Nickel oxide nanoparticles induce inflammation and genotoxic effect in lung epithelial cells

Toxicology Letters 226 (2014) 28–34

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Nickel oxide nanoparticles induce inflammation and genotoxic effect in lung epithelial cells Laura Capasso, Marina Camatini, Maurizio Gualtieri ∗ Polaris Research Centre, University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy

h i g h l i g h t s • • • •

NiONPs induce cytotoxicity and inflammation in human pulmonary A549 and BEAS-2B cells. Cytokines release depends on MAPK cascade through the induction of NF-kB pathway. Intracellular ROS increased at 45 min after exposure to NiONPs in BEAS-2B cells. NiONPs activate DNA damage signaling and promote cell cycle alterations.

a r t i c l e

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Article history: Received 29 November 2013 Received in revised form 24 January 2014 Accepted 27 January 2014 Available online 3 February 2014 Keywords: Interleukins MAP-kinases NF-kB Nickel oxide nanoparticles Pulmonary epithelial cells ROS

a b s t r a c t Nickel oxide nanoparticles (NiONPs) toxicity has been evaluated in the human pulmonary epithelial cell lines: BEAS-2B and A549. The nanoparticles, used at the doses of 20, 40, 60, 80, 100 ␮g/ml, induced a significant reduction of cell viability and an increase of apoptotic and necrotic cells at 24 h. A significant release of interleukin-6 and -8 was assessed after 24 h of treatment, even intracellular ROS increased already at 45 min after exposure. The results obtained evidenced that the cytokines release was dependent on mitogen activated protein kinases (MAPK) cascade through the induction of NF-kB pathway. NiONPs induced cell cycle alteration in both the cell lines even in different phases and these modifications may be induced by the NPs genotoxic effect, suggested by the nuclear translocation of phospho-ATM and phospho-ATR. Our results confirm the cytotoxic and pro-inflammatory potential of NiONPs. Moreover their ability in inducing DNA damage responses has been demonstrated. Such effects were present in A549 cells which internalize the NPs and BEAS-2B cells in which endocytosis has not been observed. © 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Nanoparticles (NPs) are receiving increased attention by the scientific community for their physical and chemical properties, which consent their use in a wide range of applications. Manufactured NPs of different metal oxides, such as nickel ones, are currently used for paints formulation, displayer, battery and biomedical components. As a consequence of their large use and nanometric size, NPs potential impact on human health needs to be investigated, with particular attention to inhalation, which is the major exposure route. Epidemiological studies have associated the exposure to nickel compounds with an increased risk of lung cancer (Andersen et al.,

∗ Corresponding author. Polaris Research Centre, Department of Earth and Environmental Sciences, University Milano-Bicocca. 1, Piazza della Scienza - 20126, Milan, Italy. Tel.: +39 0264482922. E-mail addresses: [email protected] (L. Capasso), [email protected] (M. Camatini), [email protected], [email protected] (M. Gualtieri). 0378-4274/$ – see front matter © 2014 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2014.01.040

1996; Goodman et al., 2009) and since 1986 nickel refinery dust and nickel subsulfide have been classified as human carcinogens (EPA Federal Register, 1986). Nickel compounds are also emitted by combustion processes such as the combustion of petroleum for steam and electricity (Huggins et al., 2011) and cigarette smoke (Smith et al., 2001). Besides these observations, the effects of nickel oxide nanoparticles (NiONPs) on human health have received poor attention and a few data are available on their mechanisms of action and molecular pathways. In vivo studies (Morimoto et al., 2011; Nishi et al., 2009) have demonstrated that NiONPs induce inflammatory effects after intratracheal instillation in rat, and in vitro studies on A549 and JB6 cells (Horie et al., 2009; Zhao et al., 2009) have shown increased toxicity of metal oxide NPs compared to micrometer particles of the same composition. One of the most common NPs outcome is the induction of oxidative stress with the production of reactive oxygen species (ROS) (AshaRani et al., 2009; Xia et al., 2006), which may also depend on the pro-inflammatory mediators production (Gillespie et al., 2010). Veranth et al. (2007) have demonstrated that BEAS-2B cells exposed to different metal oxide NPs, such as NiO, SiO2 , and TiO2 , release

