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This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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The potential toxicity of nanoparticles currently raises public and scientific discussions, and attempts for developing generally accepted handling procedures for nanoparticles are underway. The investigation of the impact of nanoparticles on human health is overdue and reliable test systems accounting for the special properties of nanomaterials must be developed. Nanoparticular zinc oxide (ZnO) may be internalised through the ambient air or topical application of cosmetics, only to name a few, with unpredictable health effects. Therefore, we analysed the determinants of ZnO nanoparticle (NP) genotoxicity. ZnO NP (15-18 nm in diameter) were investigated at concentrations of 0.1, 10, and 100 µg/mL using the cell line A549. Internalised NPs were only infrequently detectable by TEM, but strongly increased Zn 2+ levels in the cytoplasm and even more in the nuclear fraction were measured by atom absorption spectroscopy, indicative for internalised zinc and nuclear accumulation. We observed a time and dosage dependent reduction of cellular viability after ZnO NP exposure. ZnCl 2 exposure of cells induced similar impairments of cellular viability. Complexation of Zn 2+ with diethylene triamine pentaacetic acid (DTPA) resulted in loss of toxicity of NPs, indicating a relevant role of Zn2+ for ZnO NP toxicity. Foci analyses showed the induction of DNA double strand breaks (DSBs) by ZnO NP and increased intracellular reactive oxygen species (ROS) levels. Treatment of the cells with the ROS scavenger N-acetyl-L-cysteine (NAC) resulted in strongly decreased intracellular ROS levels and reduced DNA damages. However, slow increase of ROS after ZnO NP exposure, and reduced but not abolished DSBs after NAC-treatment suggest that Zn2+ may exert genotoxic activities without the necessity of preceding ROS-induction. Our data indicate that ZnO NP toxicity is a result of cellular Zn 2+ intake. Subsequently increased ROS-levels cause DNA damages. However, we found evidence for the assumption that DNA-DSBs could be caused by Zn2+ without the involvement of ROS.
Introduction Zinc oxide nanoparticles (ZnO NP) are used in a variety of applications including cosmetics and coatings.1-6 The common utilization of ZnO NP results in an increasing environmental release (estimated ~500 tons in 2009 in the U.S.) and may lead to adverse health effects.7 The inflammatory potential of inhaled metal oxides like ZnO is well known since decades as acute metal fume fever syndrome.8 Recent studies demonstrated that ZnO NP induce multiple toxic responses in mammalian cells, bacteria, algae, fish, nematodes and plants.9-19 Several studies have been reported with the aim to decipher the role of solid ZnO NP, dissolved Zn 2+ and triggered intracellular metabolites and pathways. Whereas some authors found an impact of both, solid ZnO NP as well as dissolved Zn ions, for toxic responses of exposed cells12,20 other studies suggested that ZnO NP toxicity is mainly attributed to the intake of the particles by endocytosis, their dissolution and subsequently in mitochondria generated reactive oxygen species (ROS).21 The release of ROS has been reported in cells exposed to ZnO NPs,12,18,22-25 suggesting destructive oxidation of cellular components and structures as possible downstream mechanism of ZnO NP exposure. More recently, DNAdouble strand breaks (DSBs) have been observed after ZnO NP-
This journal is © The Royal Society of Chemistry 2013
exposure, i.e., genotoxicity has turned up as another possible feature of ZnO toxicity.26,27 This appears comprehensible as the observed intracellular high ROS-levels after ZnO NP-exposure could be DNA-damaging. However, beside the most likely involvement of ROS in ZnO NP genotoxicity, other genotoxic mechanisms of ZnO NP or dissolved Zn2+ ions like direct interaction of internalised ZnO NP or Zn2+ with vital intracellular structures remain possible.2125,28,29 In accordance with the assumed role of Zn for DNA damages, Hackenberg et al demonstrated the cellular uptake and nuclear accumulation of BSA-coated and thereby stabilized ZnO NPs in nasal mucosa cells.27 These results of ZnO NP uptake are in agreement with several previous data obtained with different cell lines such as primary human epidermal keratinocytes, acute monocytic leukemia cells and nasal mucosa cells.24,25,28,29 In contrast to this result Valdiglesias et al showed no cellular uptake of ZnO NPs in neuronal cells by flow cytometric analysis.29 The later data support the hypothesis of Koa et al. who suggested an endocytotic ZnO NP uptake and subsequent dissociation into Zn ions in the acidic environment of early endosomes.21 Overall, the data remain controversial. Therefore, we here analysed in detail the DNA-damaging properties of ZnO NP at concentrations
J. Name., 2013, 00, 1-3 | 1
Nanoscale Accepted Manuscript
Published on 16 April 2015. Downloaded by University of Sussex on 21/04/2015 14:21:15.
