Non-functionalized multi-walled carbon nanotubes alter the paracellular permeability of human airway epithelial cells

Toxicology Letters 178 (2008) 95–102

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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet

Non-functionalized multi-walled carbon nanotubes alter the paracellular permeability of human airway epithelial cells Bianca Maria Rotoli a , Ovidio Bussolati a,∗ , Massimiliano G. Bianchi a , Amelia Barilli a , Chidambara Balasubramanian c , Stefano Bellucci c , Enrico Bergamaschi b a

Department of Experimental Medicine, Unit of General and Clinical Pathology, University of Parma, via Volturno 14, 43100 Parma, Italy Department of Clinical Medicine, Nephrology and Health Sciences, Unit of Occupational Medicine, University of Parma, Parma, Italy c INFN, Frascati, Rome, Italy b

a r t i c l e

i n f o

Article history: Received 14 January 2008 Received in revised form 26 February 2008 Accepted 26 February 2008 Available online 4 March 2008 Keywords: Tight junctions Carbon nanotubes Airway epithelium Fibrosis Permeability

a b s t r a c t Little information is available upon the effects of carbon nanotubes (CNT) on the airway barrier. Here we study the barrier function of Calu-3 human airway epithelial cells, grown on permeable filters, after the exposure to commercial single-walled or multi-walled CNT, produced through chemical vapour deposition. To assess changes in the paracellular permeability of CNT-treated Calu-3 monolayers, we have measured the trans-epithelial electrical resistance (TEER) and the permeability to mannitol. Multi-walled CNT caused a large decrease in TEER and an increase in mannitol permeability but no substantial alteration in monolayer viability. Single-walled CNT produced much smaller changes of TEER while CNT, synthesized through the arc discharge method, and Carbon Black nanoparticles had no effect. If commercial multiwalled CNT were added during the formation of the tight monolayer, no further increase in trans-epithelial resistance was observed. Moreover, the same nanomaterials, but neither single-walled counterparts nor Carbon Black, prevented the TEER recovery observed after the discontinuation of interleukin-4, a Th2 cytokine that causes a reversible barrier dysfunction in airway epithelia. These findings suggest that commercial multi-walled CNT interfere with the formation and the maintenance of tight junctional complexes in airway epithelial cells. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The potential risks of exposure to nanoparticles (NP) or nanofibers are not predictable a priori from the bulk physicochemical characteristics of these materials. Indeed, due to their size and high surface/volume ratio, nanostructured materials have specific properties that increase their industrial value but also point to the needing for specific toxicological studies on the single nanomaterial (Oberdorster et al., 2005). To this purpose, due to the high costs associated to in vivo experiments, in vitro approaches are being developed to yield inexpensive and efficient assessments of biological effects of nanomaterials. Among nanomaterials of industrial relevance, carbon nanotubes (CNT) are produced in increasing amounts for several industrial

Abbreviations: AD, arc discharge; CB, Carbon Black; CNT, carbon nanotubes; CVD, chemical vapour deposition; EMEM, Eagle’s minimum essential medium; HUVECs, human endothelial cells from umbilical veins; IL-4, interleukin-4; MWCNT, multiwalled carbon nanotubes; NM, nanomaterials; PBS, phosphate-buffered saline; SWCNT, single-walled carbon nanotubes; TEER, trans-epithelial electrical resistance. ∗ Corresponding author. Tel.: +39 0521 033783; fax: +39 0521 033742. E-mail address: [email protected] (O. Bussolati). 0378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2008.02.007

applications, because these structures are endowed with extremely advantageous chemical and mechanical features. Moreover, it is relatively easy to functionalize CNT with various chemical groups, so as to render them efficient carriers for chemicals and drugs (Banerjee et al., 2003; Bottini et al., 2005, 2006a,c; Donaldson et al., 2006; Lam et al., 2006). At present, CNT production occurs through three distinct processes: chemical vapour deposition (CVD), the major process of industrial importance, arc discharge (AD), and laser ablation. The two latter processes lead to the production of CNT less contaminated with metals but are largely restricted to laboratories for research use. Multi-walled CNT (MWCNT) can be produced without metals, although the presence of small amount of metal catalysts helps to align the nanotubes; an increase in the metal nanoparticles-to-carbon ratio favors the formation of singlewalled CNT (SWCNT) (Iijima and Ichihashi, 1993). The consequences of the interaction of CNT with biological structures are not completely known and their potentially toxic effects are still object of debate (Donaldson et al., 2006; Lam et al., 2006; Helland et al., 2007). The available information, obtained mostly in rodents, points to lung toxicity as the major consequence of exposure to CNT, with the early formation of fibrosis and granulomas, hypertrophy of epithelial cells, and the ensuing of functional impairment (Lam et al., 2004; Warheit et al., 2004; Muller et