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interleukin-6 (IL-6), a mediator of the acute phase response, and IL-8, a chemoattractant involved in lung injury. Several studies assessed the induction of inflammatory mediators and showed their dependence on the activation of the nuclear factor-kB (NF-kB) and mitogen activated protein kinases (MAPK), such as p38 and c-Jun N-terminal kinases (JNK). Moreover Ke et al. (2008) and Ding et al. (2006) have confirmed that nickel compounds too have the ability to induce MAPK through the stimulation of JNK and NF-kB pathways. It has been demonstrated (AshaRani et al., 2009; Eom and Choi, 2010) that NPs induce not only ROS generation and inflammation but also DNA damage, a pivotal event usually associated with the activation of apoptotic cascade and cell cycle alteration. When DNA damage occurs, the proteins ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia/Rad3 related (ATR), are involved in the initial recognition of damage and in the activation of a repair mechanism. Furthermore Damrot et al. (2009) and Yang et al. (2004) have shown a possible crosstalk between the MAPK and the activation of DNA damage recognition machinery. Despite the numerous papers on NiONPs toxicity and proinflammatory potential, no in vitro data are available on the ability of such particles to induce DNA damage-recognition responses. Furthermore a study analysing the cell-particles interaction/internalization and the biological responses induced is missing. Here we present the results obtained exposing the human cell line A549, representative of type II alveolar cells, and BEAS-2B, representative of the bronchial epithelium, to commercially available NiONPs (nominal diameter <50 nm). These cell lines were selected as representative model of the lung distal portion. Moreover BEAS2B cells have an active p53 and are a suitable in vitro model to analyse DNA damage and DNA damage responses. On the other hand A549 cells are largely used and could be considered a reference model for in vitro experiments on lung toxicity. We have evaluated the cytotoxic and pro-inflammatory potential of NiONPs, with particular attention to the activation of the MAPK and NFkB pathways. Particles internalization, ROS production, cell cycle alteration and genotoxic effect have also been analysed.

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to NiONPs for 24 h, while Alamar blue (AB) test was specifically used for BEAS2B, following the manufacturer’s instructions. Moreover Hoechst 33342/propidium iodide (PI) staining was performed according to Gualtieri et al. (2010), in order to assess the amount of viable, necrotic and apoptotic cells. 2.4. ROS production The quantitative measurement of intracellular H2 O2 using H2 DCFDAfluorescence was investigated by flow cytometry, according to Gualtieri et al. (2010), after 45 min of exposure at 60 and 100 ␮g/ml of NiONPs. 2.5. Cytokine analysis IL-6 and IL-8 protein levels into the supernatants were determined by sandwich ELISA according to the manufacturer’s guidelines (Human Cytoset–CHC1303, CHC1263–Invitrogen Corporation, Camarillo, USA). Cells were treated for 24 h at different concentrations of NiONPs. The absorbance of each sample was measured by Multiskan Ascent plate reader (Thermo) at 450 nm and 570 nm. 2.6. Western blot analysis Western blot analysis was used to detect different proteins after treatment of cells to 60 (data not shown) and 100 ␮g/ml of NiONPs (see Supplementary file S1). ␤-actin, Rab5, the not-phosphorylated and the phosphorylated forms of ATR, ATM, JNK, p38, NF-kB, IkB-␣ were detected after 2 h of exposure, while Rab7 was detected after 24 h. 2.7. Immunocytochemistry Cells were seeded on glass cover slips, treated for 2 or 24 h at 60 (data not shown) and 100 ␮g/ml of NiONPs, and analysed under AxioLab II microscopy (Zeiss) in order to detect Rab5, Rab7, phospo-ATR and phospho-ATM (see Supplementary file S1). 2.8. Flow cytometry Cells were treated for 24 h at 0, 20, 40, 60, 80, 100 ␮g/ml of NiONPs and cell cycle analysis was performed as previously reported (Moschini et al., 2010). 2.9. Statistical analysis Mean and standard error of mean (SEM) of at least three independent experiments are reported (n ≥ 3). Statistical analyses were performed by Sigma Stat 3.1 software, using one-way ANOVA with Bonferroni multiple comparison test (when variance were uniform), Dunnett’s as a post hoc test. Values of p < 0.05 were considered statistically significant.