Julia Heim, a Eva Felder, a Muhammad Nawaz Tahir, b Anke Kaltbeitzel, d Ulf Ruediger Heinrich, a Christoph Brochhausen, c Volker Mailänder, d,e Wolfgang Tremel, b Juergen Briegera*
Nanoscale
occurring in the environment and clarified whether ZnO NP, Zn2+– ions or induced metabolites exert DNA damages. Our study provides insights into the toxic mechanisms of synthesized ZnO NP in a model system of respiratory epithelia. The data suggests that ZnO NP dissolve quickly into Zn2+ prior to internalisation and nuclear accumulation, resulting in reduced cellular viability and DNA damages. Our data suggests direct genotoxic properties of Zn2+ besides those caused by Zn2+ induced ROS.
Published on 16 April 2015. Downloaded by University of Sussex on 21/04/2015 14:21:15.
Experimental Synthesis of ZnO nanoparticles The ZnO nanoparticles were synthesized following a procedure reported by Cheng et al. with slight modifications.30 Zn(Ac)2 x 2H2O (1.09 g) was dissolved in 10 mL of methanol. (5+15) mL of the tetra methyl ammonium hydroxide (25% w/w in high-purity H2O and tetramethyl ammonium hydroxide 25% w/w in methanol) were added under stirring at room temperature. The solution was transferred to a Teflon-lined stainless steel autoclave and heated at 50 °C for 24 h. The white precipitate was collected and purified by washing with water and dried in air at room temperature. Prior to use in cell culture experiments particles were weighted and solubilized in high-purity H2O as described under cell studies. Sample characterization ζ-potential Measurements. The measurement of the ζ-potential of the ZnO particles was performed using a Zetasizer Nano ZS from Malvern Instruments using a disposable capillary cell. Typically, 1 mL aliquots of each sample were injected into the capillary cell and 5−10 measurements per sample were performed at 25°C. Electron microscopy. The size and morphology of the ZnO NP were investigated using transmission electron microscopy (TEM, Philips EM 420, Philips International B.V., Amsterdam, Netherlands; instrument with an acceleration voltage of 120 kV). Samples for transmission electron microscopy were prepared by placing a drop of dilute nanoparticle solution in hexane on a carbon coated copper grid. Low-resolution TEM images and ED patterns were recorded on a Philips EM420 microscope (Firma) operating at an acceleration voltage of 120kV. X-ray diffraction. X-ray powder diffraction measurements were performed on a Bruker D8 Advance powder diffractometer operating with Mo-Kα radiation and a Sol-X energy-dispersive detector. Samples were prepared as loose powder on nearly background free Sisingle crystal plates. Background, lattice parameters, crystallite sizes, scale factors and the partial occupation factors of Zn and O were refined. Cell studies Nanoparticle treatment conditions. ZnO NPs were synthesized with a defined size of 15-18 nm. ZnO NP or ZnCl2 (Sigma-Aldrich, St. Louis, Missouri, USA) were weighted on the day of experiment and suspended in high-purity H2O (Cayman Chemical Company, Ann Arbor, Michigan, USA) at a concentration of 1 mg/mL. The ZnO NP dispersion or ZnCl2 solution were sonicated using a Sonorex Super RK 510 H (BANDELIN electronics, Berlin, Germany) for 5 min at 32 W amplitude. The cells were treated immediately in a final concentration of 0.1 µg/mL, 10 µg/mL or 100 µg/mL for 1, 15,
2 | J. Name., 2012, 00, 1-3
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Journal Name 30 min, and 1, 4, 24, and 48 h. Controls were cells cultured in standard medium without nanoparticle treatment. Concentrations were chosen according to values measured in sun protection (up to 100mg/ml and drinking water 10 µg/ml.44,45 Preparation of ZnO NP free supernatant. ZnO NP, in a final concentration of 100 µg/mL were added for 10 min to 8 mL cell culture medium and stirred at RT. Afterwards the suspension was centrifuged at 16,000 g for 10 min. The supernatant was used for cell treatments. Cell culture conditions. We used the alveolar epithelial-like type-II cell line A549 (ATCC, Manassas, VA, USA), maintained in DMEM/Ham’s F12 (Sigma-Aldrich), supplemented with 5 % fetal calf serum (FCS, Sigma-Aldrich) and 1 % Penicillin-Streptomycin (Penicillin 100 U/mL and Streptomycin 100 µg/mL; Sigma-Aldrich) at 