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al., 2005; Shvedova et al., 2005). Interestingly, these pathological changes are observed with SWCNT but not with Ultrafine Carbon Black (CB) used at comparable mass doses (Lam et al., 2004; Shvedova et al., 2005). Although several toxicological studies have investigated CNT effects on various cell models in vitro (Shvedova et al., 2003; Cui et al., 2005; Monteiro-Riviere et al., 2005; Bottini et al., 2006b; De Nicola et al., 2007; Zhang et al., 2007), the consequences of CNT exposure on airway cells are only incompletely known. In particular, SWCNT are more toxic than MWCNT for alveolar macrophages (Jia et al., 2005), while, most recently, rat alveolar epithelial cells have been described to exhibit no cytotoxic effect after exposure to SWCNT (Yacobi et al., 2007). Airway epithelial cells represent the first body barrier for inhaled particles and constitute a major determinant of the interaction of the potentially toxic compound with other compartments of the organism. The barrier function of these cells is due to the formation and maintenance of tight junctional complexes that allow strictly polarized secretory functions and prevent the entry of xenobiotics and pathogens, lowering the paracellular permeability of the epithelium to very low levels. Tight junctions are dynamic multiprotein structures, which exhibit a peculiar sensitivity to cytokines, toxins, and pathogens (see for review Schneeberger and Lynch, 2004; Aijaz et al., 2006; Shin et al., 2006). On the other hand, alterations in the paracellular permeability of airway epithelia have been implicated in enhanced sensitivity to pathogens or exaggerated inflammatory reactions (Reed and Kita, 2004; Landau, 2006). Airway epithelial cells grown in culture maintain the capability to form tight junctional complexes. Therefore, in vitro cell models of airway epithelium are currently adopted to predict the behaviour of the respiratory barrier in vivo (Sakagami, 2006). We have used one of these models, the human epithelial cell line Calu-3, to assess the consequences of the exposure to various types of CNT on the paracellular permeability of the airway epithelial cells. We have found that commercial MWCNT and, at a much lesser degree, SWCNT impair the barrier function of airway epithelial cell monolayers. Neither nanotubes synthesized with the arc discharge method (ADCNT), nor amorphous carbon nanoparticles exhibit the same effects.

