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 ARTICLE IN PRESS G Model JTEMB-25884; No. of Pages 8 Journal of Trace Elements in Medicine and Biology xxx (2017) xxx–xxx Contents lists available at ScienceDirect Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.com/locate/jtemb 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 b 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 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%. Pre- liminarily, 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 redox- active 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 oxida- tive 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 respi- ratory system following inhalation. Inhaled single and multi-walled carbon nanotubes (SWCNTs and MWCNTs, respectively), penetrate Abbreviations: CNT, carbon nanotubes; pMWCNT, pristine multi-walled car- bon 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)2Fe(SO4)2; NTA, nitrilotriacetic acid; calcein-AM, calcein- acetoxymethyl ester; ym, mitochondrial transmembrane potential; DCF-DA, 2 ′ ,7 ′ -dichlorofluorescein-diacetate; FAU, fluorescence arbitrary units; 8-oxo-dG, 8- Oxo-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: gvisalli@unime.it (G. Visalli), alessiof1976.af@gmail.com (A. Facciolà), diannazzo@unime.it (D. Iannazzo), apiperno@unime.it (A. Pistone), adipietr@unime.it, pistone@unime.it (A. Di Pietro). deeply, inducing pulmonary inflammation, cytotoxicity and car- cinogenesis [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 bioper- sistence [9,10]. Similar to asbestos, CNTs are also able to cause mesothelioma [11] as well as alveolitis, pulmonary fibrosis, granu- loma 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 http://dx.doi.org/10.1016/j.jtemb.2017.01.005 0946-672X/© 2017 Elsevier GmbH. All rights reserved.