37 °C in 5 % CO2. Cell preparation for TEM-imaging. For the ultra structural analysis of ZnO NP uptake, cell pellets of ZnO NP (100 µg/mL; exposure time 15 min, 30 min and 1 h) treated A549 cells were applied. They were washed with PBS and centrifuged at 300 g for 5 min. This step was repeated three times. Afterwards, cell pellets were incubated for 15 min in fixation buffer (4 % paraformaldehyde, 0.1 % glutardialdehyde, and 0.2 % picric acid, in 0.1 mol Sørensen buffer). Pellets were washed two times again for 5 min in 0.1 M Sørensen buffer. Pellets were dehydrated in a concentration series of EtOH (70, 80, 90 and 100 %), for 5 min each. Afterwards, they were embedded for 10 min in a 1:1 suspension of 100 % EtOH and LR-White™ (London Resin Company Ltd, London, United Kingdom) and further two times for 30 min in LR-White™. After embedding the pellets were polymerised over night at 58 °C. Ultrathin slides were prepared (80100 nm) and the intracellular deposition of NPs was evaluated by electron microscopy (LEO 906: Zeiss, Oberkochen, Germany). Measurement of Zn2+ concentrations in the cytoplasmatic and nuclear fractions. For atomic absorption spectroscopy (AAS) measurement of Zn, 600,000 cells were incubated with 100 µg/mL ZnO NP for 4, 24 and 48 h and counted (CASY TT®, Roche, Mannheim, Germany) prior to fractionation. Cells were washed with ice cold PBS and centrifuged for 10 min at 400 g. The supernatants were removed and each pellet was resuspended in 250 µL of hypotonic buffer (20 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2; pH 7) including 10 µL DTT (0.5 mM) and incubated for 15 min on ice. Afterwards, 12.5 µL NP-40 detergent was added and mixed for 1 min. The homogenate was centrifuged at 4 °C at 1000 g for 10 min for separation of the cytoplasmatic fraction (supernatant) and the nuclear fraction (pellet). The nuclear fraction was resuspended in 30 µL of cell extraction buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1 % Triton X-100, 10 % Glycerol, 0.1 % SDS, 0.5 % Na-deoxycholate) and incubated for 30 min on ice. Nuclear fractions were sonicated for 5 min and further centrifuged by 4 °C at 16,000 g for 30 min. Finally, plasma and nuclear fractions were filled up with aqua dest. to a total volume of 4 mL. Zn2+-concentrations in the cytoplasmatic and nuclear fractions were measured by atomic absorption spectrometry (AAS). The samples were prepared by dissolving the aliquots in 2N HNO3 solution. The measured Zn-concentrations were allocated with the total volume of 4 mL giving the total Zn content and than divided through viable cell counts measured before giving absolute values of Zn/- nucleus and cytoplasm of a single cell.
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Journal Name Viability measurement with CASY TT®. Viability was measured by the use of the CASY TT® (Roche Diagnostics GmbH, Penzberg, Germany), an electric field multi-channel cell counting system. CASY TT® measures cell number, cell size and cell viability and is based on a pulse surface analysis. The cells were sucked with a constant speed into a capillary between to electrodes. The magnetoresistance of the capillary equates to the cell volume. The cell membrane of a dead or injured cell is more permeable than those of an intact cell and causes less change in magneto-resistance. The cells were treated with three different concentrations of ZnO NPs (0.1; 10; 100 µg/mL) or the supernatants of ZnO NPs. After exposure the cells were washed with PBS and were covered with fresh medium (5 % FCS, 2 % P/S). The cellular viability of the cells was determined after 4, 24, and 48 h of ZnO NP exposure. After incubation cells were trypsinized and centrifuged at 450 g for 8 min. The cells were resuspended with 750 µL (4 h, 24 h) or 200 µL (48 h) cell culture medium depending on the exposure time. Afterwards, 4 tubes with 10 mL of CASY-ton were supplied with 200 µL (50:1) cell suspension of each time point and were measured by CASY TT®. Chelating studies. Diethylene triamine pentaacetic acid (DTPA) was used as extracellular chelator of dissociated Zn2+. Cells were seeded in a 6-well-plate (control, 100 µg/mL ZnO NP, 100 µg/mL ZnO NP + DTPA) and cultured for 24 h. On the following day the medium of the controls was replaced by