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The role of the iron catalyst in the toxicity of multi-walled carbon nanotubes (MWCNTs)
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Toxicology
The role of the iron catalyst in the toxicity of multi-walled carbon nanotubes (MWCNTs) Giuseppa Visalli a , Alessio Facciolà a , Daniela Iannazzo b , Anna Piperno c , Alessandro Pistone b , Angela Di Pietro a,∗ a
Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Italy Department of Electronic Engineering, Industrial Chemistry and Engineering, University of Messina, Italy c Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy b
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Article history: Received 12 September 2016 Received in revised form 30 December 2016 Accepted 12 January 2017 Keywords: Iron Pristine multi-walled carbon nanotubes (pMWCNT) Acid-treated multi-walled carbon nanotubes (fMWCNT) Oxidative damage Alveolar epithelial cell line
a b s t r a c t This study aimed to investigate the role of iron, used as a catalyst, in the biological response to pristine and functionalized multi-walled carbon nanotubes (p/fMWCNTs) with an iron content of 2.5–2.8%. Preliminarily, we assessed the pro-oxidant activity of MWCNTs-associated iron by an abiotic test. To evaluate iron bioavailability, we measured intracellular redox-active iron in A549 cells exposed to both MWCNT suspensions and to the cell medium preconditioned by MWCNTs, in order to assess the iron dissolution rate under physiological conditions. Moreover, in exposed cells, we detected ROS levels, 8-oxo-dG and mitochondrial function. The results clearly highlighted that MWCNTs- associated iron was not redoxactive and that iron leakage did not occur under physiological conditions, including the oxidative burst of specialized cells. Despite this, in MWCNTs exposed cells, higher level of intracellular redox-active iron was measured in comparison to control and a significant time-dependent ROS increase was observed (P < 0.01). Higher levels of 8-oxo-dG, a marker of oxidative DNA damage, and decreased mitochondrial function, confirmed the oxidative stress induced by MWCNTs. Based on the results we believe that oxidative damage could be attributable to the release of endogenous redox-active iron. This was due to the damage of acidic vacuolar compartment caused by endocytosis–mediated MWCNT internalization. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction The impact of carbon nanotubes (CNTs) on human health has not yet been clarified, although they seem to elicit toxicity in the respiratory system following inhalation. Inhaled single and multi-walled carbon nanotubes (SWCNTs and MWCNTs, respectively), penetrate
Abbreviations: CNT, carbon nanotubes; pMWCNT, pristine multi-walled carbon nanotubes; fMWCNT, acid-treated multi-walled carbon nanotubes; CCVD, catalytic chemical vapor deposition; FCS, fetal calf serum; PBS, phosphate buffered saline; FACS, Fluorescence-activated cell sorting; DFX, deferoxamine mesylate; FAS, (NH4 )2 Fe(SO4 )2 ; NTA, nitrilotriacetic acid; calcein-AM, calceinacetoxymethyl ester; ym , mitochondrial transmembrane potential; DCF-DA, 2 ,7 -dichlorofluorescein-diacetate; FAU, fluorescence arbitrary units; 8-oxo-dG, 8Oxo-2 -deoxyguanosine. ∗ Corresponding author at: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Via C. Valeria n◦ 1, Gazzi, 98100, Messina, Italy. E-mail addresses: [email protected] (G. Visalli), [email protected] (A. Facciolà), [email protected] (D. Iannazzo), [email protected] (A. Pistone), [email protected], [email protected] (A. Di Pietro).
deeply, inducing pulmonary inflammation, cytotoxicity and carcinogenesis [1]. Numerous in vitro and in vivo studies have shown the acute and chronic inflammatory responses to CNTs [2–8]. CNTs, especially MWCNTs, have strong similarities with asbestos fibres, including a needle-like shape, pro-oxidant capability and biopersistence [9,10]. Similar to asbestos, CNTs are also able to cause mesothelioma [11] as well as alveolitis, pulmonary fibrosis, granuloma and bronchogenic carcinoma [12,13]. According to several authors [14,15], the induction of the carcinogenic asbestos-like effects of MWCNTs is driven by the presence of transition metals, especially iron, used as a catalyst in nanotube synthesis. In a previous study, we observed in A549 cells a strong pro-oxidant effect by using MWCNTs synthesized in our laboratory by the catalytic chemical vapor deposition (CCVD) method [16]. In our experiments, the role of iron apparently was confirmed by using the chelator deferoxamine mesylate (DFX); the redox imbalance was completely neutralized in cells treated with DFX-MWCNTs mixtures. However, the iron content of the tested MWCNTs was largely trapped inside the nanotubes; thus, it did not fully explain the observed redox imbalance. All this led us to
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hypothesize that oxidative damage was mainly attributable to the release of endogenous redox-active iron, due to cell damage caused by endocytosis–mediated MWCNT internalization. In order to test this hypothesis, and thus to explain the true role of the iron catalyst in the toxicity of MWCNTs, in the present study we performed further experiments using the same homemade nanomaterials.
2. Materials and methods 2.1. Pristine and functionalized MWCNTs Since the biological effects of MWCNTs are strongly linked to their physicochemical properties, which are in turn related to postsynthetic modifications, we examined both pristine and functionalized MWCNTs, designated pMWCNT and fMWCNT, respectively. pMWCNT were synthesized by catalytic chemical vapor deposition (CCVD) using Fe/Al2 O3 as the catalyst and they were successively purified as previously reported (16). fMWCNT, i.e. MWCNT-COOH, were prepared by the oxidation of purified pMWCNT using a mixture (1:1 vol ratio) of sulfuric acid and nitric acid [17]. As extensively reported in our previous study, MWCNTs were characterized by thermogravimetric analysis (TGA), UV spectra, scanning electron microscopy and high-resolution transmission electron microscopy. In pMWCNTs, the inorganic fraction, assessed by oxidative TGA analysis, was equal to 3.5-4%, almost all comprised of Fe2 O3 , showing the high quality (>95% purity) of the MWCNTs. As confirmed by atomic absorption spectroscopy analysis, the percentage of metallic iron was 2.5-2.8%. The percentages of inorganic fraction and metallic iron present in the fMWCNTs were evaluated following the same procedure and were found to be equal to 3.1–3.5% for Fe2 O3 and 2.0–2.2% for the metallic iron. Based on the synthesis method used, the iron was localized within the MWCNTs and, sometimes, at one end. This was evident by observing the images of pMWCNTs obtained by HRTEM. Fig. 1 clearly shows iron catalyst clusters encapsulated within the multi-walled nanostructure (Fig. 1a and b). These were formed of 15–20 layers and the outer surfaces were smooth and well-graphitized. Sometimes, point defects among graphitic carbon planes were observed. Other nanotubes appeared to have been formed using as a base the catalyst that, remaining at one end of the nanostructure (Fig. 1c), was largely exposed to the external environment. As previously reported, pMWCNT had an average length of 10–20 m and a diameter close to 15–30 nm, while fMWCNT were by far shorter (average length between 200 and 1000 nm) and showed an external layer eroded at many points, due to the oxidative insertion of terminal functional groups. Due to their hydrophobicity common to all CNTs, our MWCNTs, in particular pMWCNT, had a strong propensity to agglomerate in water. In order to minimize this feature, which makes MWCNT/cell interactions highly variable, we made concentrated MWCNT suspensions (100 x in PBS) that were sonicated for 20 min in an ice bath. Since proteins stabilize MWCNT suspensions [18], just before each experiment, the concentrated suspensions were diluted in cell culture medium (containing 2% FCS) and again subjected to a fast sonication (3 min). To assess the dissolution ratio of iron under physiological conditions, additional experiments were performed. A first set of tests consisted in treating cells with the supernatant obtained from cell culture medium (containing 2% FCS) preconditioned with nanotubes. Briefly, the MWCNT suspensions were incubated at 37 ◦ C for 48 h and then centrifuged (3000g for 10 min). Considering the leaning of nanotubes to the sedimentation, this treatment allowed to obtain the MWCNT-free supernatants that were used for the experiments. AAS analysis was performed on the preconditioned media and on the untreated cell medium. No relevant differ-
