Nanoscale This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online COMMUNICATION View Journal | View Issue Nitric oxide-dependent biodegradation of Cite this: Nanoscale, 2020, 12, 16730 graphene oxide reduces inflammation in the Open Access Article. Published on 10 August 2020. Downloaded on 1/7/2022 5:30:50 PM. gastrointestinal tract† Received 11th May 2020, Guotao Peng, a Marcelo F. Montenegro, b Chifundo N. M. Ntola,c Accepted 7th August 2020 Sandra Vranic,c Kostas Kostarelos, c,d Carmen Vogt,e Muhammet S. Toprak, e DOI: 10.1039/d0nr03675g Tianbo Duan,f Klaus Leifer, f Lars Bräutigam,g Jon O. Lundbergb and rsc.li/nanoscale Bengt Fadeel *a Understanding the biological fate of graphene-based materials and animal peroxidases, including myeloperoxidase (MPO), such as graphene oxide (GO) is crucial to assess adverse effects has been documented.2,3 Purified human MPO was shown to following intentional or inadvertent exposure. Here we provide degrade highly dispersed GO, but failed to metabolize aggre- first evidence of biodegradation of GO in the gastrointestinal tract gated GO.3 Furthermore, we demonstrated in a previous study using zebrafish as a model. Raman mapping was deployed to that primary human neutrophils are capable of degrading GO assess biodegradation. The degradation was blocked upon knock- when activated to undergo degranulation with release of down of nos2a encoding the inducible nitric oxide synthase (iNOS) MPO,4 while GO functionalized with a chemotactic peptide or by pharmacological inhibition of NOS using L-NAME, demon- triggered neutrophil activation with subsequent degradation of strating that the process was nitric oxide (NO)-dependent. NO- GO.5 Single- and few-layer graphene is also susceptible to enzy- dependent degradation of GO was further confirmed in vitro by matic degradation though the process was found to be less combining a superoxide-generating system, xanthine/xanthine efficient as compared to GO.6 oxidase (X/XO), with an NO donor (PAPA NONOate), or by simul- Previous studies on single- and multi-walled carbon nano- taneously producing superoxide and NO by decomposition of tubes (CNTs) have revealed the propensity of these materials to SIN-1. Finally, by using the transgenic strain Tg(mpx:eGFP) to visu- undergo peroxidase-mediated biodegradation.7–10 Furthermore, alize the movement of neutrophils, we could show that inhibition CNTs may also undergo peroxynitrite (ONOO−) dependent of the degradation of GO resulted in increased neutrophil infiltra- biodegradation in macrophages11,12 and chondrocytes.13 tion into the gastrointestinal tract, indicative of inflammation. Peroxynitrite can be generated by the rapid reaction of super- oxide radicals with nitric oxide (NO) according to the following Graphene and its derivatives have been intensively explored for reaction: a multitude of biomedical and other applications. This, there- fore, mandates a comprehensive assessment of the potential O2 • þ NO• ! ONOO impact of these materials on human health.1 In recent years, attention has been focused on the biological fate of graphene- The peroxynitrite anion produced in this manner displays based materials, including their potential biodegradability. high diffusibility,14 obviating the need for a direct interaction Thus, enzymatic degradation of graphene oxide (GO) by plant of CNTs with the participating enzymes.11 However, no study has been conducted to investigate the possibility of NO-driven biodegradation of GO. Here we studied GO produced by the a Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden. Hummers’ method to investigate the in vitro and in vivo E-mail: bengt.fadeel@ki.se degradability. The physicochemical characterization results are b Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, summarized in Table S1.