before use. Human endothelial cells from umbilical veins (HUVECs) were obtained and cultured in Medium 199 supplemented with 20% FBS, heparin and endothelial cell growth supplement, as previously described (Visigalli et al., 2007). Images of cells in phase contrast were obtained with a Nikon DS5MC digital camera (Nikon Instruments SpA, Firenze, Italy). 2.2. Characterization of CNT Commercial multi-walled (MWCNT, Aldrich 659258) and single-walled (SWCNT, Aldrich 636797) nanotubes were obtained from Sigma–Aldrich, Milan, Italy. A mixture of single-walled and multi-walled CNT was obtained with the arc discharge method (AD-CNT) at the laboratories of Istituto Nazionale di Fisica Nucleare (INFN) Frascati, Rome (Bellucci et al., 2007a,b). Ultrafine Carbon Black nanoparticles (Printex 90TM , 14 nm diameter) were a generous gift of Degussa Italia SpA, Advanced Fillers & Pigments, Ravenna, Italy. The characteristics of the commercial nanotubes, as declared by the manufacturer, are as follows: MWCNT, Aldrich 659258 is largely made of multi-walled nanotubes (at least 90%), with residual amorphous carbon (metal contaminants, i.e. mostly iron, amount to less than 0.1%); the purity of commercial single-walled nanotubes, Aldrich 636797 is only >50%, where the impurities consist approximately of a 40% amount of other types of nanotubes, 3% amorphous carbon and traces of metals. In order to obtain a determination of the amount of possible contaminants in the AD-CNT samples, we carried out their elemental analysis using a JEOL JEM 2010 TEM microscope. The results indicate that only a Si contamination was detectable, while the AD-CNT samples were totally metal-free. The main characteristics of the CNT used are summarized in Table 1. 2.3. Exposure to CNT and CB Before the experiments all the nanomaterials were heated at 220 ◦ C for 3 h to eliminate possible contamination from lipopolysaccharide. After cooling at room temperature, nanomaterials were dispersed at a concentration of 1 mg/ml in sterile phosphate-buffered saline (PBS) to obtain a stock suspension for a series of experiments. Immediately before the single experiments, nanomaterials were extensively vortexed, sonicated three times for 15 min in a Bransonic Ultrasonic ultrasound bath and then added to normal growth medium to reach the desired concentration. No detergent was used to improve the solubility of nanomaterials in aqueous solutions. After the addition of nanomaterials to culture medium, they rapidly tend to precipitate and to form more or less expanded aggregates that come into contact with the cell monolayer (see the representative image shown in Fig. 5). Given the volume of the medium in the apical chamber and the filter surface, Calu-3 monolayers, grown in cell culture inserts for Falcon 24-well-multitrays and incubated with a nominal MWCNT concentration of 100 ␮g/ml, were exposed to 75 ␮g of nanomaterials per cm2 of monolayer, roughly corresponding to a maximum of 0.3 ng/cell. 2.4. Measurements of trans-epithelial resistance and paracellular permeability

2. Materials and methods 2.1. Cells and experimental treatments Calu-3 cells, obtained from a human lung adenocarcinoma and derived from serous cells of proximal bronchial airways (Finkbeiner et al., 1993), were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia (Brescia, Italy). Cells were routinely cultured in 10-cm diameter dishes in Eagle’s Minimum Essential Medium (EMEM) supplemented with 1 mM sodium pyruvate, 10% FBS, streptomycin (100 ␮g/ml) and penicillin (100 U/ml) in a humidified atmosphere of 5% CO2 in air. For the experiments, Calu-3 cells were seeded into cell culture inserts with membrane filters (pore size of 0.4 ␮m) for Falcon 24-well-multitrays (Cat. N◦ 3095, Becton, Dickinson & Company, Franklin Lakes, NJ, USA), at a density of 75 × 103 cells/300 ␮l. Cells were allowed to grow for 10–14 d, as detailed for the single experiments. NM were added in the apical chamber from a 1 mg/ml stock solution without changing the medium, while basolateral chamber was used for IL-4 supplementation. THP-1 macrophages were obtained from the Istituto Zooprofilattico Sperimentale della Lombardia, cultured in RPMI1640 medium supplemented with 10% FBS and antibiotics, and differentiated for 3 d with 50 ng/ml phorbol-12,13-myristate

Measurements of trans-epithelial electrical resistance (TEER) were made on monolayers of Calu-3 cells, grown for 10–14 d on permeable supports (Falcon), using an epithelial voltohmeter (EVOM, World Precision Instruments Inc., Sarasota, FL, USA). Changes in the paracellular permeability to extracellular solutes were assessed with measurements of the apical-to-basolateral flux of mannitol, as previously described (Rotoli et al., 2000). d-[1-14 C]-Mannitol was added at a final concentration of 0.1 mM and at the activity of 5 ␮Ci/ml to the apical side. At the indicated times, aliquots of 50 ␮l were sampled from the basolateral compartment to determine the radioactivity with a liquid scintillation spectrometer Wallac Trilux2 (Perkin Elmer SpA, Monza, Italy). 2.5. Cell viability Cell viability was tested with the resazurin method (O’Brien et al., 2000). According to this method, viable cells reduce the non-fluorescent compound resazurin into the fluorescent resorufin that accumulates into the medium. Since the culture inserts did not allow direct fluorescence reading from the wells, these experiments were performed on cells cultured on standard plasticware. Cells were incubated for 1 h