ence in terms of iron content were observed. The values were 50, 52 and 51 mg/L−1 for the untreated, pMWCNT- and fMWCNTpreconditioned media respectively (P > 0.05). To assess the possible iron leakage from the nanotubes subjected to the action of reactive oxygen species, we performed further abiotic experiments. Briefly, the MWCNTs were placed in contact with the ROS generated in situ by treating peroxides in the presence of Cu2+ catalyst. Both p-MWCNT and f-MWCNT samples (50 g mL−1 ) were separately treated with hydrogen peroxide or peroxyacetic acid (400 M), in deionized water at 37 ◦ C, in the presence of a catalytic amount of CuSO4 (50 M) and incubated for 6 h, as in in vitro experiments. Then, the mixtures were centrifuged (3500g for 10 min) and the supernatants were analyzed by AAS spectroscopy while MWCNTs were washed by PBS and analyzed to assess the presence of redox active iron. 2.1. Cells and exposure conditions As an in vitro cell model, we used the human alveolar cell line A549, derived from a lung carcinoma (ATCC, Rockville, USA). Cells were cultured in RPMI medium with 2 mM L-glutamine, 10% (v/v) fetal calf serum (FCS), 100 IU mL−1 penicillin and 100 g mL−1 streptomycin at 37 ◦ C in a humidified 5% CO2 atmosphere. All reagents for cell cultures were purchased from Gibco (Invitrogen Gibco, Milan, Italy). For all experiments, we used subconfluent monolayers grown in 6-well cell plates. In parallel, A549 cells were exposed to pMWCNT and fMWCNT (50 g mL−1 ), to their respective supernatants, as reported above, and to an iron solution at the same concentration present in pMWCNTs. This was obtained from (NH4 )2 Fe(SO4 )2 (final concentration 54.4 M) and used as a positive control. Monolayers treated with PBS were included in each experiment as a negative control (i.e. control cells). To confirm MWCNT uptake, previously quantified in the same cell model [16], we performed a qualitative analysis by microscopic observation of A549 semiconfluent monolayers. The cells were grown on cell slides (Invitrogen Gibco) and treated as reported above for 180 min. 2.2. Assessment of iron bioavailability To study the role of iron in the toxicological effect of MWCNTs, we used two different analyses aimed to assess the bioavailability of MWCNTs-associated iron and to measure the intracellular concentration of redox-active iron in exposed cells. The first analysis was performed by using a fluorimetric abiotic assay, based on the method devised by Esposito et al. [19].Briefly, in MWCNT suspensions (50 g mL−1 ), both as such and subjected to the action of ROS, and in the supernatant of MWCNT pre-treated cell culture medium, we measured the oxidation of the non-fluorescent probe dihydrorhodamine (DHR) to its fluorescent form rhodamine, a reaction catalyzed by redox-active iron. To exclude other DHR oxidation mechanisms not caused by the presence of redox-active iron, the samples were analyzed in parallel by the addition of the iron chelator deferoxamine mesylate (DFX),which quenches only iron-induced fluorescence. Briefly, samples were assayed in quadruplicate in 96-well plates by adding the DHR (50 M) in reagent solution (pH 7.3) containing 40 M of ascorbate to regenerate Fe(II) after its oxidation to Fe(III). DFX (50 M) was added to this solution in two of the wells. A Fe:NTA (nitrilotriacetic acid) (1:7 mM) complex, starting from freshly prepared FAS (NH4 )2 Fe(SO4 )2 and NTA (pH 7.0), was used to build a calibration curve (1–54.4 M). To assess the kinetics of the reaction, emitted fluorescence was recorded every 2 min, starting from 15 min up to 40 min, by using 485/535 nm excitation/emission filters (plate reader Tecan, Brescia, Italia). The differences between the samples with and without DFX, due to redox-active iron, were used to calculate fluorescence units
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Fig. 1. HRTEM images of pMWCNT that are formed of 15–20 layers and show outer surfaces smooth and well-graphitized. (A and B) Iron catalyst clusters are encapsulated within the multiwalled nanostructures. (C) The iron catalyst cluster is exposed to the external environment as present at one end of the nanostructure.
per minute (FU/min). Iron values (M) were extrapolated from the calibration curves. The intracellular concentration of redox-active iron was measured by using an argon laser flow cytometry instrument (Dako Galaxy Cytomation, Glostrup, Denmark) and calceinacetoxymethyl ester (calcein-AM) as a probe. This non-fluorescent non-chelating lipophilic ester easily penetrates cellular membranes and diffuses into cells. There, it is rapidly cleaved by unspecific cytosolic esterases, producing the fluorochromic alcohol calcein ( ex 488 nm; em 518 nm) that chelates intracellular uncomplexed iron (free iron) [20]. This reaction quenches the green fluorescence of calcein in a dose-dependent manner, allowing the detection of bioavailable iron. To validate Calcein assay, we used the specific iron chelator deferiprone (3-idrossi-1,2-dimetilpiridin-4(1H)-one: DFP). In presence of redox-active iron, DFP restores calcein fluorescence by the probe dequenching. DFP is a well-tested drug that, displaying a high binding affinity for the iron, is widely used in the therapy of siderosis. It is a bidentate ligand fat-soluble and therefore capable to complexing intracellular iron. Briefly, A549 monolayers were treated for 300 min as reported above and then cells were harvested by trypsinization (0.25% trypsin–1 mM EDTA). For each sample two cell suspensions (1 × 105 mL−1 in PBS containing 10 mM D-glucose at pH 7.4) were loaded with calcein-AM (final concentration 60 nM) and, after incubation at 37 ◦ C for 5 min, only in one was added DFP (final dose: 0.3 M), incubating again for 10 min. Cell suspensions were then submitted to FACS analysis collecting signals in the fluorescence channel 1. Intracellular iron was assessed by the differences in emission values between cell suspensions treated with the mix (calcein+ DFP) and the ones treated with calcein only. 2.3. Assessment of MWCNT-induced toxicity In treated A549 cells we used FACS to assess cell viability and the intracellular production of ROS. Moreover, to assess oxidative damage in cell compartments, we evaluated genotoxicity by measuring 8-oxo-dG and mitochondrial function by assessing m . After treatment of monolayers and trypsinization, as reported above, cell suspensions (1 × 105 mL−1 in PBS added by 10 mM Dglucose at pH 7.4) were separately loaded by the probes. These were propidium iodide (3 g mL−1 ), 2 ,7 -dichlorofluorescein-diacetate (DCF-DA) (1 M), FITC-labelled avidin (0.2 M) and rhodamine 123 (R123: 10 M) (Invitrogen) to detect cell viability, the intracellular production of ROS, 8-oxo-dG and m , respectively. Starting from 1 h to 6 h, at intervals of 1 h, a time-course was performed. For each treatment, at each time point, four different cell aliquots were taken and loaded with the respective probes as previously reported [21,22]. Specifically, we used a FITC-labelled avidin probe, which is
known to bind with high specificity to 8-oxo-dG, to assess oxidative DNA damage [23].Since the interference of MWCNTs prevented us from performing the more commonly used colorimetric viability tests (e.g. MTT), we detected dead cells stained with a DNA intercalating probe by measuring emission signals in the FL3 channel. For intracellular ROS and 8-oxo-dG detection, emission signals were recorded in the FL1 channel while the emitted fluorescence of R123 was collected in the FL2 channel. FACS analyses were performed in triplicate and separately for each fluorochrome. The weighted average of emission values for 100 cells was calculated and expressed in arbitrary fluorescence units (AFU). 2.4. Statistical analyses All data are presented as mean ± standard error (SE) based on at least triplicate observations from three independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) and multiple comparisons of the means were performed by the Tukey-Kramer test (GrafPAD Software for Science). The relationships between different parameters were assessed by the Pearson correlation coefficient. Significance was accepted at P < 0.05. 3. Results 3.1. Iron bioavailability of MWCNTs The results of the fluorimetric assay to measure the pro-oxidant potential of MWCNT-associated iron are reported in Fig. 2A. The cell-free method, allowing us to exclude probe oxidation not caused by iron, clearly highlighted the absence of redox-active iron in the assayed MWCNTs. In particular, the emission values due to the oxidation of DHR almost overlapped in the two sets of wells (with and without the chelator DFX). Similar results were also obtained by testing the supernatant obtained from cell culture medium pretreated with nanotubes and used to evaluate iron dissolution under physiological conditions (Fig. 2A). Moreover we used this abiotic test to assess the possible iron leakage from the nanotubes subjected to the action of reactive oxygen species present in specific cell compartments. As shown by the graph (Fig. 2A), the treatment with in situ generated ROS not made the iron content of MWCNTs more accessible and bioavailable. Similar results were recorded by the analysis of the supernatants obtained by this MWCNT treatment for 6 and 24 h. Indeed, the AAS spectroscopy analysis did not detect traces of iron ions in the supernatants, excluding the iron leakage from the nanomaterials (data not shown). Despite these results, exhibiting the lack of MWCNT-associated bioavailable iron, the FACS analysis of intracellular redox active iron underlined its higher presence in A549 cells exposed for 6 h
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Fig. 2. Iron bioavailability of MWCNTs. (A) Results of fluorimetric assay to assess the pro-oxidant potential of MWCNT-associated iron. pMW: pristine MWCNT, fMW: functionalized MWCNT, pMW* and fMW*: MWCNTs treated with in situ generated ROS, SpMW and SfMW: supernatant obtained from cell medium preconditioned (at 37 ◦ C for 48 h) with pMWand fMWrespectively. Calibration curve was built by using a Fe:NTA (complex and the iron content was in the range 1.7–54.4 M, reported in the graph as St 1–6 respectivly. The results are expressed as the average values of FU/min in the interval 15–40 min. (B) Cytofluorimetric analysis to measure intracellular concentration of redox-active iron by using calcein-AM as a probe and DFP to restore calcein fluorescence by breaking of the complex Fe-calcein. The presence of bioavailable intracellular iron quenched the calcein fluorescence while the probe dequenching, in presence of redox-active iron, was obtained by DFP. Intracellular iron is highlighted by the differences between cell suspensions treated with the mix (calcein+ DFP) and the ones treated with calcein only. Cells exposed to (NH4 )2 Fe(SO4 )2 (54.4 M) were used as positive control. (All data are reported as means ± SE of three separate experiments). In pristine and oxidized MWCNTs and in Fe (II)- treated cells the differences in emission values were significant (*P < 0.05).
