In vitro evaluation of the toxicity induced by nickel soluble and particulate forms in human airway epithelial cells

Toxicology in Vitro 25 (2011) 454–461

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In vitro evaluation of the toxicity induced by nickel soluble and particulate forms in human airway epithelial cells Efrat Forti a, Susan Salovaara a, Yuksel Cetin a, Anna Bulgheroni a, Richard Tessadri b, Paul Jennings c, Walter Pfaller c, Pilar Prieto a,⇑ a In-Vitro Methods Unit, European Centre for the Validation of Alternative Methods, Institute for Health and Consumer Protection, European Commission Joint Research Centre, TP 580, Via Fermi 2749, 21027 Ispra (VA), Italy b Institute of Mineralogy & Petrography, Faculty of Geo- and Atmospheric Sciences, University of Innsbruck, Innrain 52, Bruno-Sander-Haus, A-6020 Innsbruck, Austria c Division of Physiology, Department of Physiology and Medical Physics, Innsbruck Medical University, Fritz-Preglstr. 3, A-6020 Innsbruck, Austria

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Article history: Received 24 February 2010 Accepted 17 November 2010 Available online 25 November 2010 Keywords: Airway epithelial cells NiCl2 Ni particles In vitro Barrier integrity Oxidative stress

a b s t r a c t Epidemiological studies show that exposure to nickel (Ni) compounds is associated with a variety of pulmonary adverse health effects, such as lung inflammation, fibrosis, emphysema and tumours. However, the mechanisms leading to pulmonary toxicity are not yet fully elucidated. In the current study we used Calu-3, a well differentiated human bronchial cell line, to investigate in vitro the effect of Ni in soluble form (NiCl2) and in the form of micro-sized Ni particles on the airway epithelium. For this purpose, we evaluated the effect of Ni compounds on the epithelial barrier integrity by monitoring the transepithelial electrical resistance (TEER) and on oxidative stress pathways by measuring reactive oxygen species (ROS) formation and induction of stress-inducible genes. Our results showed that exposure to NiCl2 and Ni particles resulted in a disruption of the epithelial barrier function observed by alterations in TEER, which occurred prior to the decrease in cell viability. Moreover, Ni compounds induced oxidative stress associated with ROS formation and up-regulation of the stress-inducible genes, Metallothionein 1X (MT1X), Heat shock protein 70 (HSP70), Heme oxygenase-1 (HMOX-1), and gamma-glutamylcysteine synthetase (cGCS). Furthermore, we have demonstrated that the induced effects by Ni compounds can be partially attributed to the increase in Ni ions (Ni2+) intracellular levels. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Nickel (Ni) compounds are important in modern industry and are widely used in many processes, such as electroplating, electroforming, and for the production of electronic equipment and nickel–cadmium batteries. Nickel normally occurs at very low levels in the environment. Recently, EPA estimated that the average nickel concentration in air in the United States is 2.2 ng/m3 (ATSDR, 2005). However, the high consumption of Ni-containing products inevitably leads to environmental pollution by this metal and its by-products at all the stages of production, recycling and disposal. Nickel concentrations in workroom air, particularly in the refining industry may be significantly increased compared to those in ambient air. Values ranging from 110 to 180 ng/m3 have been reported from heavily industrialized areas (Bennett, 1994). The main routes of Ni intake for humans are inhalation, ingestion and absorption through the skin (ATSDR, 2005). Ni is selectively concentrated in the lung independently of the route of exposure

⇑ Corresponding author. Tel.: +39 0332 785534; fax: +39 0332 785336. E-mail address: [email protected] (P. Prieto). 0887-2333/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2010.11.013

(Tjalve et al., 1984; Denkhaus and Salnikow, 2002). Moreover, pulmonary absorption is a major route of concern for Ni-induced toxicity and can result in adverse health effects, such as pulmonary inflammation, alveolar epithelium hyperplasia, fibrosis, asthma, bronchitis and damage to the epithelial cell layer, often resulting in increased epithelial permeability (Barchowsky and O’Hara, 2003; Das et al., 2008). Little is known about risk groups in the general population, although smokers and those exposed at work have higher exposures than the other groups within the population. Environmental exposure to inhaled Ni particles has been also linked to increased mortality in the United States (Laden et al., 2000). Lung diseases associated with Ni can result from the inhalation of either soluble forms or insoluble particulate forms of Ni existing in the atmosphere. While ionic Ni is transported to the cells by diffusion or through calcium channels (Refsvik and Andreassen, 1995; Davidson et al., 2005; Lu et al., 2005), insoluble particulate Ni can be taken up via endocytosis by the cells and generate Ni ions (Ni2+) after gradual dissolution in the lysosomes (Costa and Mollenhauer, 1980; Costa et al., 2005; Lu et al., 2005; Beyersmann and Hartwig, 2008). At the cellular level, Ni may promote the formation of reactive oxygen species (ROS), resulting in oxidative stress and depletion