Table 1 Principal characteristics of CNT preparations Type

Dimensions (diameter × length)

MWCNT SWCNT AD-CNTa

110–170 nm × 5–9 ␮m 0.7–1.2 nm × 2–20 ␮m 10–40 nm × 1–5 ␮m

Density (g/cm3 )

Surface area (m2 /g)

Preparation method

1.35 2.25 ND

1.3 × 10 1.7 × 103 ND

CVD CVD Electric arc discharge

2

ND, not determined. a AD-CNT are a mixture of, approximately, 30% SWCNT, 50% MWCNT, and 20% amorphous C and fullerene. The dimensions refer to the MWCNT component.

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with fresh, serum-free medium supplemented with 44 ␮M resazurin; fluorescence was then measured at 572 nm with a fluorimeter (Wallac 1420 Victor2 Multilabel Counter, Perkin Elmer). 2.6. Statistical analysis Values of TEER of control and NM-treated cultures were compared with a t-test for unpaired data. 2.7. Materials Fetal bovine serum and culture media were purchased from Euroclone (Celbio, Pero, Milan, Italy). Recombinant human interleukin-4 (rIL-4) was obtained from Vinci-Biochem, Firenze, Italy. [14 C]Mannitol (51 mCi/mmol) was obtained from Perkin Elmer Life and Analytical Sciences, Boston, MA, USA. Sigma–Aldrich (Milan, Italy) was the source of all other chemicals.

3. Results 3.1. Effects of CNT on the paracellular permeability of Calu-3 cell monolayers When seeded on permeable filters in a two-chamber culture system, Calu-3 cells spend 8–10 d to reach confluence. After this period TEER progressively increases up to 13–15 d and reaches values as high as 2000  cm2 . The addition of MWCNT, SWCNT, AD-CNT, or CB, at a concentration of 100 ␮g/ml, to the apical chamber of tight Calu-3 cell monolayers (TEER ≥ 1000  cm2 ) had very different effects on TEER (Fig. 1, Panel A). AD-CNT and CB were completely ineffective and the TEER of monolayers exposed to these nanomaterials (NM) was not significantly different from that of control monolayers. In contrast, MWCNT produced a progressive decrease in TEER. The decrease was already detectable after 3 d of incubation and, after 6 d, the TEER value was less than 30% of the control TEER, determined at the same time in untreated monolayers. At later times of treatment (>4 d), also commercial SWCNT produced a statistically significant decrease of TEER, although at a much lesser degree than MWCNT. The effect of increasing concentrations of MWCNT on the TEER of tight monolayers was monitored for 4 d (Fig. 1, Panel B). Starting from Day 2 of treatment, concentrations as low as 10 ␮g/ml prevented further increases of TEER, so as to yield TEER values sig-

Fig. 2. MWCNT increase monolayer permeability to mannitol. Calu-3 cells were cultured for 10 d on membrane filters. At the end of this period, monolayers were incubated for 4 d in the absence or in the presence of 100 ␮g/ml MWCNT. d[1-14 C]-Mannitol (0.1 mM, 5 ␮Ci/ml) was then added to the apical chamber and 50 ␮l-aliquots of the basolateral medium were sampled at the indicated times and counted for radioactivity. Three filters were used for each condition. Data are means ± S.D. Lines represent the best-fit linear regression of the mean values. R2 = 0.909 and 0.908 for control and MWCNT-treated monolayers, respectively. A representative experiment, performed three times with comparable results, is shown.

nificantly lower than control, while even 5 ␮g/ml were able to lower TEER at Day 4. At a nominal concentration of 50 ␮g/ml, the TEER value was intermediate between the values measured at 10 and 100 ␮g/ml. If the decrease in TEER caused by MWCNT were due to the opening of tight junctions, it should correspond to an increase in the paracellular permeability of Calu-3 cell monolayers to cellexcluded solutes. The experiment shown in Fig. 2 demonstrates that this is the case. In this experiment, labelled mannitol, an extracellular solute (m.w. 180) that cannot be transported through the plasma membrane, was added to the apical chamber of Calu-3 monolayers pre-incubated for 4 d in the absence or in the presence of 100 ␮g/ml MWCNT. The radioactivity in the basolateral chamber was then measured after increasing periods of incubation. The results indicate that the paracellular permeability to mannitol increased from 11.3 ± 1.03 to 49.6 ± 4.99 cpm/min in control and MWCNT-treated monolayers, respectively. In the same cultures, TEER was 57% lower in MWCNT-treated than in control epithelial monolayers.