to MWCNTs. To assess the intracellular iron, emission values of calcein were recorded in cell suspensions loaded with only this metal sensor and in the ones treated also with the iron chelator DFP which restored calcein fluorescence. As shown in Fig. 2B, there was no difference between the two measurements in control cells and in cells exposed to both MWCNT preconditioned medium, highlighting the lack of intracellular free iron. Instead, the cells treated with the assayed pristine and oxidized nanostructures showed a quenching of calcein fluorescence which was almost entirely restored by DFP. In particular, the differences between the measurements obtained in the presence and in the absence of DFP were equal to 28 and 25% in cells treated with pristine and oxidized MWCNTs respectively (P < 0.05). A similar difference was observed in the positive control (i.e. Fe (II)- treated cells). 3.2. MWCNT-induced toxicological effect The qualitative assessment of MWCNT uptake confirmed previous results (Fig. 3). In A549 semiconfluent monolayers exposed for 3 h to either pristine or oxidized MWCNTs, uptake was highlighted by the presence of dark aggregates in the cytoplasm that were bulkier and more numerous in pMWCNT treated cells (Fig. 3B and C). Moreover, consistent spaces were observed, indicating the detachment of dead cells. The monolayers exposed to
the Fe (II) solution (54.4 M) had large and numerous spaces, indicating high Fe-induced cytotoxicity to the tested dose/time (Fig. 3.F).Conversely, cells exposed to the MWCNT preconditioned cell medium were similar to control cells (Fig. 3A, D and E), underlining the absence of iron leakage from the assayed MWCNTs under physiological conditions. In order to compare the biological effect of MWCNTs to those caused by iron at the same concentration present in the assayed nanostructures, cell viability and ROS production were detected. The used doses were 50 g mL−1 and 54.4 M for MWCNTs and the iron solution, respectively. The same analyses were performed by using MWCNTs-pre-treated medium for cells exposure. The time course of cell viability, detected using propidium iodide to stain dead cells, showed significant cytotoxicity elicited by MWCNTs. Fig. 4 A shows the percent increase ( %) of dead cells in comparison to the control cells. The % was time-dependent, and in cells treated with both MWCNT preparations, on average it was equal to 18 and 17% for pristine and oxidized MWCNT, respectively (P < 0.05 at 4 and 6 h). In cells treated with Fe (II) solution the % were higher and the differences were significant at all-time points examined in comparison with control cells (P < 0.05). On the contrary, dead cells were almost absent in cells exposed to culture medium pre-treated with MWCNTs, confirming the microscopic observations. The time-course of ROS production is reported in Fig. 4 B. FACS analysis highlighted the time-dependent pro-oxidant effect of the assayed nanostructures. In comparison with control cells, the emission values of the oxidized probe at all-time points examined were significantly higher (P < 0.01) in MWCNT exposed cells. On average, the AFU values were10.7- and 6.8-fold greater than in control cells in response to pristine and oxidized MWCNTs, respectively; similarly, in positive control (i.e. cells exposed to redox active iron), the value was 8.2-fold higher than in cell control, further indicating the strong oxidative potential of the assayed MWCNTs. Instead, the values of emitted fluorescence were similar to control cells after exposure to cell culture medium preconditioned with MWCNTs. To confirm the pro-oxidant effect of MWCNTs, we assessed the effect of oxidative damage in cell compartments. As expected, significantly related to ROS overproduction (P < 0.01), we observed mitochondrial impairment and enhanced DNA oxidative damage. In particular, the time course of the R123 emission values, used to assess mitochondrial transmembrane potential, highlighted progressive mitochondrial failure, emphasizing the metabolic impairment induced by our tested carbon nanotubes. Fig. 4C reports the changes of mitochondrial potential in cells exposed to both MWCNT suspensions, to iron solution and to preconditioned cell medium. The opposite trend to ROS overproduction was observed in cells treated with MWCNTs and iron solution. In these cells the mitochondrial impairment was evident from the start even though the differences were significant only at latest time points examined (at 4 and 6 h: P < 0.05). In comparison to control cells, at 6 h m was 88.2,82.9 and 86.0% in cells treated with pristine and oxidized nanotubes and iron solution, respectively. Similar to ROS detection, no effect on mitochondrial function was observed in cells exposed to pretreated medium. In these cells the values of m were roughly overlapped to negative control (±5%). In the tested times the average values of percentage changes in comparison to negative control were 100.8 and 101.3 in pristine and functionalised MWCNT preconditioned media respectively), confirming the absence of leakage from the assayed MWCNTs. The outcomes of free radical-initiated oxidation upon cellular exposure to MWCNTs were also observed in the nuclear compartment of A549 cells. As indicated by FACS analysis using as probe FITC-labelled avidin, treated cells showed a time-dependent increase in 8-oxo-dG (free radical-induced oxidative lesions) compared to untreated cells. Fig. 4 D reports the average values in
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Fig. 3. Phase contrast microscopy images of A549 semiconfluent monolayers to show MWCNTs uptakein cells treated for 3 h. (A) control cells, (B and C)cells exposed to pristine and functionalized MWCNT. (D and E) cells exposed to S pMW and S fMW. (F) cells exposed to (NH4 )2 Fe(SO4 )2 solution (54.4 M).
Fig. 4. Toxicological effects of MWCNT exposure assessed by FACS analysis. Cells exposed to (NH4 )2 Fe(SO4 )2 solution (54.4 M) were used as positive control. (A) Time course of cell viability. The graph reports the percent increase (% ) of dead cells in comparison to the control cells. The% were significant at 4 and 6 h for pristine and oxidized MWCNT, respectively (P < 0.05). In cells treated with Fe (II) solution the % were significant at all-time points examined in comparison with control cells (P < 0.05) In the graph are reported the linear trend lines. (B) Time-course of ROS production assessed by emission values of DCF. Considering the very fast ROS production, the interval 30 min–6 h was evaluated. Data are reported as% and the linear trend lines are drawn. In comparison with control cells, the emission values of the oxidized probe at all-time points examined were significantly higher (P < 0.05 and P < 0.01) in MWCNT exposed cells and in positive control. (C) Time course of m . Data of R123 emission (expressed as AFU) show, compared to control cells, the reduction of mitochondrial potential in cells exposed to both MWCNT suspensions and to iron solution. In pristine and oxidized nanotubes and in iron solution, the differences were significant only at latest (4 and 6) time point examined (P < 0.05). The graph reports the logarithmic trend lines (D) 8-oxo-dG detection by FITC-labelled avidin emission to assess oxidative DNA damage. The graph reports the percent increase ( %) of 8-oxo-dG in nanotubes and iron solution treated cells (positive control) respect to control cells (*: P < 0.05). Data are expressed as average values of % in the time interval 1–6 h. (Data of graphs A, B, C and D are reported as means ± SE of three separate experiments.
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the time interval 1–6 h of the percent increase ( %). The emitted fluorescence values were significantly higher (P < 0.05)in MWCNT exposed cells than in control cells, suggesting that synthetic nanoparticles induced DNA oxidative damage in pneumocytes. The 8-oxo-dG increases were slightly higher in pMWCNTs treated cells in comparison to cells treated with oxidized nanotubes and iron solution (1.95 vs. 1.60 and 1.50 fold respectively). Similar to the ROS and m analyses, negative results were obtained after cell exposure to preconditioned medium.