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of the antioxidant system. Moreover, Ni has also been shown to induce the activation of hypoxic signalling pathways and disruption of calcium homeostasis (Denkhaus and Salnikow, 2002; Barchowsky and O’Hara, 2003; Valko et al., 2006; Das et al., 2008). Furthermore, exposure to heavy metals in general activates transcription of a number of genes, including Metallothionein 1X (MT1X), Heat shock protein 70 (HSP70), Heme oxygenase-1 (HMOX-1), and gamma-glutamylcysteine synthetase (cGCS), which play a role in cellular protection and defence of the lung against oxidant-induced injury (Delmas et al., 1996; McDowell et al., 2000; Cheng et al., 2003; Wesselkamper et al., 2006; Stark et al., 2009; Forti et al., 2010). Although several toxicological studies have investigated the effects of Ni in various lung cell models (Bajpai et al., 1999; Barchowsky et al., 2002; Zhang et al., 2003; Riley et al., 2005; Ding et al., 2006; Peters et al., 2007; Schmid et al., 2007; Horie et al., 2009), the mechanisms by which Ni in soluble and particulate forms cause damage to airway epithelium are still poorly understood. Therefore, the aim of this study was to investigate the effects of Ni in soluble form (NiCl2) and in the form of micro-sized Ni particles (Ni powder type 210, Ni t210) on cultured airway epithelial cells. For this purpose, the human bronchial cell line Calu-3 was chosen, as these cells differentiate into a polarized monolayer and are able to express in vitro many of the characteristics of the human native airway epithelium as they readily form tight junctions and produce mucous (Finkbeiner et al., 1993; Berger et al., 1999; Forbes, 2000; Wan et al., 2000; Steimer et al., 2005; Grainger et al., 2006). In this study we focused on the effects of soluble and microsized Ni particles on the epithelial barrier of airway epithelial cells by monitoring the transepithelial electrical resistance (TEER), and also by investigating the effect on oxidative stress pathways including ROS formation and mRNA alteration of MT1X, HSP70, HMOX-1 and cGCS. 2. Materials and methods 2.1. Materials All compounds unless otherwise mentioned, were purchased from Sigma (Milan, Italy). 2.2. Cell culture and treatment with Ni 2.2.1. Maintenance The Calu-3 human bronchial epithelial cell line was purchased from the American Type Culture Collection (ATCC, USA) and used for experiments between passages 20 and 45. The cells were routinely maintained in Minimum essential Medium Eagle (MEM) supplemented with 10% Fetal bovine serum (FBS, Lonza, Milan, Italy), 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin and 100 lg/ml streptomycin. The cells grown in 75 cm2 tissue culture flasks (Corning, Pero, Italy) at 37 °C in 5% CO2 humidified incubator were subcultured when 90% confluence was reached. Prior to experiments, cells were seeded either in 96-well plates (Corning) or on transwells membrane filters (0.33 cm2 polyester, 0.4 lm pore size, Corning). 2.2.2. Calu-3 in 96-well plates Calu-3 cells were seeded in 96-well plates at a cell density of 3  105 cells/ml (100 ll/well) and were cultured for 14 days at 37 °C in humidified atmosphere of 5% CO2.