Fig. 1. Carbon nanotubes lower the trans-epithelial electrical resistance (TEER) of Calu-3 cell monolayers. (Panel A) Calu-3 cells were cultured for 10 d on 0.4 ␮m membrane filters. At the end of this period, MWCNT, SWCNT, AD-CNT, or CB were added to the apical chamber of the culture system at a concentration of 100 ␮g/ml and TEER was determined at the indicated times. Empty symbols, control filters maintained in the absence of CNT. (Panel B) Calu-3 cells were cultured for 10 d on 0.4 ␮m membrane filters. At the end of this period, different concentrations of MWCNT were added to the apical chamber of the culture system, as indicated, and TEER was determined at the indicated times. For both panels, four filters were used for each condition. The figure shows representative experiments repeated three times with comparable results. Data are means ± S.D. * p < 0.05, ** p < 0.01 vs. control, untreated cultures.

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Fig. 3. MWCNT prevent the establishment of high TEER in Calu-3 cell monolayers. (Panel A) The mean TEER was determined in 15 Calu-3 monolayers cultured for 2 d on membrane filters. (Panel B) The incubation was then prolonged in the absence (none) or in the presence of MWCNT, SWCNT, AD-CNT or CB in the apical chamber, as indicated. The nanomaterials were added at a concentration of 100 ␮g/ml. TEER was determined after 4 and 7 d of exposure to the indicated nanomaterial. Three filters were used for each condition. The figure shows a representative experiment repeated twice with comparable results. Data are means ± S.D. n.s. not significantly different vs. TEER of control, untreated cultures measured 2 d after the passage (Panel A); * p < 0.05, ** p < 0.01 vs. TEER of control, untreated cultures measured on the same day (Panel B).

If CNT were added during the early phase of Calu-3 culture, when a tight monolayer had not yet been established, their effect was strictly dependent on the type of nanomaterial used (Fig. 3). As expected, the TEER value measured after 2 d from the passage was very low (Panel A) while exhibited a marked and progressive increase after further 4 d or 7 d of culture (Panel B). However, TEER did not increase at all if MWCNT had been added to the incubation medium (Panel B), indicating that these nanomaterials substantially suppressed the formation of a high-resistance epithelium. On the contrary, at the same times of incubation, TEER markedly increased in the monolayers incubated in the presence of CB, SWCNT, or AD-CNT although, in SWCNT- and AD-CNT-treated cultures, TEER values were significantly lower than in cultures incubated in unsupplemented medium. 3.2. Effects of CNT on the changes in TEER induced by IL-4 It is known that the Th2 cytokine interleukin-4 (IL-4) lowers the TEER of Calu-3 cell monolayers (Ahdieh et al., 2001). In the experiment shown in Fig. 4, Calu-3 cell monolayers were incubated at the basolateral side with IL-4 (5 ng/ml) in the absence or in the presence of MWCNT, SWCNT, AD-CNT, or CB, pre-added for 24 h to the apical medium at the concentration of 100 ␮g/ml. The decrease in TEER induced by IL-4 was comparable in any condition and roughly corresponded to 50% of the initial value after 24 h of treatment. If at this time the basolateral medium was replaced by IL-4-free fresh medium, the TEER decreased for further 24 h but then exhibited a significant restoration at later times. Indeed, after 72 h of IL-4-free incubation TEER was rescued by over 50% compared to control values. However, if the rescue from IL-4 treatment was performed in the presence of MWCNT in the apical compartment, TEER restoration was completely suppressed. On the contrary, TEER recovery in SWCNT and CB-treated monolayers was comparable to that observed in control, untreated cells. 3.3. Effects of CNT on the viability of Calu-3 cells The treatment with MWCNT did not seem to modify substantially the morphology of Calu-3 cell monolayers (Fig. 5). MWCNT appeared as ropes of various dimensions adherent to the monolayer. However, to assess quantitatively if CNT affected Calu-3 cell viability, we used a viability test based on the fluorescent vital dye resazurin (O’Brien et al., 2000). For this test, we had to use cultures grown in standard plasticware since cells grown on per-