4. Discussion Thorough knowledge of MWCNT-induced pathogenic mechanisms would allow a change in the synthesis process to reduce their bioreactivity. The design of safer nanofibres is extremely important considering the ease with which lightweight nano-sized MWCNTs aerosolize, increasing pulmonary disorders after their inhalation. Several authors have attributed a key role of MWCNT-induced toxicity to the iron present as residual catalyst in these nanomaterials, demonstrating that iron-free particles, including synthetic asbestos fibres were biologically inactive [14,15]. In a previous study [16] we obtained similar results observing that, in cells exposed to MWCNT–chelating agent mixtures, oxidative damage was almost completely reset even if the harmful effect related to MWCNT-induced mechanical damage retained unchanged. The results obtained in the present study have enabled us to better define the MWCNT-triggered pathogenic pathways, excluding the iron bioavailability of the homemade tested nanoparticles. Despite non-negligible levels of iron (54.4 and 43.9 M in pMWCNT and fMWCNT, respectively), its content was not leached in physiological conditions and it was not bioavailable. Contrary to that reported by Vales et al. [24], who observed a pro-oxidant effect in the supernatant obtained from CNT-preconditioned cell medium, our nanomaterials were devoid of easily leachable iron. Moreover, the complete absence of biological effects in cells exposed to preconditioned medium suggested that even amorphous carbon, in addition to iron, was not present in our homemade MWCNTs, highlighting their high degree of purity. More importantly, we did not detect redox-active (i.e. bioavailable) iron in either MWCNT in physiological conditions, despite residual catalyst also being present on the surface in pMWCNT at one end of the nanostructure and, therefore, potentially active toward cells. Unlike functionalized MWCNTs, pristine ones were not subjected to strong oxidizing acids (HNO3 and H2 SO4 ) able to leach the superficial residual catalyst. Independently of being encapsulated or present on the surface, the entire iron content was not bioavailable and therefore harmful in our cell model. This is easily explained, as iron(III) oxide, present in the tested MWCNTS, is extremely stable, typically insoluble in aqueous solutions and, consequently, unable to trigger the well-known Haber-Weiss and Fenton reactions that produce strongly reactive hydroxyl radicals. This feature substantially differentiates these homemade MWCNTs from asbestos fibres, especially amphiboles such as crocidolite and amosite, in which the iron, as well as being plentiful and always located on the surface, is in a redox and coordination state to allow a pro-oxidant effect [14,25]. The lack of bioavailable iron in our MWCNTs only apparently contradicted previous findings showing the antioxidant effect of the chelating agent. The significant increase of intracellular iron highlighted in this study would seem to confirm the hypothesis that oxidative damage in cells exposed to nanotubes is mainly caused by the release of endogenous redox-active iron. This is due to damage to the acidic vacuolar compartment, previously observed in the same cell model, caused by MWCNT internalization. As confirmed here, MWCNT uptake in pneumocytes is very
efficient and may occur through passive diffusion across the cellular membrane [26], favoured by the strong hydrophobicity of CNTs [27]. An alternative mechanism is clathrin- and/or caveolaemediated endocytosis/phagocytosis [28]. MWCNT internalization would seem to cause the release of intralysosomal redox-active low molecular mass iron, produced during autophagocytic degradation of metalloproteins such as cytochromes [29]. This would explain the increase of redox-active intracellular iron observed in MWCNT-exposed cells; instead, the possible release of iron from the nanotubes should be excluded as the phagolysosome pH is not low enough. As observed by using phagolysosomal simulant fluid [30], metals in CNTs were stable for up to two months [31]. This is certainly true for our CNTs, considering the strong mineral acids treatment (i.e. HCl for the purification step of pMWCNT and H2 SO4 /HNO3 for the synthesis of fMWCNT) and sonication procedures, involving also the removal of the released material at each steps (16), to which they were subjected. Moreover our results highlighted as the iron content of the tested nanomaterials was not released even in “extreme physiological conditions” such as those present exclusively in specific cell compartments of specialized cells. Due to oxidative burst, professional phagocytic cells as macrophages, neutrophils etc. produces highly reactive oxygen and nitrogen derivatives to kill engulfed pathogens. Despite we used alveolar epithelial cells as biological model, in the abiotic experiments simulating oxidative burst, we did not observe an iron release of our MWCNTs in presence of ROS. On this basis, we can reasonably rule out the iron release from the assayed MWCNTs in the biological conditions and the time considered in our study. Therefore, the increase of redox-active iron could be related to the release of endogenous iron, caused by endocytosis–mediated MWCNT internalization. In addition to the effect of redox-active iron, likely released by MWCNT-induced cell damage, we may presume that the strong cytotoxicity was due to features of MWCNTs. In comparison to isometric particles, the fibrous shape with nano-sized diameter of CNTs plays a key role in nanotube-induced toxicity, causing phagolysosomal dysfunction. Previously, we observed that damage in the cellular acid compartment was inversely related to cell vitality, thus strongly underlining the importance that this pathway may have in the pathogenicity of CNTs. Due to increased lysosomal permeability, several hydrolytic enzymes are released in the cytoplasm. This can induce cell death by apoptosis, similarly to what has been shown by silica particles [32], or autophagic activation, as suggested for fullerenes [33]. Alongside the fibrous aspect, the surface properties should be considered as essential parameters in CNTinduced cytotoxicity [34,35]. The presence of oxygenated groups and of several surface defects that modify the graphene structure in our functionalized MWCNTs would explain the similar toxicity elicited by pristine (extremely long) and oxidized MWCNTs. Regardless of the presence of metals, almost all studies report redox imbalance in SWCNT- and MWCNT-exposed cells [36–40]. This common nanotube-induced pathogenic effect was assessed by the detection of ROS overproduction and/or oxidative damage to macromolecules (i.e. lipid hydroperoxides, oxidized DNA, etc.), as well as the depletion of antioxidant molecules (i.e. reduced glutathione). Our results confirmed that ROS overproduction in exposed cells was strongly related to mitochondrial failure and to significantly increased levels of 8-oxo-dG. This marker of oxidative DNA damage strengthens the marked genotoxicity, previously observed by using the micronuclei test (MNi) and Comet assay [16]. Both these effects, in addition to phagolysosomal dysfunction, clearly demonstrate the broad spectrum of toxicological effects of the tested MWCNTs. Overall, the toxicological effects of the homemade tested MWCNTs, devoid of biavalable iron, unlike many commercially available