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with 1 ml of medium added to the bottom compartment. On the following day, the air interfaced culture (AIC) was established by the removal of the medium from the apical compartment and cells were grown for 14 days at 37 °C in 5% CO2 humidified incubator, in order to reach a tight and stable barrier with TEER values higher than 150 X cm2. 2.2.4. Exposure to Ni compounds NiCl2 (99.95%, Alfa Aesar, Karlsruhe, Germany) was dissolved in PBS to 100 mM stock solution and was stored at 4 °C. Ni powder type 210 (Ni t210) was produced by Inco and obtained from Particle Technology Labs, Ltd (Downers Grove, IL, USA). Ni t210 particles were suspended in PBS to 100 mM (5.87 mg/ml) stock solution, then vortexed and sonicated for 25 min in an ultrasonic bath (ELMAÒ Transsonic T420, Singen, Germany). For experiments, the working solutions (10 mM) were freshly prepared in PBS, diluted to appropriate concentrations ranging from 0.1 to 5 mM (5.87–293.5 lg/ml), which were immediately applied to the cells. For cells grown in 96-well plates, NiCl2 or Ni t210 were added in 100 ll of complete medium. For cells grown on transwells, NiCl2 or Ni t210 were added in 250 ll of PBS to the apical compartment. Particle suspensions were shaken prior to administration to each cell culture. The solubility of Ni t210 particles was measured by inductively coupled plasma optical emission spectrometry (Jobin-Yvon ACTIVA) using standard conditions after 24 and 72 h. 2.2.5. Ni t210 particles characterisation The characteristics of the commercial Ni t210 particles, as declared by the manufacturer, are as follows: composition of 99% Ni, particles size 0.5–1 lm, density of 0.8 g/cm3, and surface area of 1.5–2.5 m2/g. As the quantities of material available did not allow for an in depth particle size analysis the characterisation of the Ni particles available was restricted to an X-ray powder diffraction analysis, laser diffraction and scanning electron microscopy (SEM). Measurement of particle size distribution was performed using a Mastersizer2000-Malvern, laser particle sizer. Two measurement series with different sample preparation using ultrasonic were performed. Stable size distribution was achieved after 3 min of sonication. Ni particles were prepared for SEM in two different manners. In the first procedure, nickel particles were brought to a thin conductive silver layer on a metal specimen holder and dried over night in vacuum at a pressure of 1.3 Pa (Pascal) sputter coated with a 0.1 nm thin gold layer and examined with a Jeol Superprobe 8100 at 10 kV as well as with a JSM 35 SEM. In the second procedure, Ni particles were washed 3 times in isopropanol (2 min each wash) prior to drying in vacuum over night. Sputter coating was identical. 2.3. Trans-epithelial electrical resistance measurement To assess the integrity of the barrier, TEER was measured by placing the transwells in an Endohm-6 chamber electrode (World Precision Instruments, Berlin, Germany) connected to an epithelial voltohmmeter (EVOM, World Precision Instruments). Before measuring, 1.4 ml of pre-warmed medium was added to the chamber electrode. For each set of experiments, TEER was also measured in a cell-free filter (blank) and the value obtained was subtracted from the raw TEER data of the cell seeded filters. TEER expressed as X cm2 was obtained by multiplying TEER values (corrected by the background resistance of the blank filter) by the surface area of the filter (0.33 cm2). 2.4. Neutral red uptake (NRU) assay

2.2.3. Calu-3 on transwells Calu-3 cells were seeded on the transwell membrane filters at a cell density of 1  105 cells/cm2 in 250 ll of complete medium

Cell viability was assessed by NRU as previously described (NIH, 2006). Briefly, after exposure to Ni compounds, cells were washed

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twice with 250 ll PBS and incubated for 3 h at 37 °C, 5% CO2, with 250 ll of 25 lg/ml neutral red (NR) solution in PBS. NR solution was then removed and the cells were washed with 250 ll of PBS. Two hundred microliters of NR destaining solution (5 parts ethanol, 4.9 parts distilled water and 0.1 part glacial acetic acid) was added and the plates were placed on an orbital shaker for 45 min. For measurement, the volume in the apical compartment (200 ll) was transferred into a 96-well plate and absorbance was recorded at 540 nm using a spectramax 250 plate reader (Molecular Devices, Sunnyvale CA, USA). 2.5. Intracellular reactive oxygen species measurement 0