Fig. 4. MWCNT interfere with TEER restoration after IL-4 treatment. Calu-3 cells were cultured for 12 d on membrane filters. At the end of this period, NM (MWCNT for Panel A; SWCNT for Panel B; AD-CNT for Panel C; CB for Panel D) were added to the apical chamber of the culture system at a concentration of 100 ␮g/ml. After 24 h (Day 0), IL-4 (5 ng/ml) was added to the basolateral chamber and the incubation prolonged for a further day. The basolateral surface of the filters was then washed twice in fresh, unsupplemented medium and transferred to new wells with a IL-4free medium in the basolateral chamber. For the sake of clarity, the same data from control filters (no pre-incubation with CNT, empty symbols) have been shown in all the Panels. TEER was determined at the indicated times. Three filters were used for each condition. Data are means ± S.D. ** p < 0.01 vs. control monolayer not exposed to nanomaterials. The figure shows a representative experiment repeated twice with comparable results.

meable filters cannot be used in the microplate reader required for the assay. Due to the experimental evidence pointing to possible interference between CNT and dyes used in viability tests (Worle-Knirsch et al., 2006), preliminary experiments (Fig. 6) were performed so as

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The incubation with 100 ␮g/ml MWCNT markedly affected the viability of THP-1 and, even more markedly, of HUVEC cells. In contrast, Calu-3 cells were not significantly affected by MWCNT. The different consequences of cell exposure to MWCNT were also confirmed by direct microscopic observation, which showed the substantial integrity of the Calu-3 cell monolayer, the heavy damage of THP-1 cultures and the almost complete destruction of the HUVEC population. At the same concentration of 100 ␮g/ml, SWCNT had significant effects on the cell viability of HUVEC and THP-1 cells but no effect on Calu-3 cells. CB produced no significant effect on cell viability in HUVEC and Calu-3 cultures, while THP-1 cells exhibited only a moderate viability decrease.

4. Discussion

Fig. 5. MWCNT do not affect grossly the morphology of Calu-3 cell monolayers. Confluent Calu-3 cell monolayers (TEER ∼ 1200  cm2 ) were incubated for 4 d in complete growth medium in the absence (upper Panel) or in the presence (lower Panel) of 100 ␮g/ml MWCNT. Images are microphotographs of representative fields. ×100.

to ascertain that the nanomaterials did not interfere with resazurin. The results obtained indicate that the incubation with MWCNT, SWCNT, or CB did not increase the low background level of resazurin detected after 1 h of incubation in the absence of cells (Panel A). Moreover, the addition of CNT or CB immediately before the measurement did not quench grossly the high cell-dependent fluorescence, although a significant decrease in the signal (∼15–20%) was detected when CB or MWCNT were present during fluorescence detection (Panel B). In Fig. 7 the effect of CNT on cell viability was examined in cultures of three different human cells: Calu-3 cells, endothelial cells derived from umbilical veins (HUVECs), and THP-1 macrophages.

This study demonstrates for the first time that MWCNT impair the barrier function of human airway epithelial cells, as indicated by both a decrease in TEER and an increase of the paracellular permeability of mannitol. The effect of MWCNT is time- and dose-dependent, with significant changes observed at relatively low doses (5–10 ␮g/ml) while the exposure to 100 ␮g/ml MWCNT caused a very marked TEER decrease. The changes in paracellular permeability, induced by the exposure to MWCNT, are not due to artefacts, referable to detergents used to solubilize CNT, since no detergent has been used in this study. LPS contamination of the materials can be excluded, since a LPS-inactivating protocol has been regularly adopted. Moreover, LPS per se has no significant effect on the TEER of Calu-3 monolayers up to a concentration of 1 ␮g/ml (B.M. Rotoli and O. Bussolati, unpublished observation). Although data on changes in cell viability caused by CNT are highly variable, some cell types appear severely affected by these nanomaterials (Cui et al., 2005; Bottini et al., 2006b; Zhang et al., 2007). Moreover, very recent data indicate that, after a few days of exposure, genotoxic effects are detectable in epithelial cells treated with MWCNT (Muller et al., 2008). Hence, the possibility exists that the MWCNT effect on the paracellular permeability may be referable to toxic effects on Calu-3 cells. However, this does not seem to be the case, since neither resazurin assay results nor direct observation demonstrate evident changes in viability after a 48 h-exposure to MWCNT, when TEER alteration is already clearly detectable (see Fig. 1B). Rather, Calu-3 cells, cultured on plastic dishes, proved quite resistant to the direct exposure to MWCNT (see Fig. 7), although it should be stressed that their behaviour when grown differentiated on membrane filters may be different. However, when evaluated with the same method, other cell types grown on plasticware, such