† Based on the evaluation of GO by Sweden c National Graphene Institute, and Faculty of Biology, Medicine and Health, TEM, SEM, optical microscopy and AFM, we found that 90% of University of Manchester, Manchester, UK the sheets ranged between 0.1 to 15 µm in lateral dimensions d Catalan Institute of Nanoscience and Nanotechnology (ICN2), Barcelona, Spain (representative TEM images are shown in Fig. 1a). AFM ana- e Department of Applied Physics, KTH Royal Institute of Technology, Stockholm, lysis revealed that the thickness of the GO sheets was between Sweden f 1 to 2 nm, allowing us to conclude that GO was mainly present Department of Engineering Sciences, Uppsala University, Uppsala, Sweden g Comparative Medicine, Karolinska Institutet, Stockholm, Sweden as single- or few-layer sheets in water and after incubation for † Electronic supplementary information (ESI) available. See DOI: 10.1039/ 24 h in E3 medium (Fig. 1b and c). Note that the spikes in d0nr03675g thickness following incubation in E3 medium are due to 16730 | Nanoscale, 2020, 12, 16730–16737 This journal is © The Royal Society of Chemistry 2020 View Article Online Nanoscale Communication This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 10 August 2020. Downloaded on 1/7/2022 5:30:50 PM. Fig. 1 Structural characterization of GO sheets after incubation in E3 medium versus water using transmission electron microscopy (TEM) (a) and atomic force microscopy (AFM) (b) height image and (c) cross section analysis highlighted by the line in the height image. wrinkling and folding of GO sheets as a result of incubation in employed two approaches: (i) downregulation of the inducible a high salt containing medium. NO synthase (iNOS) by using morpholino oligonucleotides Inhalation is the main route of unintentional exposure to directed against nos2a; and (ii) inhibition of NOS by using the engineered nanomaterials, and the lungs therefore represent cell-permeable inhibitor, N-omega-nitro-L-arginine methyl ester the most immediate target organ for their toxic effects. hydrochloride (L-NAME). The successful downregulation of However, extrapulmonary effects of inhaled nanomaterials nos2a was documented by RT-qPCR (Fig. 2b). As demonstrated including effects on the gastrointestinal (GI) tract have been in Fig. 2c, GO accumulated in the GI tract and could be detected reported; additionally, nanomaterials can be ingested leading to on the basis of its characteristic Raman signature, i.e., the D a direct exposure of the GI tract.15 Despite the potential impli- band (1354 cm−1), G band (1582 cm−1), and 2D band cations on human health, the impact of nanomaterials on the (2690 cm−1).19 The background signal of the fish tissues did not GI tract has been largely overlooked.16 We focused here on oral interfere with the detection of GO by Raman (data not shown). exposure to GO. It is well known that NO is produced in the GI Importantly, our interventions (shown schematically in Fig. 2a) tract through enzymatic and non-enzymatic reactions,17 and we resulted in a pronounced change in the intensity of the D and G hypothesized that GO may undergo NO-dependent bio- band when compared to untreated, GO-exposed fish, indicating degradation (Fig. 2a). To address this question, we used zebra- that GO was biodegraded in the latter case through an NO- fish embryos that have emerged in recent years as a robust dependent mechanism (Fig. 2d). It is worth noting that a pre- in vivo model in toxicology and drug discovery.18 The degra- vious study showed no degradation of GO when immersed in dation of GO in zebrafish larvae (5 days postfertilization, dpf) simulated digestive juices representative of the GI tract, as eval- was evaluated using confocal Raman mapping. To investigate uated by Raman analysis.20 This allows us to rule out a role of the mechanism of biodegradation of GO in the GI tract, we gastrointestinal fluids in the degradation of GO. This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 16730–16737 | 16731 View Article Online Communication Nanoscale This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 10 August 2020. Downloaded on 1/7/2022 5:30:50 PM. Fig. 2 NO-dependent biodegradation of GO in vivo. (a) Schematic diagram showing the generation of nitric oxide (NO) by iNOS followed by the reaction of superoxide radicals and NO to form the potent oxidant, peroxynitrite. (b) Relative mRNA expression level of nos2a following knockdown of nos2a. (c) Raman confocal mapping of GO in zebrafish. GO was found to accumulate in