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The production of ROS was measured using 2 ,7 -dichlorofluorescin diacetate (DCFH-DA, Molecular Probes, Leiden, The Netherlands) (Wang and Joseph, 1999). DCFH-DA passively enters the cells, then cellular esterases act on the molecule to form the non-fluorescent moiety DCFH, which is ionic in nature and, therefore, trapped inside the cell. A reaction with ROS leads to an oxidation of DCFH to the highly fluorescent compound dichloroflourescein (DCF). Briefly, stock solution of DCFH-DA (200 mM, DMSO) was prepared and stored at 20 °C. Cells grown in 96-well plates were washed twice with PBS before the addition of DCFH-DA working solution (20 lM, complete medium) for 45 min at 37 °C. Cells were then washed once with PBS and incubated with NiCl2, Ni particles (0.1, 1, 2 mM) or the positive control hydrogen peroxide (H2O2, 1 mM) for 24 h at 37 °C. Fluorescence was determined at 485-nm excitation and at 520-nm emission using a microplate reader (spectramax 250). 2.6. Gene expression analysis Alterations in the mRNA expressions of MT1X, HSP70, HMOX-1 and cGCS were assessed after 24 h and 48 h of the cells exposure to NiCl2 and Ni t210 (1, 2 mM). Cells were lysed and the total RNA was extracted according to the instructions of applied RNeasy Mini Kit (Qiagen, Milan, Italy). For reverse transcriptase reaction (final volume of 20 ll), 25 ng/ll RNA were incubated with 2.5 mM PCR Nucleotide Mix (Promega, Milano, Italy) and 12.5 ng/ll random primers (Promega) for 5 min at 65 °C. M-MLV buffer (Promega) was added to the samples together with 10 U/ll of M-MLV reverse transcriptase (Promega) and 2 U/ll of RNaseOUT (Invitrogen). The termocycling conditions of the reverse transcription reaction were the following: annealing at 25 °C for 10 min, cDNA synthesis at 37 °C for 1 h and enzymes inactivation at 70 °C for 15 min. mRNA levels of different genes were determined by TaqMan real-time PCR using ABI PRISM 7000 sequence detection system according to the instructions of the manufacturer (Applera Italia, Monza, Italy). The primers used were (gene symbol, assay ID): b-actin (ACTB, Hs99999903_m1), Metallothionein 1X (MT1X, Hs00745167_sH), Heat shock 70KDA protein (HSP70, Hs00271229_s1), Heme oxygenase (decycling) 1 (HMOX-1, Hs00111251_m1), gamma-glutamylcysteine synthetase (cGCS, Hs00155249_m1). Data analysis was performed by DDCt method with b-actin as the house-keeping gene. 2.7. Newport green dye staining Cells grown on transwells were exposed to NiCl2 and Ni particles (0.5 mM) for 24 h. At the end of treatment, cells were washed twice with PBS and were incubated for 45 min in HBSS/ 5% FBS containing 5 lM of the Newport Green DCF diacetate (NPG-Ac, cell permeant) dye mixture [1:1 ratio of dye/F-127 Pluoronic acid (Molecular Probes)] at 37 °C. Cells were then washed three times with HBSS/5% FBS and incubated in the same buffer for 45 min. At the end of incubation, intracellular fluorescence

was observed by a fluorescence microscope (Olympus IX70, Hamburg, Germany). 2.7.1. Microplate-based fluorescent detection of intracellular Ni2+ Cells grown in 96-well plates for 14 days were exposed to NiCl2 and Ni particles for 24 h and 48 h, respectively. At the end of exposure, Newport green (NPG) dye staining was performed as described above and NPG fluorescence signal was determined at 506-nm excitation and at 535-nm emission using a microplate reader (spectramax 250). 2.8. Statistical analysis Prism 5.0 (GraphPad Software, San Diego, CA, USA) was used for data plotting and statistical analysis. All results are expressed as mean ± SEM. Data groups were compared using one-way ANOVA analysis with Dunnett’s multiple comparison post test. Results were considered significant when P-values were less than 0.05 (P < 0.05). 3. Results 3.1. Ni t210 particles characterisation Ni t210 particles analysed by X-ray diffraction displayed narrow reflexes (peaks), which are indicative of well crystallised particles (Fig. 1A). SEM showed that the particles are forming dense networks and have cluster morphology (Fig. 1B). No morphological differences were observed using the two different SEM preparations. These results are almost consistent with the information provided by the manufacturer. The mean ‘‘sphere diameter’’ (d50) of the particles measured with laser diffraction was 12–13 lm. This value differs from the one reported by the manufacturer in their certificate (the ‘‘Fisher Sub-Sieve Size’’ = 0.5–1.0 lm). This value stems from blowing a constant gas flow through the particles produced and the resistance to flow or the flow volume, respectively, gives the grain size, while in our procedure the scattering of a laser beam was used to determine the d50. The solubility of Ni t210 particles amounts to 3% of the mass placed into PBS. This amount is still recovered after 72 h, which means that the maximum solubility is reached after 24 h and then plateaus off. 3.2. Time- and concentration-dependent effect of Ni compounds on epithelial barrier integrity and cell viability The effect of increasing concentrations of NiCl2 and Ni t210 on the epithelial barrier integrity was monitored after 24, 48 and 72 h of exposure. Results showed a statistically significant increase in TEER after 72 h incubation with 1 mM and 2 mM of NiCl2 (increased by 71% ± 6% and 64% ± 8%, respectively) (Fig. 2A). A decrease in TEER was observed starting from 24 h incubation with 5 mM. The smallest TEER values were observed after 72 h incubation with 5 mM (reduced by 93% ± 0.5%). A statistically significant increase in TEER was observed after 48 h starting from 1 mM of Ni t210 (increased by 31% ± 3%), and reached maximal TEER values after 72 h treatment with 3.5 mM (increased by 94% ± 16%). Conversely, increasing concentrations of Ni t210 did not cause a decrease in TEER (Fig. 2B). Empty wells treated with the same amount of Ni particles show the same TEER values as the blank (cell-free filter), which rule out a possible contribution of particle sedimentation to the increase in TEER recorded after treatment with Ni t210 suspensions. Treatment with either NiCl2 or Ni t210 did not show a pronounced effect on cell viability up to 72 h of incubation (Fig. 2C).