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CNTs, highlighted how further studies are still necessary to prevent potential health risks related to MWCNT exposure. In fact, it is not enough to reduce the presence of iron, by post-synthetic processes of purification, to make “safer” these nanomaterials, whose cyto-genotoxicity seems mainly due to the cell mechanical damage. Conflict of interests None. Funding This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. References [1] C. Monteiller, L. Tran, W. MacNee, S. Faux, A. Jones, B. Miller, K. Donaldson, The pro-inflammatory effects of low-toxicity low-solubility particles, nanoparticles and fine particles, on epithelial cells in vitro: the role of surface area, Occup. Environ. Med. 64 (September (9)) (2007 Sep) 609–615. [2] A.A. Shvedova, E. Kisin, A.R. Murray, V.J. Johnson, O. Gorelik, S. Arepalli, A.F. Hubbs, R.R. Mercer, P. Keohavong, N. Sussman, et al., Inhalation vs. aspiration of single-walled carbon nanotubes in C57BL/6 mice: inflammation, fibrosis, oxidative stress, and mutagenesis, Am. J. Physiol. Lung Cell. Mol. Physiol. 295 (2008) L552–L556. [3] E.A. Thompson, B.C. Sayers, E.E. Glista-Baker, K.A. Shipkowski, M.D. Ihrie, K.S. Duke, A.J. Taylor, J.C. Bonner, Role of signal transducer and activator of transcription 1 in murine allergen-induced airway remodeling and exarcerbation by carbon nanotubes, Am. J. Respir. Cell Mol. Biol. 53 (2015) 625–636. [4] R.F. Hamilton, Z. Wu, S. Mitra, P.K. Shaw, A. Holian, Effect of MWCNT size, carboxylation, and purification on in vitro and in vivo toxicity, inflammation and lung pathology, Part. Fibre Toxicol. 13 (2013) 13–57. [5] B.W. Lee, C. Kadoya, M. Horie, Y. Mizuguchi, M. Hashiba, T. Kambara, T. Okada, T. Myoho, T. Oyabu, A. Ogami, et al., Analysis of pulmonary surfactant in rat lungs after intratracheal instillation of short and long multi-walled carbon nanotubes, Inhal. Toxicol. 25 (2013) 609–620. [6] L. Ma-Hock, S. Treumann, V. Strauss, S. Brill, F. Luizi, M. Mertler, K. Wiench, A.O. Gamer, R. van Landdiedel, Inhalation toxicity of multiwall carbon nanotubes in rats exposed for 3 months, Toxicol. Sci. 112 (2009) 468–481. [7] R.R. Mercer, J. Scabilloni, L. Wang, E. Kisin, A.R. Murray, D. Schwegler-Berry, A.A. Shvedova, V. Castranova, Alteration of deposition pattern and pulmonary response as a result of improved dispersion of aspirated single-walled carbon nanotubes in a mouse model, Am. J. Physiol. Cell Mol. Physiol. 294 (2008) L87–L97. [8] J. Muller, M. Delos, N. Panin, V. Rabolli, F. Huaux, D. Lison, Absence of carcinogenic response to multiwall carbon nanotubes in a 2-year bioassay in the peritoneal cavity of the rat, Toxicol. Sci. 110 (2009) 442–448. [9] C.A. Poland, Carbon nanotubes introduced into the abdominal cavity of mice show asbestos-like pathogenicity in a pilot study, Nat. Nanotechnol. 3 (2008) 423–428. [10] E. Miozzi, V. Rapisarda, A. Marconi, C. Costa, I. Polito, D.A. Spandidos, M. Libra, C. Fenga, Fluoro-edenite and carbon nanotubes: the health impact of ‘asbestos-like’ fibres, Exp Ther Med. 11 (1) (2016 Jan) 21–27. [11] R.R. Mercer, A.F. Hubbs, J.F. Scabilloni, L. Wang, L.A. Battelli, D. Schwegler-Berry, V. Castranova, D.W. Porter, Distribution and persistence of pleural penetrations by multi-walled carbon nanotubes, Part Fibre Toxicol. 7 (October (4)) (2010) 28, http://dx.doi.org/10.1186/1743-8977-7-28. [12] F. Guo, N. Ma, Y. Horibe, S. Kawanishi, M. Murata, Y. Hiraku, Nitrative DNA damage induced by multi-walled carbon nanotube via endocytosis in human lung epithelial cells, Toxicol. Appl. Pharmacol. 260 (April (2)) (2012) 183–192, http://dx.doi.org/10.1016/j.taap.2012.02.010. [13] E.M. Rydman, M. Ilves, E. Vanhala, M. Vippola, M. Lehto, P.A. Kinaret, L. Pylkkänen, M. Happo, M.R. Hirvonen, D. Greco, K. Savolainen, H. Wolff, H. Alenius, A single aspiration of rod-like carbon nanotubes induces asbestos-like pulmonary inflammation mediated in part by the IL-1 receptor, Toxicol. Sci. 147 (September (1)) (2015) 140–155, http://dx.doi.org/10.1093/ toxsci/kfv112. [14] E. Gazzano, F. Turci, E. Foresti, M.G. Putzu, E. Aldieri, F. Silvagno, I.G. Lesci, M. Tomatis, C. Riganti, C. Romano, B. Fubini, N. Roveri, D. Ghigo, Iron-loaded synthetic chrysotile: a new model solid for studying the role of iron in asbestos toxicity, Chem. Res. Toxicol. 20 (March (3)) (2007) 380–387. [15] E. Aldieri, I. Fenoglio, F. Cesano, E. Gazzano, G. Gulino, D. Scarano, A. Attanasio, G. Mazzucco, D. Ghigo, B. Fubini, The role of iron impurities in the toxic effects exerted by short multiwalled carbon nanotubes (MWCNT) in murine alveolar macrophages, J. Toxicol. Environ. Health A 76 (18) (2013) 1056–1071, http:// dx.doi.org/10.1080/15287394.2013.834855.
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Toxicology
The role of the iron catalyst in the toxicity of multi-walled carbon nanotubes (MWCNTs) Giuseppa Visalli a , Alessio Facciolà a , Daniela Iannazzo b , Anna Piperno c , Alessandro Pistone b , Angela Di Pietro a,∗ a
Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Italy Department of Electronic Engineering, Industrial Chemistry and Engineering, University of Messina, Italy c Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina, Italy b
a r t i c l e
i n f o
Article history: Received 12 September 2016 Received in revised form 30 December 2016 Accepted 12 January 2017 Keywords: Iron Pristine multi-walled carbon nanotubes (pMWCNT) Acid-treated multi-walled carbon nanotubes (fMWCNT) Oxidative damage Alveolar epithelial cell line
a b s t r a c t This study aimed to investigate the role of iron, used as a catalyst, in the biological response to pristine and functionalized multi-walled carbon nanotubes (p/fMWCNTs) with an iron content of 2.5–2.8%. Preliminarily, we assessed the pro-oxidant activity of MWCNTs-associated iron by an abiotic test. To evaluate iron bioavailability, we measured intracellular redox-active iron in A549 cells exposed to both MWCNT suspensions and to the cell medium preconditioned by MWCNTs, in order to assess the iron dissolution rate under physiological conditions. Moreover, in exposed cells, we detected ROS levels, 8-oxo-dG and mitochondrial function. The results clearly highlighted that MWCNTs- associated iron was not redoxactive and that iron leakage did not occur under physiological conditions, including the oxidative burst of specialized cells. Despite this, in MWCNTs exposed cells, higher level of intracellular redox-active iron was measured in comparison to control and a significant time-dependent ROS increase was observed (P < 0.01). Higher levels of 8-oxo-dG, a marker of oxidative DNA damage, and decreased mitochondrial function, confirmed the oxidative stress induced by MWCNTs. Based on the results we believe that oxidative damage could be attributable to the release of endogenous redox-active iron. This was due to the damage of acidic vacuolar compartment caused by endocytosis–mediated MWCNT internalization. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction The impact of carbon nanotubes (CNTs) on human health has not yet been clarified, although they seem to elicit toxicity in the respiratory system following inhalation. Inhaled single and multi-walled carbon nanotubes (SWCNTs and MWCNTs, respectively), penetrate
Abbreviations: CNT, carbon nanotubes; pMWCNT, pristine multi-walled carbon nanotubes; fMWCNT, acid-treated multi-walled carbon nanotubes; CCVD, catalytic chemical vapor deposition; FCS, fetal calf serum; PBS, phosphate buffered saline; FACS, Fluorescence-activated cell sorting; DFX, deferoxamine mesylate; FAS, (NH4 )2 Fe(SO4 )2 ; NTA, nitrilotriacetic acid; calcein-AM, calceinacetoxymethyl ester; ym , mitochondrial transmembrane potential; DCF-DA, 2 ,7 -dichlorofluorescein-diacetate; FAU, fluorescence arbitrary units; 8-oxo-dG, 8Oxo-2 -deoxyguanosine. ∗ Corresponding author at: Department of Biomedical and Dental Sciences and Morphofunctional Imaging, University of Messina, Via C. Valeria n◦ 1, Gazzi, 98100, Messina, Italy. E-mail addresses: [email protected] (G. Visalli), [email protected] (A. Facciolà), [email protected] (D. Iannazzo), [email protected] (A. Pistone), [email protected], [email protected] (A. Di Pietro).
deeply, inducing pulmonary inflammation, cytotoxicity and carcinogenesis [1]. Numerous in vitro and in vivo studies have shown the acute and chronic inflammatory responses to CNTs [2–8]. CNTs, especially MWCNTs, have strong similarities with asbestos fibres, including a needle-like shape, pro-oxidant capability and biopersistence [9,10]. Similar to asbestos, CNTs are also able to cause mesothelioma [11] as well as alveolitis, pulmonary fibrosis, granuloma and bronchogenic carcinoma [12,13]. According to several authors [14,15], the induction of the carcinogenic asbestos-like effects of MWCNTs is driven by the presence of transition metals, especially iron, used as a catalyst in nanotube synthesis. In a previous study, we observed in A549 cells a strong pro-oxidant effect by using MWCNTs synthesized in our laboratory by the catalytic chemical vapor deposition (CCVD) method [16]. In our experiments, the role of iron apparently was confirmed by using the chelator deferoxamine mesylate (DFX); the redox imbalance was completely neutralized in cells treated with DFX-MWCNTs mixtures. However, the iron content of the tested MWCNTs was largely trapped inside the nanotubes; thus, it did not fully explain the observed redox imbalance. All this led us to
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hypothesize that oxidative damage was mainly attributable to the release of endogenous redox-active iron, due to cell damage caused by endocytosis–mediated MWCNT internalization. In order to test this hypothesis, and thus to explain the true role of the iron catalyst in the toxicity of MWCNTs, in the present study we performed further experiments using the same homemade nanomaterials.