the gut upon oral exposure, as demonstrated by optical and Raman confocal images. (d) Zebrafish larvae at 5 dpf were exposed to GO at 5 µg mL−1 for 24 h. Morpholino oligonucleotides (MO) were injected into one-cell stage zebrafish embryos to downregulate nos2a expression. L-NAME was used to inhibit NOS in conventional fish. Each spec- trum represents the average of 10.000 spectra across the whole area scan. To support the role of NO in the biodegradation process, we in vitro experiments by using two peroxynitrite-generating measured NO production in vivo by using the fluorescent systems, i.e., simultaneous production of superoxide radicals probe, DAF-FM-DA. Bacterial lipopolysaccharide (LPS) was and NO by decomposition of 3-morpholinosydnonimine used to trigger NO production (Fig. 3a). NO production was hydrochloride (SIN-1), or the combination of a superoxide-gen- suppressed by L-NAME (Fig. 3b). Exposure to GO also led to an erating system, xanthine/xanthine oxidase (X/XO), with an NO increase in NO (Fig. 3b). Importantly, knockdown of nos2a donor (PAPA NONOate).11 As shown in Fig. 4a, GO degradation with morpholino oligos resulted in a decline in LPS- and GO- after 5 days of continuous incubation in the presence of SIN-1 induced NO production (Fig. 3b; representative images of zeb- or PAPA NONOate-X/XO was visually detectable as the suspen- rafish embryos are shown in panel a). We also detected peroxy- sions turned translucent in both cases. To more objectively nitrite generation in vivo by using DAX-J2 PON Green, a fluo- document the degradation, we performed Raman spec- rescent probe with high selectivity for peroxynitrite and negli- troscopy. The characteristic D and G bands of GO were remark- gible reactivity with other relevant species including NO. ably diminished in both systems suggesting a near-complete Peroxynitrite production was observed in vivo in response to loss of structure of GO (Fig. 4b). The topography and structure GO and to a lesser extent with LPS and the signal was sup- of GO incubated with the two peroxynitrite-generating systems pressed by L-NAME (Fig. S1†). was further characterized by TEM (Fig. 4c) and AFM (Fig. 4d). To further confirm the degradation of GO and provide more Specifically, after PAPA NONOate-X/XO treatment, the typical insight regarding the NO-dependent reaction, we performed morphology of the GO sheets disappeared, and only small frag- 16732 | Nanoscale, 2020, 12, 16730–16737 This journal is © The Royal Society of Chemistry 2020 View Article Online Nanoscale Communication This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. Open Access Article. Published on 10 August 2020. Downloaded on 1/7/2022 5:30:50 PM. Fig. 3 NO production in zebrafish. (a) Representative fluorescence images of NO production in zebrafish gut, represented as the corrected gut flu- orescence determined by DAF-FM-DA assay. The gut is indicated by the white dashed line. (b) Quantification of NO production determined by DAF-FM-DA assay. NO production was induced by LPS and GO, while L-NAME and downregulation of nos2a significantly inhibited NO production. Student’s t-test was used to evaluate the statistical significance between treated fish and untreated controls (* p < 0.05, ** p < 0.01, *** p < 0.001), as well as the LPS and GO-exposed fish with/without NOS inhibition/suppression (# p < 0.05, ## p < 0.01, ### p < 0.001). Fig. 4 NO-dependent degradation of GO in vitro. (a) Visual evidence of degradation upon incubation of GO with the peroxynitrite donor, SIN-1 or PAPA NONOate-X/XO for 5 days. (b) Raman analysis of GO after incubation with SIN-1 and PAPA NONOate-X/XO for 5 days showed almost com- plete degradation of GO as evidenced by loss of the characteristic D and G bands. (c) Representative TEM images of untreated GO versus GO exposed to SIN-1 and PAPA NONOate-X/XO for 5 days. Numerous defects in the GO sheets are visible in SIN-1 exposed samples, in particular, as well as in GO exposed to PAPA NONOate-X/XO. (d) AFM height contrast images of representative GO samples with or without SIN-1 and PAPA NONOate-X/XO. The surface of pristine GO is flat, with a mean square roughness (MSR) of 0.112 