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Fig. 1. Structure of Ni t210 particles. (A) X-ray diffraction plot of Ni t210 powder indicating the crystalline nature of these particles. (B) Scanning electron microscopic image of Ni t210 particles. Note the aggregation and cluster formation formed by the particles and their branched and long structure.

Fig. 2. Ni compounds induced decrease in cell viability and alteration in TEER in a time and concentration-dependent manner. A–B: Cells grown on transwells for 14 days were treated either with NiCl2 (A) or Ni particles (B). Following exposure, TEER was measured after 24, 48 and 72 h and cell viability (C) was assessed after 72 h by performing NRU assay. Ni particles concentrations ranged from 5.87 lg/ml to 293.5 lg/ml. The results were expressed as percentage (%) of control (untreated cells) ± SEM of three independent experiments (two replicates each). *Significant difference with respect to untreated control (P < 0.05).

A slight decrease of cell viability measured by reduction of NRU was observed after treatment with NiCl2 and Ni t210 starting from 1 mM (reduced by 16% ± 3% and 14% ± 3%, respectively). The highest decrease in cell viability (reduced by 33% ± 5%) was induced after treatment with 5 mM of NiCl2.

3.3. Elevation of intracellular ROS by Ni compounds As an indicator of oxidative stress, ROS production was assessed after 24 h treatment with Ni compounds. Exposure of the cells to Ni compounds prompted a concentration-dependent increase of DCF fluorescence intensity (Fig. 3). Maximal increase was obtained after treatment with 2 mM of NiCl2 and Ni t210 (increased by 28% ± 4% and 37% ± 3%, respectively) (Fig. 3A and B). The positive

control, H2O2 (1 mM) induced an increase in DCF fluorescence intensity by 161% ± 16% (Fig. 3C). 3.4. Up-regulation of stress inducible genes by Ni compounds MT1X, HSP70, HMOX-1 and cGCS were up-regulated in a timeand concentration dependent manner (Fig. 4). Treatment with 1 mM of NiCl2 for 24 h resulted in a 7.1 ± 1.8-fold increase of MT1X mRNA levels as compared to the untreated control, whereas 2 mM induced two times higher up-regulation (14.2 ± 2.4-fold) (Fig. 4A). The highest induction (37.2 ± 6-fold compared to untreated control) was observed after 48 h exposure to 2 mM. HSP70 and HMOX-1 showed similar up-regulation and were significantly elevated after 24 h and 48 h treatment with 1 mM and 2 mM NiCl2 (Fig. 4B and C). The highest induction of HSP70 and

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Fig. 3. Ni compounds elevated intracellular levels of ROS. Cells were grown in 96-well plates for 14 days, then DCFH-DA was added to the cells for 45 min before the addition of NiCl2 (A), Ni particles (B), or the positive control H2O2 (C). Ni particles concentrations ranged from 5.87 lg/ml to 117.4 lg/ml. DCF fluorescence intensity was measured at 485-nm excitation and 520-nm emission and the results were expressed as percentage (%) of control (untreated cells) ± SEM of three independent experiments (three replicates each). *Significant difference with respect to the untreated control (P < 0.05).