3. Results 2. Materials and methods For details see Supplementary files S1. See Supplementary file 1 (S1) as supplementary file. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/ 10.1016/j.toxlet.2014.01.040. 2.1. Preparation and characterization of NPs suspensions Nickel oxide nanoparticles (50 nm nominal diameter, 99.8% purity, Sigma–Aldrich, Italy) were suspended in PBS + 0.2% BSA at a final concentration of 8 mg/ml (NiONPs stock solution). The stock solution was then sonicated (20 Hz for 15 min on ice) and maintained at 4 ◦ C until use. In order to characterize the NPs their surface area has been assessed by BET (Brunauer–Emmett–Teller) analysis and their dimension by transmission electron microscopy (TEM). Dynamic light scattering (DLS) has been used to assess the particles hydrodynamic diameter after dispersion in aqueous solutions and their stability evaluated with the quantification of nickel ions released in solution (see Supplementary file S1). 2.2. Cell culture and exposure conditions The human type II alveolar epithelial cell line A549 (American Type Culture Collection, ATCC Rockville, MD, USA) and the human bronchial epithelial cell line BEAS-2B (European Collection of Cell Cultures, ECACC, Salisbury, UK) were maintained and seeded as reported in Supplementary file S1. Cells were exposed to different concentrations of NiONPs (0, 20, 40, 60, 80, 100 ␮g/ml) for 45 min to 24 h. 2.3. Cell viability As two different cell lines were used, different viability test were performed to avoid interference between the assay and the cell lines. MTT test was performed as previously reported (Gualtieri et al., 2012), to assess viability of 549 cells exposed

3.1. Nanoparticles characterization TEM analysis showed that primary NiONPs are smaller than 50 nm claimed by the manufacturer and they have the tendency to aggregate/agglomerate in aqueous solutions. Two population were observed: one formed by small agglomerates clearly formed by several NPs (up to 100 nm, Fig. 1A), and the other of larger ones with diameters up to hundreds nanometers (Fig. 1B). Similar results were observed for particles suspended in cell culture medium (Supplementary file S2, Fig. A1.1). See Supplementary file 2 (S2) as supplementary file. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2014.01.040. DLS measurements confirmed TEM observations and revealed the presence of two populations in aqueous solutions (culture media, PBS and distilled water), with a mean diameter of 80 nm and of 450 nm, respectively. No significant differences in the DLS results were observed among the aqueous solutions tested. The zetapotential measurements confirmed these results, since the negative values recorded (−12 mV/−22 mV) demonstrated that NiONPs are instable in the solutions tested (more details on DLS in Supplementary file S2, Fig. A1.1). The analysis, performed by BET techniques, determined the total specific surface area of NiONPs equal to 61.19 m2 g−1 . The Ni2+ release from NiONPs was negligible in the cell free systems since ions concentration was always lower than 0.04 ␮g/ml.

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Fig. 1. NPs characterization. NiONPs suspended in distilled water analysed by TEM. (A) scale bar = 50 nm and (B) scale bar = 100 nm. (C) Ni2+ release in cell media at 24 h of exposure to different NiONPs concentrations. The data represent the mean + SEM of 3 experiments (ANOVA, Dunnett’s). *Statistically different from the control, p < 0.05. Representative picture and diagrams are reported.

The maximal solubility was reached after 24 h of test, when the concentration reached a plateau (data not shown). The amount of Ni2+ released into cell lines supernatants, collected after 24 h of exposure to different NPs concentrations, were 2.8 ␮g/ml in A549 cells and 4.2 ␮g/ml in BEAS-2B exposed to 100 ␮g/ml (Fig. 1C).

doses. Moreover NPs induced a higher release of IL-6 in BEAS-2B than in A549 cells (Fig. 4A), while IL-8 release was quite similar in the two cell lines (Fig. 4B). IL-1␤ was also evaluated (data not shown) and no increment was present.

3.2. Cell viability

To verify if the interleukins release was dependent on NF-kB pathway, the level of the phosphorylated form of this protein, of its inhibitor binding molecule (IkB-␣) and of two relevant MAPK (JNK and p38), involved in NF-kB activation, were evaluated by western blot. The fold increase values are reported in Table 1 and representative images are presented in Supplementary file S2 (Fig. A1.2). The non phosphorylated forms were also analysed and no significant modulation was apparent (data not shown).