2. Materials and methods 2.1. Pristine and functionalized MWCNTs Since the biological effects of MWCNTs are strongly linked to their physicochemical properties, which are in turn related to postsynthetic modifications, we examined both pristine and functionalized MWCNTs, designated pMWCNT and fMWCNT, respectively. pMWCNT were synthesized by catalytic chemical vapor deposition (CCVD) using Fe/Al2 O3 as the catalyst and they were successively purified as previously reported (16). fMWCNT, i.e. MWCNT-COOH, were prepared by the oxidation of purified pMWCNT using a mixture (1:1 vol ratio) of sulfuric acid and nitric acid [17]. As extensively reported in our previous study, MWCNTs were characterized by thermogravimetric analysis (TGA), UV spectra, scanning electron microscopy and high-resolution transmission electron microscopy. In pMWCNTs, the inorganic fraction, assessed by oxidative TGA analysis, was equal to 3.5-4%, almost all comprised of Fe2 O3 , showing the high quality (>95% purity) of the MWCNTs. As confirmed by atomic absorption spectroscopy analysis, the percentage of metallic iron was 2.5-2.8%. The percentages of inorganic fraction and metallic iron present in the fMWCNTs were evaluated following the same procedure and were found to be equal to 3.1–3.5% for Fe2 O3 and 2.0–2.2% for the metallic iron. Based on the synthesis method used, the iron was localized within the MWCNTs and, sometimes, at one end. This was evident by observing the images of pMWCNTs obtained by HRTEM. Fig. 1 clearly shows iron catalyst clusters encapsulated within the multi-walled nanostructure (Fig. 1a and b). These were formed of 15–20 layers and the outer surfaces were smooth and well-graphitized. Sometimes, point defects among graphitic carbon planes were observed. Other nanotubes appeared to have been formed using as a base the catalyst that, remaining at one end of the nanostructure (Fig. 1c), was largely exposed to the external environment. As previously reported, pMWCNT had an average length of 10–20 m and a diameter close to 15–30 nm, while fMWCNT were by far shorter (average length between 200 and 1000 nm) and showed an external layer eroded at many points, due to the oxidative insertion of terminal functional groups. Due to their hydrophobicity common to all CNTs, our MWCNTs, in particular pMWCNT, had a strong propensity to agglomerate in water. In order to minimize this feature, which makes MWCNT/cell interactions highly variable, we made concentrated MWCNT suspensions (100 x in PBS) that were sonicated for 20 min in an ice bath. Since proteins stabilize MWCNT suspensions [18], just before each experiment, the concentrated suspensions were diluted in cell culture medium (containing 2% FCS) and again subjected to a fast sonication (3 min). To assess the dissolution ratio of iron under physiological conditions, additional experiments were performed. A first set of tests consisted in treating cells with the supernatant obtained from cell culture medium (containing 2% FCS) preconditioned with nanotubes. Briefly, the MWCNT suspensions were incubated at 37 ◦ C for 48 h and then centrifuged (3000g for 10 min). Considering the leaning of nanotubes to the sedimentation, this treatment allowed to obtain the MWCNT-free supernatants that were used for the experiments. AAS analysis was performed on the preconditioned media and on the untreated cell medium. No relevant differ-
ence in terms of iron content were observed. The values were 50, 52 and 51 mg/L−1 for the untreated, pMWCNT- and fMWCNTpreconditioned media respectively (P > 0.05). To assess the possible iron leakage from the nanotubes subjected to the action of reactive oxygen species, we performed further abiotic experiments. Briefly, the MWCNTs were placed in contact with the ROS generated in situ by treating peroxides in the presence of Cu2+ catalyst. Both p-MWCNT and f-MWCNT samples (50 g mL−1 ) were separately treated with hydrogen peroxide or peroxyacetic acid (400 M), in deionized water at 37 ◦ C, in the presence of a catalytic amount of CuSO4 (50 M) and incubated for 6 h, as in in vitro experiments. Then, the mixtures were centrifuged (3500g for 10 min) and the supernatants were analyzed by AAS spectroscopy while MWCNTs were washed by PBS and analyzed to assess the presence of redox active iron. 2.1. Cells and exposure conditions As an in vitro cell model, we used the human alveolar cell line A549, derived from a lung carcinoma (ATCC, Rockville, USA). Cells were cultured in RPMI medium with 2 mM L-glutamine, 10% (v/v) fetal calf serum (FCS), 100 IU mL−1 penicillin and 100 g mL−1 streptomycin at 37 ◦ C in a humidified 5% CO2 atmosphere. All reagents for cell cultures were purchased from Gibco (Invitrogen Gibco, Milan, Italy). For all experiments, we used subconfluent monolayers grown in 6-well cell plates. In parallel, A549 cells were exposed to pMWCNT and fMWCNT (50 g mL−1 ), to their respective supernatants, as reported above, and to an iron solution at the same concentration present in pMWCNTs. This was obtained from (NH4 )2 Fe(SO4 )2 (final concentration 54.4 M) and used as a positive control. Monolayers treated with PBS were included in each experiment as a negative control (i.e. control cells). To confirm MWCNT uptake, previously quantified in the same cell model [16], we performed a qualitative analysis by microscopic observation of A549 semiconfluent monolayers. The cells were grown on cell slides (Invitrogen Gibco) and treated as reported above for 180 min. 2.2. Assessment of iron bioavailability To study the role of iron in the toxicological effect of MWCNTs, we used two different analyses aimed to assess the bioavailability of MWCNTs-associated iron and to measure the intracellular concentration of redox-active iron in exposed cells. The first analysis was performed by using a fluorimetric abiotic assay, based on the method devised by Esposito et al. [19].Briefly, in MWCNT suspensions (50 g mL−1 ), both as such and subjected to the action of ROS, and in the supernatant of MWCNT pre-treated cell culture medium, we measured the oxidation of the non-fluorescent probe dihydrorhodamine (DHR) to its fluorescent form rhodamine, a reaction catalyzed by redox-active iron. To exclude other DHR oxidation mechanisms not caused by the presence of redox-active iron, the samples were analyzed in parallel by the addition of the iron chelator deferoxamine mesylate (DFX),which quenches only iron-induced fluorescence. Briefly, samples were assayed in quadruplicate in 96-well plates by adding the DHR (50 M) in reagent solution (pH 7.3) containing 40 M of ascorbate to regenerate Fe(II) after its oxidation to Fe(III). DFX (50 M) was added to this solution in two of the wells. A Fe:NTA (nitrilotriacetic acid) (1:7 mM) complex, starting from freshly prepared FAS (NH4 )2 Fe(SO4 )2 and NTA (pH 7.0), was used to build a calibration curve (1–54.4 M). To assess the kinetics of the reaction, emitted fluorescence was recorded every 2 min, starting from 15 min up to 40 min, by using 485/535 nm excitation/emission filters (plate reader Tecan, Brescia, Italia). The differences between the samples with and without DFX, due to redox-active iron, were used to calculate fluorescence units
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Fig. 1. HRTEM images of pMWCNT that are formed of 15–20 layers and show outer surfaces smooth and well-graphitized. (A and B) Iron catalyst clusters are encapsulated within the multiwalled nanostructures. (C) The iron catalyst cluster is exposed to the external environment as present at one end of the nanostructure.
per minute (FU/min). Iron values (M) were extrapolated from the calibration curves. The intracellular concentration of redox-active iron was measured by using an argon laser flow cytometry instrument (Dako Galaxy Cytomation, Glostrup, Denmark) and calceinacetoxymethyl ester (calcein-AM) as a probe. This non-fluorescent non-chelating lipophilic ester easily penetrates cellular membranes and diffuses into cells. There, it is rapidly cleaved by unspecific cytosolic esterases, producing the fluorochromic alcohol calcein ( ex 488 nm; em 518 nm) that chelates intracellular uncomplexed iron (free iron) [20]. This reaction quenches the green fluorescence of calcein in a dose-dependent manner, allowing the detection of bioavailable iron. To validate Calcein assay, we used the specific iron chelator deferiprone (3-idrossi-1,2-dimetilpiridin-4(1H)-one: DFP). In presence of redox-active iron, DFP restores calcein fluorescence by the probe dequenching. DFP is a well-tested drug that, displaying a high binding affinity for the iron, is widely used in the therapy of siderosis. It is a bidentate ligand fat-soluble and therefore capable to complexing intracellular iron. Briefly, A549 monolayers were treated for 300 min as reported above and then cells were harvested by trypsinization (0.25% trypsin–1 mM EDTA). For each sample two cell suspensions (1 × 105 mL−1 in PBS containing 10 mM D-glucose at pH 7.4) were loaded with calcein-AM (final concentration 60 nM) and, after incubation at 37 ◦ C for 5 min, only in one was added DFP (final dose: 0.3 M), incubating again for 10 min. Cell suspensions were then submitted to FACS analysis collecting signals in the fluorescence channel 1. Intracellular iron was assessed by the differences in emission values between cell suspensions treated with the mix (calcein+ DFP) and the ones treated with calcein only. 2.3. Assessment of MWCNT-induced toxicity In treated A549 cells we used FACS to assess cell viability and the intracellular production of ROS. Moreover, to assess oxidative damage in cell compartments, we evaluated genotoxicity by measuring 8-oxo-dG and mitochondrial function by assessing m . After treatment of monolayers and trypsinization, as reported above, cell suspensions (1 × 105 mL−1 in PBS added by 10 mM Dglucose at pH 7.4) were separately loaded by the probes. These were propidium iodide (3 g mL−1 ), 2 ,7 -dichlorofluorescein-diacetate (DCF-DA) (1 M), FITC-labelled avidin (0.2 M) and rhodamine 123 (R123: 10 M) (Invitrogen) to detect cell viability, the intracellular production of ROS, 8-oxo-dG and m , respectively. Starting from 1 h to 6 h, at intervals of 1 h, a time-course was performed. For each treatment, at each time point, four different cell aliquots were taken and loaded with the respective probes as previously reported [21,22]. Specifically, we used a FITC-labelled avidin probe, which is