nm and a thickness of 1–2 nm, while in GO sheets exposed to SIN-1, several defects of lateral sizes ranging from 50 nm to 200 nm appear, which could be attributed to the degradation of GO. Furthermore, GO incubated with PAPA NONOate-X/XO shows greater roughness (MSR = 1.655 nm) and an increase of the thickness of GO, which could be due to the aggregation of the degraded smaller flakes of GO. This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 16730–16737 | 16733 View Article Online Communication Nanoscale ments could be observed. In addition, SIN-1 treatment led to generated a negligible signal (Fig. 5c). Collectively, these numerous perforations of the GO sheets, as shown by TEM, results suggest that the degradation of GO by PAPA NONOate- and these defects were also seen by AFM. To confirm that the X/XO may be attributed to NO, but not to superoxide radicals, two systems were functional, we monitored NO and peroxy- and that the decomposition of SIN-1 produces superoxide and This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. nitrite production based on real-time chemiluminescence and NO resulting in rapid formation of ONOO−, which ‘digests’ fluorescence detection, respectively. PAPA NONOate induced a GO. One may ask what the potential advantages or disadvan- robust and stable NO signal, and the addition of X/XO was tages would be of NO-driven degradation of GO. One impor- able to decrease the NO signal, likely due to the rapid reaction tant aspect is that NO-driven degradation does not require between NO and the superoxide radicals (Fig. 5a). Additionally, direct interaction with the participating enzymes which means continuous treatment of GO revealed that PAPA NONOate that cellular uptake of the nanomaterial is not required. alone was able to decrease the optical density of the GO sus- Instead, NO released from gastrointestinal cells may diffuse to Open Access Article. Published on 10 August 2020. Downloaded on 1/7/2022 5:30:50 PM. pensions, indicative of GO degradation, while X/XO alone did the gut lumen and ‘attack’ GO, leading to its degradation. On not exert observable effects (Fig. S2a†). Raman analysis con- the other hand, a possible limitation is that the kinetics of firmed these results (Fig. S2b†). On the other hand, the peroxy- NO-driven degradation are evidently slower than those of nitrite donor SIN-1 did not generate a NO signal (at 300 µM), MPO.4 though there was a slight increase when a higher dose Only a few attempts have been made to assess the biological (900 µM) was applied (Fig. 5b). Instead, the fluorescence inten- consequences of nanomaterial degradation or the lack thereof. sity of DAX-J2 PON Green increased in a time- and dose-depen- Kagan et al.21 found that ex vivo degraded CNTs induced less dent manner upon reacting with SIN-1, clearly showing that pulmonary inflammation when aspirated into the lungs of peroxynitrite was formed, while PAPA NONOate (an NO donor) mice whereas non-degraded CNTs triggered granuloma for- Fig. 5 Detection of NO and peroxynitrite using in vitro systems. (a) The NO donor, 1-propamine 3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA) NONOate, elicited a significant NO signal and the introduction of the superoxide-generating system xanthine/xanthine oxidase (X/XO) resulted in a decrease of the signal, indicative of peroxynitrite generation. (b) SIN-1 triggered a very low NO signal when compared with PAPA NONOate. (c) SIN-1 generated peroxynitrite in a dose- and time-dependent manner, while no peroxynitrite was observed when adding the NO donor PAPA NONOate (at 10 mM). 