A549 cells showed a dose dependent decrease in MTT-value (Fig. 2A), with a reduction of 40% at 100 ␮g/ml of NiONPs, at 24 h of treatment. Similar results were obtained for BEAS-2B cells, even the viability reduction is above 30%, according the AB assay (Fig. 2A). Moreover, in A549, Hoechst/PI nuclear staining evidenced above 30% of necrotic cells at 100 ␮g/ml of NiONPs and 10% of apoptotic cells (Fig. 2B), while BEAS-2B cells presented above12% of both necrotic and apoptotic cells (Fig. 2C).

3.5. Possible inflammatory pathways involved

3.6. DNA damage signaling

3.3. ROS production

Immunocytochemistry analyses showed a significant nuclear translocation of phospho-ATM and phospho-ATR in both the cell

BEAS-2B cells exposed to NiONPs showed a significant increase of H2 DCFDA-fluorescence intensity, demonstrating an increased level of ROS (Fig. 3), not detected in A549 cells.

Table 1 Protein expression.

3.4. Cytokine release In both cell lines, after 24 h of exposure, a release of IL-6 and IL-8 was detected. The results, expressed as fold increase in comparison with control cells, showed a peak of release at 40 and 60 ␮g/ml

A549 BEAS-2B

p-NF-kB

p-IkB-␣

p-p-38

p-JNK

p-ATR

p-ATM

1.34 1.31

1.67 1.65

1.96 2.52

1.48 1.97

1.63 1.91

1.34 1.34

Representative values obtained by western blot analysis in A549 and BEAS-2B cells after 2 h of 100 ␮g/m NiONPs treatments. Densiometric data normalized to untreated cells as fold-control are presented.

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lines at 2 h of treatment with 100 ␮g/ml of NiONPs (Fig. 5). Western blot proteins quantification confirmed these data (Table 1 and Supplementary file S2: Fig. A1.3). 3.7. Cell cycle At 24 h of exposure, A549 cells showed a dose dependent decrease of G1 phase cell population (from 73% to 53%) and a corresponding increase of cells in G2/M (from 10% to 26.7%), with a not significant increase of cells in S phase (from 16.9% to 19.3%) (Fig. 6A). BEAS-2B cells showed a significant increase of G1 phase cell population (from 56.3% in the control to 76%) and a corresponding decrease of cells in G2/M (from 25.2% to 10%) and in S phase (from 18.1% to 11.8%) (Fig. 6B). A significant increase of BEAS-2B cells in subG1 phase (from 0.4% to 2.2%) was detected at the highest doses, confirming the data obtained with Hoechst/PI staining. 4. Discussion

Fig. 2. NiONPs cytotoxic effects at 24 h of exposure. Cell viability assays: (A) MTT assay (A549 cells) and Alamar Blue (BEAS-2B cells). Hoechst/PI analysis in A549 (B) and BEAS-2B cells (C). The data represent the mean + SEM of 3 experiments (ANOVA, Dunnett’s). *Statistically different from the control, p < 0.05.

Fluorescence (a.u.)

2

A549

1,5

BEAS-2B

*

*

1

0,5

0 C

60 μg/ml

100 μg/ml

Fig. 3. ROS production at 45 min of exposure. ROS production evidenced by flow cytometry analysis after 45 min of NiONPs exposure. The data represent the mean + SEM of 3 experiments (ANOVA, Dunnett’s) *Statistically different from the control, p < 0.05.