known to bind with high specificity to 8-oxo-dG, to assess oxidative DNA damage [23].Since the interference of MWCNTs prevented us from performing the more commonly used colorimetric viability tests (e.g. MTT), we detected dead cells stained with a DNA intercalating probe by measuring emission signals in the FL3 channel. For intracellular ROS and 8-oxo-dG detection, emission signals were recorded in the FL1 channel while the emitted fluorescence of R123 was collected in the FL2 channel. FACS analyses were performed in triplicate and separately for each fluorochrome. The weighted average of emission values for 100 cells was calculated and expressed in arbitrary fluorescence units (AFU). 2.4. Statistical analyses All data are presented as mean ± standard error (SE) based on at least triplicate observations from three independent experiments. Data were analyzed by one-way analysis of variance (ANOVA) and multiple comparisons of the means were performed by the Tukey-Kramer test (GrafPAD Software for Science). The relationships between different parameters were assessed by the Pearson correlation coefficient. Significance was accepted at P < 0.05. 3. Results 3.1. Iron bioavailability of MWCNTs The results of the fluorimetric assay to measure the pro-oxidant potential of MWCNT-associated iron are reported in Fig. 2A. The cell-free method, allowing us to exclude probe oxidation not caused by iron, clearly highlighted the absence of redox-active iron in the assayed MWCNTs. In particular, the emission values due to the oxidation of DHR almost overlapped in the two sets of wells (with and without the chelator DFX). Similar results were also obtained by testing the supernatant obtained from cell culture medium pretreated with nanotubes and used to evaluate iron dissolution under physiological conditions (Fig. 2A). Moreover we used this abiotic test to assess the possible iron leakage from the nanotubes subjected to the action of reactive oxygen species present in specific cell compartments. As shown by the graph (Fig. 2A), the treatment with in situ generated ROS not made the iron content of MWCNTs more accessible and bioavailable. Similar results were recorded by the analysis of the supernatants obtained by this MWCNT treatment for 6 and 24 h. Indeed, the AAS spectroscopy analysis did not detect traces of iron ions in the supernatants, excluding the iron leakage from the nanomaterials (data not shown). Despite these results, exhibiting the lack of MWCNT-associated bioavailable iron, the FACS analysis of intracellular redox active iron underlined its higher presence in A549 cells exposed for 6 h
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Fig. 2. Iron bioavailability of MWCNTs. (A) Results of fluorimetric assay to assess the pro-oxidant potential of MWCNT-associated iron. pMW: pristine MWCNT, fMW: functionalized MWCNT, pMW* and fMW*: MWCNTs treated with in situ generated ROS, SpMW and SfMW: supernatant obtained from cell medium preconditioned (at 37 ◦ C for 48 h) with pMWand fMWrespectively. Calibration curve was built by using a Fe:NTA (complex and the iron content was in the range 1.7–54.4 M, reported in the graph as St 1–6 respectivly. The results are expressed as the average values of FU/min in the interval 15–40 min. (B) Cytofluorimetric analysis to measure intracellular concentration of redox-active iron by using calcein-AM as a probe and DFP to restore calcein fluorescence by breaking of the complex Fe-calcein. The presence of bioavailable intracellular iron quenched the calcein fluorescence while the probe dequenching, in presence of redox-active iron, was obtained by DFP. Intracellular iron is highlighted by the differences between cell suspensions treated with the mix (calcein+ DFP) and the ones treated with calcein only. Cells exposed to (NH4 )2 Fe(SO4 )2 (54.4 M) were used as positive control. (All data are reported as means ± SE of three separate experiments). In pristine and oxidized MWCNTs and in Fe (II)- treated cells the differences in emission values were significant (*P < 0.05).
to MWCNTs. To assess the intracellular iron, emission values of calcein were recorded in cell suspensions loaded with only this metal sensor and in the ones treated also with the iron chelator DFP which restored calcein fluorescence. As shown in Fig. 2B, there was no difference between the two measurements in control cells and in cells exposed to both MWCNT preconditioned medium, highlighting the lack of intracellular free iron. Instead, the cells treated with the assayed pristine and oxidized nanostructures showed a quenching of calcein fluorescence which was almost entirely restored by DFP. In particular, the differences between the measurements obtained in the presence and in the absence of DFP were equal to 28 and 25% in cells treated with pristine and oxidized MWCNTs respectively (P < 0.05). A similar difference was observed in the positive control (i.e. Fe (II)- treated cells). 3.2. MWCNT-induced toxicological effect The qualitative assessment of MWCNT uptake confirmed previous results (Fig. 3). In A549 semiconfluent monolayers exposed for 3 h to either pristine or oxidized MWCNTs, uptake was highlighted by the presence of dark aggregates in the cytoplasm that were bulkier and more numerous in pMWCNT treated cells (Fig. 3B and C). Moreover, consistent spaces were observed, indicating the detachment of dead cells. The monolayers exposed to
the Fe (II) solution (54.4 M) had large and numerous spaces, indicating high Fe-induced cytotoxicity to the tested dose/time (Fig. 3.F).Conversely, cells exposed to the MWCNT preconditioned cell medium were similar to control cells (Fig. 3A, D and E), underlining the absence of iron leakage from the assayed MWCNTs under physiological conditions. In order to compare the biological effect of MWCNTs to those caused by iron at the same concentration present in the assayed nanostructures, cell viability and ROS production were detected. The used doses were 50 g mL−1 and 54.4 M for MWCNTs and the iron solution, respectively. The same analyses were performed by using MWCNTs-pre-treated medium for cells exposure. The time course of cell viability, detected using propidium iodide to stain dead cells, showed significant cytotoxicity elicited by MWCNTs. Fig. 4 A shows the percent increase ( %) of dead cells in comparison to the control cells. The % was time-dependent, and in cells treated with both MWCNT preparations, on average it was equal to 18 and 17% for pristine and oxidized MWCNT, respectively (P < 0.05 at 4 and 6 h). In cells treated with Fe (II) solution the % were higher and the differences were significant at all-time points examined in comparison with control cells (P < 0.05). On the contrary, dead cells were almost absent in cells exposed to culture medium pre-treated with MWCNTs, confirming the microscopic observations. The time-course of ROS production is reported in Fig. 4 B. FACS analysis highlighted the time-dependent pro-oxidant effect of the assayed nanostructures. In comparison with control cells, the emission values of the oxidized probe at all-time points examined were significantly higher (P < 0.01) in MWCNT exposed cells. On average, the AFU values were10.7- and 6.8-fold greater than in control cells in response to pristine and oxidized MWCNTs, respectively; similarly, in positive control (i.e. cells exposed to redox active iron), the value was 8.2-fold higher than in cell control, further indicating the strong oxidative potential of the assayed MWCNTs. Instead, the values of emitted fluorescence were similar to control cells after exposure to cell culture medium preconditioned with MWCNTs. To confirm the pro-oxidant effect of MWCNTs, we assessed the effect of oxidative damage in cell compartments. As expected, significantly related to ROS overproduction (P < 0.01), we observed mitochondrial impairment and enhanced DNA oxidative damage. In particular, the time course of the R123 emission values, used to assess mitochondrial transmembrane potential, highlighted progressive mitochondrial failure, emphasizing the metabolic impairment induced by our tested carbon nanotubes. Fig. 4C reports the changes of mitochondrial potential in cells exposed to both MWCNT suspensions, to iron solution and to preconditioned cell medium. The opposite trend to ROS overproduction was observed in cells treated with MWCNTs and iron solution. In these cells the mitochondrial impairment was evident from the start even though the differences were significant only at latest time points examined (at 4 and 6 h: P < 0.05). In comparison to control cells, at 6 h m was 88.2,82.9 and 86.0% in cells treated with pristine and oxidized nanotubes and iron solution, respectively. Similar to ROS detection, no effect on mitochondrial function was observed in cells exposed to pretreated medium. In these cells the values of m were roughly overlapped to negative control (±5%). In the tested times the average values of percentage changes in comparison to negative control were 100.8 and 101.3 in pristine and functionalised MWCNT preconditioned media respectively), confirming the absence of leakage from the assayed MWCNTs. The outcomes of free radical-initiated oxidation upon cellular exposure to MWCNTs were also observed in the nuclear compartment of A549 cells. As indicated by FACS analysis using as probe FITC-labelled avidin, treated cells showed a time-dependent increase in 8-oxo-dG (free radical-induced oxidative lesions) compared to untreated cells. Fig. 4 D reports the average values in
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Fig. 3. Phase contrast microscopy images of A549 semiconfluent monolayers to show MWCNTs uptakein cells treated for 3 h. (A) control cells, (B and C)cells exposed to pristine and functionalized MWCNT. (D and E) cells exposed to S pMW and S fMW. (F) cells exposed to (NH4 )2 Fe(SO4 )2 solution (54.4 M).
