (i.e. high glycolysis rate), we could verify a significant mitochondrial impairment only at the highest dose assayed. The increased residence time of electrons in complexes I and III of the respiratory chain (Trifunovic and Larsson, 2008), causes a Dwm decrease that can further amplify the redox imbalance MWCNT-induced, through the production of endogenous ROS. Mitochondrial damage, by releasing caspase-activating proteins, can trigger the intrinsic apoptotic pathway, as observed in the same cell line exposed to airborne particles (Upadhyay et al., 2003), to asbestos (Kamp et al., 2002) and to oil fly ash (Di Pietro et al., 2011). Besides the significant direct DNA damage, by using the FPG, we confirmed the oxidative DNA breakage, previously observed (Migliore et al., 2010; Kato et al., 2013). The early DNA damage highlights the carcinogenic potential of MWCNTs in lung cells. Efficient repair mechanisms are able to re-establish the macromolecule integrity in most cases. This is congruent with the lack of increase in MNi frequency observed by us in MWCNT-COOH-treated cells, therefore hypothesising that the functionalised MWCNTinduced DNA breakage is poorly related to potential carcinogene-

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sis. On the contrary, pristine nanotubes showed a significant genotoxicity, as observed by Kato et al. (2013) and by Migliore et al. (2010). Rather than oxidative DNA damage, the pMWCNT-induced genotoxicity could be due to an alternative mechanism. The significant MNi presence, also in mononucleated cells, strongly suggests aneugenic effects caused by mechanical damage. The bulky pristine nanotubes, due to their needle-shape can cause irreversible nucleus damages in addition to disruption of the mitotic spindle or kinetochore proteins. This genomic instability, transmitted to daughter cells, reinforces the strong similarity between pMWCNT and asbestos in causing pathogenic and carcinogenic mechanisms (Donaldson et al., 2013). The observed ROS overproduction can be ascribed to the large surface area of MWCNTs. However, the toxicological assessments of CNTs should take into consideration the effect of redox-active iron, used as a catalyst during their synthesis and present as metallic clusters, entrapped within the pMWCNT, or as metal residues in their surface (Bello et al., 2010). We demonstrated the pivotal role of iron in MWCNTs-induced cytotoxicity and genotoxicity and we highlighted the HO production via metal-dependent Fenton reactions. Compared to CNTs used by Pulskamp et al. (2007) and by Shvedova et al. (2003) which contained 30% and 26%, in our MWCNTs, the iron level was low (3.5–4.0%). At CNTs doses tested, the metal amounts were not sufficient to determine the pro-oxidant effects observed. This leads us to attribute the redox imbalance, entirely or partially, to intralysosomal low-molecular-mass iron. This redox-active iron, produced during autophagocytic degradation of metalloproteins such as cytochromes (Antunes et al., 2001), is released as a result of lysosomal damage causing the observed redox imbalance. 5. Conclusion Our home-made MWCNTs trigger several toxicological pathways that could be activated in respiratory epithelium. Upon inhalation, the biological impacts of nano-needles are mainly imputable to their efficient uptake and to the resulting mechanical damages. In particular, a key role has the lysosomal dysfunction, able to trigger further toxicity. This emphasises the need to improve the harmless use of these engineered nanomaterials without any health impacts, as yet recorded for asbestos. Funding The study was supported by a grant from Department of Hygiene, University of Messina, (Fondi di Dipartimento 2010). Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.

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