The data here presented have shown the particles tendency to agglomerate when dispersed in aqueous solutions, in accordance with Siddiqui et al. (2012). Previous studies demonstrated that the agglomeration state of NPs may affect their biological effects (Gualtieri et al., 2012; Sharma et al., 2013). Beside their behaviour in water suspensions, metallic NPs can release ions and this event may influence their resulting biological effects. Forti et al. (2011) and Pietruska et al. (2011) demonstrated that the different effects induced by Ni compounds (NiCl2 and Ni particles) can be partially attributed to the difference in Ni2+ intracellular levels and their bioavailability. However Mazinanian et al. (2013) reported that NiO is insoluble also in lysosomal like acid fluid, while Huang et al. (2002) showed that insoluble Ni3 S2 appears to be more effective in promoting a biochemical response in comparison to soluble NiCl2 in BEAS-2B cells. Our data showed that NiONPs were almost insoluble in water and only a low amount of ions was released in culture media, suggesting that their biological effects are principally produced by particles themselves. NiONPs caused cytotoxicity in both the cell lines here examined, in agreement with the study of Ahamed (2011), who demonstrated that A549 cells, treated with metallic nickel NPs at concentrations similar to the ones here used, were affected. The dose dependent reduction of cell metabolic activity, assessed with MTT and AB assays, can be generally related to an increase of cells death. Our data obtained with Hoechst/PI staining confirmed the presence of necrotic and/or apoptotic cells. These results are in accordance with Lee et al. (2001) and Li et al. (2008), who demonstrated that different nickel compounds (Ni3 S2 and NiSO4 ) induced apoptosis in BEAS-2B and in other cell lines. A549 cells showed an amount of necrotic cells higher than that observed in BEAS-2B. This difference may be related to the different resistance of the two cell lines to pro-apoptotic stimuli. A549 cells are resistant to radiotherapyand chemotherapy-induced apoptosis (Yun et al., 2013) for their origin from cancerous lung tissue. Oxidative stress and ROS formation have already been related to NiONPs exposure and associated to different biological effects in HEp-2 and MCF-7 cells (Siddiqui et al., 2012). In our experiments ROS increased only in BEAS-2B after NiONPs exposure, suggesting that A549 cells are more resistant to oxidative stress. In fact this cell line has a basal high level of antioxidants, as reported by Chang et al. (2006). Our data (Supplementary file S3) also suggested that NPs enter or interact with the two cell lines through different mechanisms, since A549 cells showed an increase in endocytosisrelated trafficking, not evidenced in BEAS-2B. Thus the interaction between NiONPs and cell membrane may involve different mechanism although the biological responses observed in the two cell lines are rather similar.

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Fig. 4. Pro-inflammatory cytokines release at 24 h of exposure. IL-6 (A) and IL-8 release (B) measured by ELISA. The data represent the mean ± SEM of 5 experiments and are presented as fold increase (ANOVA, Dunnett’s). Statistically different from the control: *(p < 0.05).

Fig. 5. Immunocytochemistry of the proteins phospho-ATR or phospho-ATM at 2 h of exposure. Cell lines treated for 2 h with 100 ␮g/ml of NiONPs immunostained for DAPI (blue) and phospho-ATR (green) or phospho-ATM (green). Representative images are presented. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

See Supplementary file 3 (S3) as supplementary file. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet.2014.01.040. Indeed the pro-inflammatory mediators production was increased in both the cell lines after NiONPs treatment, in accordance with recent in vitro studies (Hsiao and Huang, 2011; Park et al., 2008) on various metal oxide NPs. The release of interleukins reached a peak at intermediate doses and decreased at higher doses, in accordance with the reduction of cell viability. The difference between the amounts of interleukins release in the two cell lines may be related with the different levels of ROS produced (Gillespie et al., 2010), as well as with the different sensitivity of cells to nickel compounds, as previously reported by Giunta et al. (2012) and Veranth et al. (2008). The analysis of the molecular pathway leading to the release of interleukins showed that IkB-␣ phosphorylation, and the consequent NF-kB activation, were significantly increased after NiONPs treatment, suggesting a possible involvement of NFkB complex. Similarly, Ding et al. (2006) described an increased phosphorylation of both NF-kB and IkB-␣ in BEAS-2B cells exposed for 24 h to NiCl2 and Ni3 S2 . The activation of the inflammatory pathway was already evident at 2 h in our experiments, confirming that nickel, when present in a non-soluble particulate form, is able to activate the pro-inflammatory cascade. Considering the different ability of the two cell lines to internalize NPs, it can be deduced that internalization process is not required to activate the signalling cascade that triggers the release of pro-inflammatory proteins. Xu et al. (2011) suggested that nickel promotes the activation of TLR4 (TollLike receptor 4) and, if this is the case, our hypothesis of membrane receptor-mediated effects may be confirmed, even further experiments are needed. According to O’Neill et al. (2013) we can suppose that, after NPs treatment, in A549 cells part of TLR4 is endocytosed activating alternative pathway to the NF-kB cascade, resulting in