Fig. 4. Induced up-regulation of MT1X, HSP70, HMOX-1 and cGCS by Ni compounds. Cells grown on transwells for 14 days were treated either with NiCl2 or Ni particles for 24 h and 48 h. Ni particles concentrations ranged from 5.87 lg/ml to 117.4 lg/ml. Total RNA was then isolated and transcript levels of MT1X (A), HSP70 (B), HMOX-1 (C), and cGCS (D) were determined by real-time PCR and normalized to internal house keeping control gene levels (b-actin). The results were expressed relatively to control (untreated cells) ± SEM of three independent experiments (two replicates each). *Significant difference from the untreated control, +significant difference of 48 h versus 24 h treatment at the same concentration of Ni compounds (P < 0.05).

HMOX-1 was observed after 48 h treatment with 2 mM (by 9.2 ± 1-fold and 10.2 ± 0.6-fold, respectively). mRNA levels of cGCS were only slightly increased after 48 h treatment with 2 mM NiCl2 (5.7 ± 0.5-fold) (Fig. 4D). Ni t210 induced a less pronounced elevation of MT1X, HSP70, HMOX-1 and cGCS compared to NiCl2. All genes were up-regulated following treatment with the highest concentration tested, at 2 mM. MT1X mRNA levels were significantly up-regulated following 24 h and 48 h treatment with 2 mM of Ni t210 (1.8 ± 0.2-fold and 3.4 ± 1.1-fold, respectively) (Fig. 4A). HSP70 and HMOX-1 were up-regulated to a similar extent with maximal values obtained after 48 h treatment with 2 mM (2.6 ± 0.3-fold and 2.8 ± 0.4-fold, respectively) (Fig. 4B and C). mRNA levels of cGCS were only

slightly increased after 48 h treatment with 2 mM (1.7 ± 0.3-fold) (Fig. 4D). 3.5. Detection of intracellular level of Ni2+ following exposure to Ni compounds In order to determine the intracellular level of Ni2+, NPG-Ac a sensitive cell permeant dye was used as it exhibits a fluorescent signal when intracellular Ni2+ binds to it. As shown in Fig. 5 there was no fluorescent signal in untreated Calu-3 cells (control cultures). However, exposure to Ni compounds for 24 h resulted in intracellular fluorescent signal indicating the presence of Ni2+ in the cells both in the cytoplasm and the nucleus (Fig. 5). The

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Fig. 5. Intracellular Ni2+ accumulation following treatment with Ni compounds. Cells grown on transwells for 14 days were treated either with NiCl2 (0.5 mM) or Ni particles (29.35 lg/ml) for 24 h, then NPG dye mixture was added to the cells for 45 min. Following washing, cells were incubated for 45 min in HBSS/5% FBS, washed again and visualised under a fluorescent microscope (10).

Fig. 6. Ni compounds elevated intracellular levels of Ni2+ in a concentration-dependent manner. Cells grown in 96-well plates for 14 days were treated either with NiCl2 or Ni particles for 24 h and 48 h, then NPG dye mixture was added to the cells for 45 min. Ni particles concentrations ranged from 5.87 lg/ml to 117.4 lg/ml. Following washing, cells were incubated for 45 min in HBSS/5% FBS, washed again. NPG fluorescence intensity was measured at 506-nm excitation and 535-nm emission and the results were expressed as percentage (%) of control (untreated cells) ± SEM of three independent experiments (three replicates each). *Significant difference with respect to the untreated control (P < 0.05). +Significant difference of 48 h versus 24 h treatment at the same concentration of Ni compounds (P < 0.05).

strength of the signal was slightly higher in cells exposed to NiCl2 compared to Ni t210. Elevation of intracellular Ni2+ after 24 and 48 h exposure to Ni compounds was quantified by the measurement of NPG fluorescence intensity in a microplate reader. Results showed a concentration-dependent increase in the fluorescent signal following an exposure to NiCl2 (Fig. 6A). A time-dependent increase of 269% ± 6% and 454% ± 18% was observed after an exposure to 2 mM NiCl2 for 24 and 48 h, respectively (Fig. 6A). Exposure to Ni t210 resulted in a moderate concentrationdependent increase of the fluorescent signal with maximal values 202% ± 9% observed after the exposure to 2 mM for 48 h (Fig. 6B).