Fig. 4. Toxicological effects of MWCNT exposure assessed by FACS analysis. Cells exposed to (NH4 )2 Fe(SO4 )2 solution (54.4 M) were used as positive control. (A) Time course of cell viability. The graph reports the percent increase (% ) of dead cells in comparison to the control cells. The% were significant at 4 and 6 h for pristine and oxidized MWCNT, respectively (P < 0.05). In cells treated with Fe (II) solution the % were significant at all-time points examined in comparison with control cells (P < 0.05) In the graph are reported the linear trend lines. (B) Time-course of ROS production assessed by emission values of DCF. Considering the very fast ROS production, the interval 30 min–6 h was evaluated. Data are reported as% and the linear trend lines are drawn. In comparison with control cells, the emission values of the oxidized probe at all-time points examined were significantly higher (P < 0.05 and P < 0.01) in MWCNT exposed cells and in positive control. (C) Time course of m . Data of R123 emission (expressed as AFU) show, compared to control cells, the reduction of mitochondrial potential in cells exposed to both MWCNT suspensions and to iron solution. In pristine and oxidized nanotubes and in iron solution, the differences were significant only at latest (4 and 6) time point examined (P < 0.05). The graph reports the logarithmic trend lines (D) 8-oxo-dG detection by FITC-labelled avidin emission to assess oxidative DNA damage. The graph reports the percent increase ( %) of 8-oxo-dG in nanotubes and iron solution treated cells (positive control) respect to control cells (*: P < 0.05). Data are expressed as average values of % in the time interval 1–6 h. (Data of graphs A, B, C and D are reported as means ± SE of three separate experiments.
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the time interval 1–6 h of the percent increase ( %). The emitted fluorescence values were significantly higher (P < 0.05)in MWCNT exposed cells than in control cells, suggesting that synthetic nanoparticles induced DNA oxidative damage in pneumocytes. The 8-oxo-dG increases were slightly higher in pMWCNTs treated cells in comparison to cells treated with oxidized nanotubes and iron solution (1.95 vs. 1.60 and 1.50 fold respectively). Similar to the ROS and m analyses, negative results were obtained after cell exposure to preconditioned medium.
4. Discussion Thorough knowledge of MWCNT-induced pathogenic mechanisms would allow a change in the synthesis process to reduce their bioreactivity. The design of safer nanofibres is extremely important considering the ease with which lightweight nano-sized MWCNTs aerosolize, increasing pulmonary disorders after their inhalation. Several authors have attributed a key role of MWCNT-induced toxicity to the iron present as residual catalyst in these nanomaterials, demonstrating that iron-free particles, including synthetic asbestos fibres were biologically inactive [14,15]. In a previous study [16] we obtained similar results observing that, in cells exposed to MWCNT–chelating agent mixtures, oxidative damage was almost completely reset even if the harmful effect related to MWCNT-induced mechanical damage retained unchanged. The results obtained in the present study have enabled us to better define the MWCNT-triggered pathogenic pathways, excluding the iron bioavailability of the homemade tested nanoparticles. Despite non-negligible levels of iron (54.4 and 43.9 M in pMWCNT and fMWCNT, respectively), its content was not leached in physiological conditions and it was not bioavailable. Contrary to that reported by Vales et al. [24], who observed a pro-oxidant effect in the supernatant obtained from CNT-preconditioned cell medium, our nanomaterials were devoid of easily leachable iron. Moreover, the complete absence of biological effects in cells exposed to preconditioned medium suggested that even amorphous carbon, in addition to iron, was not present in our homemade MWCNTs, highlighting their high degree of purity. More importantly, we did not detect redox-active (i.e. bioavailable) iron in either MWCNT in physiological conditions, despite residual catalyst also being present on the surface in pMWCNT at one end of the nanostructure and, therefore, potentially active toward cells. Unlike functionalized MWCNTs, pristine ones were not subjected to strong oxidizing acids (HNO3 and H2 SO4 ) able to leach the superficial residual catalyst. Independently of being encapsulated or present on the surface, the entire iron content was not bioavailable and therefore harmful in our cell model. This is easily explained, as iron(III) oxide, present in the tested MWCNTS, is extremely stable, typically insoluble in aqueous solutions and, consequently, unable to trigger the well-known Haber-Weiss and Fenton reactions that produce strongly reactive hydroxyl radicals. This feature substantially differentiates these homemade MWCNTs from asbestos fibres, especially amphiboles such as crocidolite and amosite, in which the iron, as well as being plentiful and always located on the surface, is in a redox and coordination state to allow a pro-oxidant effect [14,25]. The lack of bioavailable iron in our MWCNTs only apparently contradicted previous findings showing the antioxidant effect of the chelating agent. The significant increase of intracellular iron highlighted in this study would seem to confirm the hypothesis that oxidative damage in cells exposed to nanotubes is mainly caused by the release of endogenous redox-active iron. This is due to damage to the acidic vacuolar compartment, previously observed in the same cell model, caused by MWCNT internalization. As confirmed here, MWCNT uptake in pneumocytes is very
efficient and may occur through passive diffusion across the cellular membrane [26], favoured by the strong hydrophobicity of CNTs [27]. An alternative mechanism is clathrin- and/or caveolaemediated endocytosis/phagocytosis [28]. MWCNT internalization would seem to cause the release of intralysosomal redox-active low molecular mass iron, produced during autophagocytic degradation of metalloproteins such as cytochromes [29]. This would explain the increase of redox-active intracellular iron observed in MWCNT-exposed cells; instead, the possible release of iron from the nanotubes should be excluded as the phagolysosome pH is not low enough. As observed by using phagolysosomal simulant fluid [30], metals in CNTs were stable for up to two months [31]. This is certainly true for our CNTs, considering the strong mineral acids treatment (i.e. HCl for the purification step of pMWCNT and H2 SO4 /HNO3 for the synthesis of fMWCNT) and sonication procedures, involving also the removal of the released material at each steps (16), to which they were subjected. Moreover our results highlighted as the iron content of the tested nanomaterials was not released even in “extreme physiological conditions” such as those present exclusively in specific cell compartments of specialized cells. Due to oxidative burst, professional phagocytic cells as macrophages, neutrophils etc. produces highly reactive oxygen and nitrogen derivatives to kill engulfed pathogens. Despite we used alveolar epithelial cells as biological model, in the abiotic experiments simulating oxidative burst, we did not observe an iron release of our MWCNTs in presence of ROS. On this basis, we can reasonably rule out the iron release from the assayed MWCNTs in the biological conditions and the time considered in our study. Therefore, the increase of redox-active iron could be related to the release of endogenous iron, caused by endocytosis–mediated MWCNT internalization. In addition to the effect of redox-active iron, likely released by MWCNT-induced cell damage, we may presume that the strong cytotoxicity was due to features of MWCNTs. In comparison to isometric particles, the fibrous shape with nano-sized diameter of CNTs plays a key role in nanotube-induced toxicity, causing phagolysosomal dysfunction. Previously, we observed that damage in the cellular acid compartment was inversely related to cell vitality, thus strongly underlining the importance that this pathway may have in the pathogenicity of CNTs. Due to increased lysosomal permeability, several hydrolytic enzymes are released in the cytoplasm. This can induce cell death by apoptosis, similarly to what has been shown by silica particles [32], or autophagic activation, as suggested for fullerenes [33]. Alongside the fibrous aspect, the surface properties should be considered as essential parameters in CNTinduced cytotoxicity [34,35]. The presence of oxygenated groups and of several surface defects that modify the graphene structure in our functionalized MWCNTs would explain the similar toxicity elicited by pristine (extremely long) and oxidized MWCNTs. Regardless of the presence of metals, almost all studies report redox imbalance in SWCNT- and MWCNT-exposed cells [36–40]. This common nanotube-induced pathogenic effect was assessed by the detection of ROS overproduction and/or oxidative damage to macromolecules (i.e. lipid hydroperoxides, oxidized DNA, etc.), as well as the depletion of antioxidant molecules (i.e. reduced glutathione). Our results confirmed that ROS overproduction in exposed cells was strongly related to mitochondrial failure and to significantly increased levels of 8-oxo-dG. This marker of oxidative DNA damage strengthens the marked genotoxicity, previously observed by using the micronuclei test (MNi) and Comet assay [16]. Both these effects, in addition to phagolysosomal dysfunction, clearly demonstrate the broad spectrum of toxicological effects of the tested MWCNTs. Overall, the toxicological effects of the homemade tested MWCNTs, devoid of biavalable iron, unlike many commercially available
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Please cite this article in press as: G. Visalli, et al., The role of the iron catalyst in the toxicity of multi-walled carbon nanotubes (MWCNTs), J Trace Elem Med Biol (2017), http://dx.doi.org/10.1016/j.jtemb.2017.01.005