a lower release of interleukins. On the contrary in BEAS-2B cells, lacking the endocytic pathway, the membrane receptor activation is followed principally by the NF-kB cascade, which may be responsible of the higher amount of IL-6 release. Since NF-kB activation may be triggered by MAPK cascade, we assessed the activation/phosphorylation of JNK and p38 proteins in response to NiONPs treatment. Although in both the cell lines the MAPK increased, their activation may determine different biological responses. Ke et al. (2008) and Xu et al. (2011) showed that Ni compounds activate the MAPK/NF-kB pathway in A549 cells and related this event to epigenetic modification and tumour invasiveness. MAPK activation in BEAS-2B cells may be responsible of the apoptotic cell death induced by NiONPs. Although this correlation has to be further analysed, Hsin et al. (2008) demonstrated that the apoptotic effect of nanosilver was mediated by a JNK-dependent mechanism in NIH3T3 cells, while Li et al. (2008) showed that different nickel compounds were able to induce apoptosis in BEAS-2B cells. Our results demonstrated that NiONPs were also potent activators of the DNA-damage signaling cascade (DSC) induced in response to genotoxic stress: the phospho-ATR nuclear localization at 2 h of treatment is an important event for DNA damage-sensing and for the consequent activation of the signaling cascade, as reported by Yang et al. (2004). Our data are in accordance with Zang et al. (2002), who showed that in human fibroblast different DNA damaging stimuli triggered an ATM/ATR-dependent JNK activation, resulting in apoptosis. However more focused research is required to identify the proteins involved in DSC, such as ␥-H2AX or Chk1/2, and better elucidate the factors involved in NiONPs-induced genotoxic response. ATM/ATR regulates a plethora of proteins responsible for cell cycle arrest at specific check points (Branzei and Foiani, 2008). Our results evidenced that NiONPs-exposed cells

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Fig. 6. Analysis of cell cycle at 24 h of exposure. Relative amount of cells in different phases of the cell cycle >at exposure to 100 ␮g/ml of NiONPs for 24 h, examined by flow cytometry: (A) A549 cells, (B) BEAS-2B cells. The data represent the mean + SEM of 3 experiments (ANOVA, Dunnett’s). Statistically different from the control, *p < 0.05.

accumulated in different phases of the cell cycle, suggesting that DNA damages may activate different response pathways according to the cell line used. Our data are in accordance with several studies (Huang et al., 2009; Saquib et al., 2012) on metal oxide NPs: TiO2 induced M or G2/M arrest in NIH 3T3 cells and WISH cells, respectively. The differences in biological responses (cell death and cell cycle behaviour) here reported may be due to cell-type specificity; in fact it must be taken into account that A549 cells are a cancer derived cell line and consequently some pathways regulating the cell cycle arrest, such as p53, may be altered. BEAS-2B is an immortalized cell line with a mutated p53, which maintains its functional activity (Lehman et al., 1993). Consequently the pivotal role of p53, preserving its functional form, may orient its final outcome and deserves future analyses.

quality of our life even nano-safety remains an issue to be further explored. Our results may represent a step in the understanding of the mechanisms involved in the health outcomes related to NPs exposure. Conflict of interest statement The authors report no conflicts of interest. Author Contributions MG and LC designed and performed the in vitro experiments, MC supervised in vitro analyses. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

5. Conclusions NiONPs are able to induce the activation of significant biological pathways in two human cultured cells and the cytotoxic and inflammatory effects can be attributed to the particles themselves rather than to the release of nickel ions. NiONPs are able to induce the pro-inflammatory cascade through the activation of NF-kB and to activate the proteins related to DNA damage sensing (ATM and ATR). The induction of pro-inflammatory stimuli and the DNA damages have been related to pro-oncogenic transformation of human cells. Considering the epidemiological data on workers exposed to nickel compounds and the increased environmental exposure to nickel-containing particles, attention has to be paid to better define cell signaling induced by NiONPs. NPs are certainly improving the

Acknowledgements The authors want to thank Holme A. Jorn for the useful critical point of view in preparing the paper; the authors also acknowledge Piero Sozzani and Angelina Comotti (Dept. of Materials Science, University of Milan-Bicocca, Italy) for the BET analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.toxlet. 2014.01.040.

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