4. Discussion Ni particles have been implicated in the adverse effects of ambient particulate air pollution and were found to trigger respiratory pathological effects, such as lung inflammation, fibrosis, emphysema and cancer (Kilburn, 1984; Haber et al., 2000; Ilic et al., 2007). In addition, the toxicological impact of micro- and nanosized particles has recently come under increased scrutiny due to the increased pulmonary and cardiovascular morbidity and mortality associated with the exposure to particulate matter (PM)

(Laden et al., 2000; Peters et al., 2000; Sorensen et al., 2005; Hogg and van Eeden, 2009). Therefore, in the last decade the interest of the scientific community to further investigate the effects of inhaled particles and the underlying mechanisms has increased resulting in a strong demand for alternative methods and testing strategies for studying in vitro their potential toxic effects. In particular, Ni compounds have been evaluated and their role in oxidative stress and inflammatory responses was shown by several authors (Denkhaus and Salnikow, 2002; Barchowsky and O’Hara, 2003; Chen et al., 2003; Valko et al., 2006; Peters et al., 2007; Schmid et al., 2007; Das et al., 2008; Li et al., 2008). However, the underlying mechanisms of the effects induced by Ni particles on the airway epithelium have not been elucidated. New Ni powders have been commercially used in the past years by major battery manufacturers in both positive and negative electrodes of Ni based batteries (Yang et al., 1999). Among them, nickel powder Type 110 has proven to be ideal for use in thin conductive coatings, such as those found in multilayer capacitors. In industries that process and use these powders workers maybe exposed to higher levels of these Ni particles compared to non-occupational settings. In our study, we have investigated the effect of Ni micro-sized particles (Type 210) and the corresponding ionic form (NiCl2) on the airway epithelium, using the well differentiated human bronchial epithelial cell line, Calu-3. The concentrations of Ni compounds used in our study

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(0.1–5 mM) are within the range observed for human occupational exposure. As shown by Svenes and Andersen, the amount of Ni in the lungs of industry workers was around 50 ± 150 lg/g of lung tissue, which corresponds to 0.85–2.6 mM (Svenes and Andersen, 1998). The maintenance of the integrity and function of the airway epithelium is crucial for the defence against foreign compounds, but is also itself a site for deposition and toxicity of inhaled particles. Our results showed, to the best of our knowledge for the first time, an interference of NiCl2 and Ni particles with the barrier function observed by alterations in TEER. This disruption in the barrier function occurs prior to the decrease in cell viability and, therefore, is an early indication of the toxic effect. Our in vitro results are supported by an in vivo study that demonstrated an increase of soluble proteins and sialic acid in the lavage following injury to the alveolar capillary barrier in rats exposed for 72 h to similar concentrations (0.2–5 mM) of NiCl2 (Bajpai et al., 1999). It has previously been shown that Ni2+ can catalyze the generation of hydroxyl radicals from hydrogen peroxide in a Fenton type reaction and to generate oxidative stress (Valko et al., 2006; Beyersmann and Hartwig, 2008; Das et al., 2008). Moreover, generation of oxidative stress and ROS has been shown to play a major role in pulmonary toxicity caused by inhaled metal particles (McNeilly et al., 2004; Sorensen et al., 2005; Li et al., 2008). We have shown that both Ni particles and NiCl2 were able to induce an increase in ROS to a similar extent in a concentration-dependent manner. The increase in ROS was already observed after 24 h while changes in other endpoints such as TEER and NRU required longer exposure (48 h and 72 h, respectively). These findings suggest the suitability of ROS as an early indicator of the toxicity induced by various metals and particles. The role of Ni compounds in the induction of oxidative stress was further demonstrated by the transcriptional activation of the stress-inducible genes MT1X, HMOX-1, HSP70 and cGCS by both Ni particles and NiCl2. We found a concentration and timedependent induction of these genes with a more pronounced effect observed after the exposure to NiCl2 than Ni particles. It has been shown by others that low levels of oxidative stress caused by particles may induce protective effects that are critical for defending against more severe effects like inflammation and cell death (Li et al., 2008). These protective effects are mediated by the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), which regulates transcriptional activation of HMOX-1 and cGCS and other antioxidant and detoxification enzymes (Jaiswal, 2004). Importantly, we have shown that both Ni particles and NiCl2 induced the expression of these two genes being the induction of HMOX-1 compared to cGCS slightly higher. In support of our findings, it has been recently demonstrated that NiCl2 activates the Nrf2 signaling pathway in human monocytic cells (Lewis et al., 2006). Furthermore, we have demonstrated that HSP70, a cellular chaperon involved in cell protection from oxidative stress (Wang and Fowler, 2008) was up-regulated by Ni compounds. Interestingly, we have observed that HMOX-1 and HSP70 were upregulated to a similar extent and this result further supports the suggestion that HMOX-1 and HSP70 share a common molecular mechanism in heavy metal-induced transcriptional activation (Koizumi et al., 2007). We also have shown an induction of MT1X by Ni compounds. MTs are cysteine-rich proteins that are involved in detoxification and homeostasis of metals and in the protection against ROS (Colangelo et al., 2004; Klaassen et al., 2009). MT protective role against Ni-induced acute lung injury was previously demonstrated in Mt-transgenic mice (Mt1/2(+/+)) (Wesselkamper et al., 2006). It was shown before that MTs have low affinity for Ni compared to other metals (Waalkes et al., 1984), thus suggesting that their protective effect does not involve a direct sequestering of Ni.