16734 | Nanoscale, 2020, 12, 16730–16737 This journal is © The Royal Society of Chemistry 2020 View Article Online Nanoscale Communication mation. In a subsequent study, inflammatory and fibrotic dependent degradation of GO which is suppressed by L-NAME responses to CNTs were aggravated in MPO-deficient mice serves to reduce the inflammatory response triggered by when compared to wild-type mice.22 We recently reported that ingested GO. We found that the recruited neutrophils were MPO-degraded GO was non-cytotoxic and did not exert DNA mainly located in the lamina propria (beneath the epithelium) This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. damage in cultured human lung cells,4 but in vivo responses while very few GFP-positive cells infiltrated into the gut lumen were not evaluated in the latter study. Here we asked whether (Fig. S3†). Therefore, direct interactions between neutrophils biodegradation would alter inflammatory responses induced and GO seem unlikely. by GO in the GI tract. To address this, we employed transgenic The fact that the major organ systems are conserved in zeb- zebrafish, Tg(mpx:eGFP), expressing fluorescent GFP under a rafish18 provides an opportunity to decipher potential health neutrophil-specific promoter to visualize neutrophil infiltra- effects of nanomaterials in humans. Specifically, zebrafish tion,23 an acknowledged marker of inflammation. As illus- may serve as an alternative to mammalian models for the Open Access Article. Published on 10 August 2020. Downloaded on 1/7/2022 5:30:50 PM. trated in Fig. 6a, the GFP-positive neutrophils were mostly study of inflammation, as the cellular and humoral machinery located in the blood vessels in control fish. Following GO that regulates immune responses is conserved. In the early exposure, neutrophils were found to infiltrate the GI tract stages of development, zebrafish rely on the innate immune (Fig. 6a). Interestingly, in zebrafish exposed to GO in the pres- system to protect against infections or injuries. The present ence of L-NAME, significantly more neutrophils migrated study shows that the presence of GO is ‘sensed’ in the gut towards the GI tract as compared to fish exposed to GO alone leading zebrafish larvae to mount a response to the offending (Fig. 6b and c). However, L-NAME alone had no impact on neu- agent. However, the implications of the present findings for trophil migration (Fig. 6c). These results suggest that NO- other species including humans can only be speculated upon Fig. 6 Inflammation in the gastro-intestinal tract of zebrafish is enhanced when NO-dependent GO degradation is blocked. (a) The transgenic zebrafish strain Tg(mpx:eGFP) with GFP-labeled neutrophils was used to visualize the movement of neutrophils. Fluorescence microscopy shows that GO exposure resulted in the migration of cells to the gut, while inhibition of NOS with L-NAME led to more neutrophil infiltration. The gut is indicated by the white dashed line. (b) Confocal images of zebrafish larvae after exposure to GO ± L-NAME. (c) Quantification of neutrophils migrated to the gut. L-NAME alone had no effect on neutrophil migration. The numbers were quantified based on 10 fish per treatment. Student’s t-test was used to evaluate statistical significance between the treated fish and untreated control fish (* p < 0.05, ** p < 0.01, *** p < 0.001), as well as GO- exposed fish co-exposed or not to the NOS inhibitor, L-NAME (# p < 0.05, ## p < 0.01, ### p < 0.001). This journal is © The Royal Society of Chemistry 2020 Nanoscale, 2020, 12, 16730–16737 | 16735 View Article Online Communication Nanoscale at this stage. Indeed, the overall role of NO in inflammation in C. Martín, L. G. Delogu, T. Buerki-Thurnherr, P. Wick, mammals remains unresolved as both pro- and anti-inflamma- D. Beloin-Saint-Pierre, R. Hischier, M. Pelin, F. Candotto tory effects of NO and related nitrogen species have been Carniel, M. Tretiach, F. Cesca, F. Benfenati, D. Scaini, reported.24,25 Anti-inflammatory effects of NO in mammals L. Ballerini, K. Kostarelos, M. Prato and A. Bianco, ACS This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. involve, for instance, inhibition of leucocyte activation and Nano, 2018, 12, 10582–10620. recruitment by endothelial cell NO.26 It should be noted that 2 G. P. Kotchey, B. L. Allen, H. Vedala, N. Yanamala, we used both pharmacological and genetic approaches to A. A. Kapralov, Y. Y. Tyurina, J. Klein-Seetharaman, inhibit or silence iNOS in the zebrafish model. This, coupled V. E. Kagan and A. Star, ACS Nano, 2011, 5, 2098–2108. with our in vitro results, has revealed a role for NO in the 3 R. Kurapati, J. Russier, M. A. Squillaci, E. Treossi, degradation of GO. However, we have not explored the