Fig. 6. Validation of resazurin viability test in the presence of CNT. (Panel A) Resazurin was added to 24-well dishes in the presence of HUVECs (left bar) or in the absence of HUVECs and the incubation prolonged for 1 h at 37 ◦ C. Empty bars, incubation in the absence of nanomaterials. Solid bars, incubation in the presence of the indicated nanomaterials at a concentration of 100 ␮g/ml. (Panel B) HUVECs were incubated for 1 h with resazurin. Immediately before fluorescence reading, the indicated nanomaterials were added at a concentration of 100 ␮g/ml to the incubation medium. Open bars, no addition of nanomaterials. For both Panels, three wells were used for each condition and data are means ± S.D. * p < 0.05, ** p < 0.01 vs. control, NM-untreated cultures. The figure shows representative experiments repeated twice with comparable results.

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Fig. 7. Effect of carbon nanoparticles on cell viability. Calu-3 cells (upper Panel), THP-1 human macrophages (middle Panel) and human endothelial cells from umbilical vein (HUVEC, lower Panel) were cultured for 48 h (Calu-3) or 24 h (THP-1 and HUVECs) on standard plasticware in the absence or in the presence of CB, SWCNT and MWCNT, at a concentration of 100 ␮g/ml. At the end of the incubation, cell viability was assessed with resazurin test (see Section 2). At the right of each panel, microphotographs of control and MWCNT-treated cells (×100), taken just after the resazurin viability test, are shown. Data are means ± S.D. of six independent determinations. ** p < 0.01, *** p < 0.001 vs. control, untreated cultures.

as human macrophages and endothelial cells, exhibit a marked toxicity upon treatment with MWCNT. Therefore, MWCNT seem to interfere specifically with the formation of tight junctional complexes, thus preventing the development of cell polarization and the establishment of a highresistance epithelial barrier. This hypothesis is consistent with the impressive effect of MWCNT on the development of tight junctions in growing Calu-3 monolayers (see Fig. 3) as well as with the behaviour exhibited by MWCNT-treated monolayers when tight junctional efficiency is reversibly hindered by the Th2 cytokine IL4 (Fig. 4). In this case, while IL-4 withdrawal leads to a fairly rapid restoration of TEER in control cells, no recovery of resistance occurs in monolayers exposed to MWCNT. The barrier dysfunction induced by the cytokine has been attributed to a marked repression of the expression of some components of tight junctions, such as ZO-1 and occludin (Ahdieh et al., 2001). However, examples exist in which marked changes in paracellular permeability are not associated with evident modifications of tight junction proteins. For instance, asbestos microfibers raise the paracellular permeability of cultured airway epithelial cells in the absence of obvious changes in occludin expression (Peterson et al., 1993; Peterson and Kirschbaum, 1998). Further experimental work is needed to assess if this is also the case for MWCNT effect. What are the structural determinants of carbon nanotubes responsible for their effect on paracellular permeability? As for