Toxicity of Ni compounds appears to be mainly associated with the bioavailability of Ni2+ at the intracellular sites (Ke et al., 2007; Horie et al., 2009), however, the ion release properties of Ni particles (e.g. Ni t210) are still unclear. Our results using the fluorescent cell permeant dye (NPG-Ac) showed an increase of Ni2+ intracellular levels in the cytoplasm and nucleus after the exposure of both NiCl2 and Ni particles. Moreover, we showed that the increase of intracellular Ni2+ is concentration-dependent, although similar levels were observed after 24 h and 48 h of exposure with the exception of 2 mM of NiCl2. The elevation of intracellular Ni2+ levels after the exposure to Ni particles (2 mM) together with the increased ROS levels and elevated TEER observed after 24 h and 48 h, respectively, suggest that the toxicity induced by Ni particles may be attributed to the release of Ni2+ and its bioavailability. The main advantage of using fluorescent-based techniques, in comparison with the more common used analytical methods, is the possibility for a dynamic assessment of intracellular metal ions in living cells. The diverse effect of NiCl2 and Ni particles that we have observed may be attributed to the difference in Ni2+ intracellular levels and their bioavailability. Indeed, our results on Ni particles solubility showed that only 3% of their mass is dissolved (Ni++ ions) when they are brought into an aqueous solvent and can be taken up by cells. It was previously demonstrated that highly soluble Ni particles were more toxic than less soluble particles and the ionic form (Peters et al., 2007; Horie et al., 2009). Therefore, characterising the solubility and ion release properties could be useful in the hazard assessment of Ni particles (and potentially of other metallic particles). In summary, we have demonstrated that exposure to NiCl2 and micro-sized Ni particles resulted in a disruption of the barrier function and induction of oxidative stress as observed by increased ROS levels and up-regulation of stress-inducible genes. The effect of NiCl2 both at the barrier level and at the molecular level was more pronounced than the effect of Ni particles. The toxic effects induced by Ni compounds can be partially attributed to the increase in Ni2+ intracellular levels and their bioavailability observed after the exposure. The alterations in TEER and gene expression (MT1X, HSP70, HMOX-1, cGCS) at non-cytotoxic concentrations demonstrate the usefulness of these endpoints to evaluate Ni and other heavy metals-induced toxic effects. Finally, we can conclude that the in vitro model used in our study consisting of Calu-3 cells grown at the air-interface has a considerable potential for the toxicity assessment of inhaled particles and to further investigate the pathways involved in the toxicity and adverse health effects observed in humans. Disclosure statement The authors declare that there are no conflicts of interest. Acknowledgments This project was funded by the Marie Curie Research Training Network ‘‘Pulmo-Net’’ (MRTN-CT-2004-512229). The authors would like to thank Mr. Gerard Bowe and Mrs. Edna Nemati for their excellent technical assistance and the Institute of Mineralogy & Petrography (Faculty of Geo- and Atmospheric Sciences, University of Innsbruck) for the X-ray diffraction analysis of Ni particles. References Agency for Toxic Substances, Disease Registry (ATSDR), 2005. Toxicological Profile for Nickel. US Department of Health and Human Services, Public Health Service, Atlanta, GA. Bajpai, R., Waseem, M., Khanna, A.K., Kaw, J.L., 1999. Comparative pulmonary toxicity of cadmium and nickel: histopathological and bronchoalveolar lavage analysis. Indian Journal of Experimental Biology 37, 541–545.

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