C. Ménard-Moyon, A. E. Del Rio-Castillo, E. Vazquez, different cell populations that may potentially produce NO, P. Samorì, V. Palermo and A. Bianco, Small, 2015, 11, 3985– Open Access Article. Published on 10 August 2020. Downloaded on 1/7/2022 5:30:50 PM. though iNOS is known to be expressed in many cell types in 3994. humans. We presumed that NO is generated by gastrointesti- 4 S. P. Mukherjee, A. R. Gliga, B. Lazzaretto, B. Brandner, nal cells in the present model and that NO ( produced by M. Fielden, C. Vogt, L. Newman, A. F. Rodrigues, W. Shao, iNOS) together with superoxide radicals ( produced by NADPH P. M. Fournier, M. S. Toprak, A. Star, K. Kostarelos, oxidase) combined to produce the highly reactive peroxynitrite K. Bhattacharya and B. Fadeel, Nanoscale, 2018, 10, 1180– anion (ONOO−). 1188. 5 C. Martín, A. Ruiz, S. Keshavan, G. Reina, D. Murera, Y. Nishina, B. Fadeel and A. Bianco, Adv. Funct. Mater., Conclusions 2019, 29, 1–11. 6 R. Kurapati, S. P. Mukherjee, C. Martín, G. Bepete, In conclusion, we have shown that GO can be degraded in vitro E. Vázquez, A. Pénicaud, B. Fadeel and A. Bianco, Angew. and in vivo in an NO-dependent manner. This study is the first Chem., Int. Ed., 2018, 11722–11727. to demonstrate biodegradation of GO in zebrafish. 7 B. L. Allen, P. D. Kichambare, P. Gou, I. I. Vlasova, Furthermore, while many deleterious reactions have been A. A. Kapralov, N. Konduru, V. E. Kagan and A. Star, Nano attributed to peroxynitrite,27 the present results suggest that Lett., 2008, 8, 3899–3903. this potent oxidant may also contribute to the innate defense 8 J. Russier, C. Ménard-Moyon, E. Venturelli, E. Gravel, against foreign intrusion insofar as it is shown here to partici- G. Marcolongo, M. Meneghetti, E. Doris and A. Bianco, pate in the degradation of GO. We also found that the degra- Nanoscale, 2011, 3, 893–896. dation of GO reduces the inflammation (evidenced as neutro- 9 K. Bhattacharya, R. El-Sayed, F. T. Andón, S. P. Mukherjee, phil infiltration) triggered upon ingestion of GO. These find- J. Gregory, H. Li, Y. Zhao, W. Seo, A. Fornara, B. Brandner, ings shed new light on the biological fate of GO and further M. S. Toprak, K. Leifer, A. Star and B. Fadeel, Carbon, 2015, support the view that GO is a biodegradable material.28 91, 506–517. 10 F. T. Andôn, A. A. Kapralov, N. Yanamala, W. Feng, A. Baygan, B. J. Chambers, K. Hultenby, F. Ye, Conflicts of interest M. S. Toprak, B. D. Brandner, A. Fornara, J. Klein- Seetharaman, G. P. Kotchey, A. Star, A. A. Shvedova, There are no conflicts to declare. B. Fadeel and V. E. Kagan, Small, 2013, 9, 2721–2729. 11 V. E. Kagan, A. A. Kapralov, C. M. St. Croix, S. C. Watkins, E. R. Kisin, G. P. Kotchey, K. Balasubramanian, Acknowledgements I. I. Vlasova, J. Yu, K. Kim, W. Seo, R. K. Mallampalli, We thank Dr Jens Sommertune (RISE Research Institute of A. Star and A. A. Shvedova, ACS Nano, 2014, 8, 5610–5621. Sweden, Stockholm) for assistance with the confocal Raman 12 D. Elgrabli, W. Dachraoui, C. Ménard-Moyon, X. J. Liu, analysis, and Dr Neus Lozano (ICN2, Barcelona) for assistance D. Bégin, S. Bégin-Colin, A. Bianco, F. Gazeau and with the analysis of the material characterization data. We also D. Alloyeau, ACS Nano, 2015, 9, 10113–10124. thank Dr Ahmed Sultan (Karolinska Institutet, Stockholm) for 13 K. Bhattacharya, C. Sacchetti, P. M. Costa, J. Sommertune, generously providing the neutrophil reporter line, and the KI B. D. Brandner, A. Magrini, N. Rosato, N. Bottini, M. Bottini zebrafish core facility for assistance with fish husbandry. This and B. Fadeel, Adv. Healthcare Mater., 2018, 7, 1–11. work was supported by the European Commission through the 14 C. Szabó, H. Ischiropoulos and R. Radi, Nat. Rev. Drug Graphene Flagship (grant no. 785219). Discovery, 2007, 6, 662–680. 15 I. S. Sohal, K. S. O’Fallon, P. Gaines, P. Demokritou and D. Bello, Part. Fibre Toxicol., 2018, 15, 29. References 16 A. Pietroiusti, E. Bergamaschi, M. Campagna, L. Campagnolo, G. De Palma, S. Iavicoli, V. Leso, 1 B. Fadeel, C. Bussy, S. Merino, E. Vázquez, E. Flahaut, A. Magrini, M. Miragoli, P. Pedata, L. Palombi and F. 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