many other cases, the paradigm of a relative independence of the biological effect on the mass concentration of the material is confirmed here. Indeed, although used at the same nominal concentration of 100 ␮g/ml, SWCNT and AD-CNT had much smaller effects than MWCNT and the amorphous nanomaterial CB had no effect at all (Fig. 1A and B). Since several metals can contaminate commercial CNT synthesized with the CVD method, the perturbation of tight junctions might be referable to the presence of metals, rather than to CNT themselves. According to the manufacturer, MWCNT present a low level of metal contamination (<0.1% Fe), while SWCNT, mainly contaminated by Co and Mo (S. Bellucci, unpublished results), have a much lesser effect on paracellular permeability. These data may point to a peculiar iron effect on tight junctions that awaits further investigations. Since MWCNT have a much smaller surface/volume ratio than SWCNT, their greater perturbing effect on paracellular permeability should not be attributed to a wider interaction with the monolayer surface. Rather, they should be also endowed with a much higher number of structural irregularities that could greatly increase their chemical reactivity. This hypothesis appears interesting, given that AD-CNT are characterized by a lower density of defects, compared to commercial CNT. Indeed, the arc discharge and laser vaporizations processes, which were the most common forms of nanotube production in the past, typically result in nanotubes with low structural defects and, hence, outstanding physical properties. CVD

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processes are more suitable for industrial applications, given the larger deposition area and mass production involved. However, this process has a major drawback, i.e. the reduced structural integrity of the resulting nanotubes. In particular, it has been pointed out by Salvetat et al. (2006) that MWCNT grown by arc discharge have much fewer structural defects and, hence, a better mechanical behaviour in terms of tensile modulus, compared to those grown by CVD. Tight junctions are dynamic structures sensitive to environmental factors, such as toxic compounds, mediators, or bacterial products. In this contribution, we used the Th2 cytokine IL-4 to perturb reversibly Calu-3 monolayer permeability, as originally described by Ahdieh et al. (2001), and to study the effects of CNT on TEER recovery. With this approach, we found that MWCNT completely suppress TEER rescue, an effect that may have important implications. Indeed, it may point to a CNT-dependent increase in the permeability of the epithelial barrier during the recovery from environmental stress conditions associated with a Th2 response. Interestingly, while Th2 cytokines have been implied in lung fibrosis (Jakubzick et al., 2004), fibrotic changes have been described after the exposure of rat lungs to SWCNT (Shvedova et al., 2005). Moreover, Th2 responses are implicated in the B cell activation and IgE production in allergic diseases. Thus, the results presented here may be consistent with the hypothesis, suggested by Murr et al. (2005), that a relationship exists between asthma incidence and indoor exposure to combustion emissions, e.g. natural gas or propane cooking stoves, where MWCNT aggregates are present at high number concentrations. On the other hand, the barrier effects of IL-4 may indicate that the activation of a Th2-response during the exposure to MWCNT could increase the likelihood of extra-pulmonary translocation of the nanomaterials from the airways to other body compartments. Nanoparticle translocation has been repeatedly investigated and recently demonstrated in vivo at least for CB (Shimada et al., 2006). Geys et al. have recently used Calu-3 cell monolayers to assess the translocation of fluorescent polystyrene particles (Geys et al., 2006, 2007). However, no translocation was observed with cells cultured on 0.4 ␮m-filters, the condition adopted in the present study, while TEER increases after incubation with the nanomaterials, possibly as a consequence of the progressive formation of tight junctional complexes observed also in the present study. On the contrary, acute and transient changes in TEER have been recently observed in rat alveolar epithelial cells exposed to SWCNT, but not to polystyrene nanoparticles (Yacobi et al., 2007). The same authors observe more evident and sustained changes in TEER after exposure to chitosan or alginate coated, metal containing quantum dots as well as to ultrafine ambient particulates, which, however, are also rich in carbonaceous aggregates including MWCNT. Thus, the changes in barrier properties of airway cell monolayers seem a specific effect of particular types of CNT, rather than an alteration generically associated to the exposure to carbon nanomaterials. Since the maintenance of the barrier efficiency is an important factor for the prevention of respiratory diseases and a determinant of the biological effects of toxic materials, CNT-induced changes may have important functional consequences. Moreover, the peculiar effect of MWCNT on the barrier properties of IL-4-pretreated epithelium suggests that the biological consequences of the exposure to these nanomaterials may depend on the simultaneous presence of immunological changes, elicited by toxic or infectious factors.

Acknowledgments This research has been funded by the Italian Ministry of University and Scientific Research (PRIN Grant No. 2006069554

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