fresh medium. The second well was treated with 100 µg/mL ZnO NP, prepared as described before and the third well was treated with (100 µg/mL ZnO NP + 0.06 mM DTPA, rate 2:1). After 48 h cells were measured by CASY TT®. ROS-measurement and NAC-treatment. The generation of ROS at different time points after ZnO NP treatment (100 µg/mL) was assessed in A549 cells by using 2,7-dichlorofluorescin diacetate (H2DFCDA, Invitrogen, Oregon, USA). The non-fluorescent compound H2DCF-DA is de-esterified and oxidized in presence of ROS to fluorescent DCF (2´,7´- dichlorofluorscein). ROS generation was studied by fluorometric analysis. Cells (200,000 per well) were seeded in 6-well culture plates and allowed to adhere for 24 h. On the following day the culture medium was replaced with fresh culture medium containing 5 mM NAC (Sigma-Aldrich). After 24 h of NAC treatment, the cells were incubated with H2DCF-DA (10 mM) for 30 min at 37 °C. The reaction mixture was removed and the cells washed with 2 mL PBS each well (Sigma-Aldrich). Afterwards, cells were covered with 2 mL culture medium containing ZnO NPs (100 µg/mL). Fluorescence intensities were measured after 1 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h using a fluorescent microplate reader (Labsystems, Ramsey, Minnesota, USA) at an excitation wavelength of 485 nm and emission wavelength of 528 nm, and values were expressed as percentage of fluorescence intensity relative to the controls. γH2A.X immunofluorescence assay. For the evaluation of γH2A.X immunofluorescence assay after ZnO NP and ZnCl2 treatment, 200,000 cells were cultivated on coverslips (IDL GmbH & Co. KG, Nidderau, Germany) in 6 well plates (Thermo Scientific, Waltham, Massachusetts, USA). We investigated three different concentrations of ZnO NP or ZnCl2 (0.1, 10, 100 µg/mL). After 1 min, 15 min, 1 h, 4 h, and 24 h cells were fixed (15 min with 4 % paraformaldehyde and 10 min methanol) and washed with PBS (Sigma-Aldrich). Nonspecific staining was blocked by incubation with 5 % BSA/PBS, 0.3 % Triton X-100 for 60 min. The cells were incubated with the pri-
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ARTICLE mary antibody, anti-phospho-Histone H2A.X (Ser139, 1:1000, Cell Signalling, Danvers, USA) diluted 1:500 in PBS with 0.3 % Triton X-100 at RT. Cells were washed two times with PBS (SigmaAldrich). The second antibody (Alexa Fluor 488 goat anti-mouse IgG diluted 1:500 in 5 % BSA/PBS with 0.3 % Triton X-100) was applied for one hour at RT. Than cells were washed two times in PBS, incubated for 2 min in TBS with 400 mM NaCl, and washed again once in PBS. The cover slips were mounted on slides using Vectashield® Hard+Set™ Mounting Medium with DAPI. All slides were examined using an inverted microscope (Nikon ECLIPSE TE2000-U). For quantitative analysis foci were counted using the software Image J 1.47f (NIH, Bethesda, USA). Each sample was analysed with at least 60 cells. Statistics. To analyse treatment responses, obtained data were compared to the controls using ANOVA followed by Tukey post tests. For comparison of single treatments, one way paired t-tests were used. Shown are the means +/- SEM of at least three independent experiments.
Results ZnO particle characterization. Transmission electronic micrographs (TEM) show the presence of spherical non-aggregated uniform particles homogeneously dispersed in water. Fig. 1 shows a representative TEM image. The size distribution as obtained from TEM shows that the average size value of the NPs is centered at 1518 nm (Fig. S1a, Supporting Information). The crystallinity and phase identity of the nanoparticles was confirmed using X-ray diffraction (XRD). Representative XRD patterns of as synthesized ZnO NP are presented in Fig. S1b (Supporting Information). All reflections could be indexed to the ZnO structure (wurtzite) with the lattice parameters a = 3.24 Å and c = 5.20 Å and space group (SG) P63mc (No. 186). Evaluation of ZnO toxicity determinants. TEM showed in most cells the absence of solid ZnO NP. Occasionally, agglomerated ZnO NPs were detectable in endosomes and single NPs in the cytoplasm, and the nucleus after NP-exposure (Fig. 1). The intracellular measurement of Zn2+ by AAS indicated Zn-internalisation as well. Intracellular Zn-concentrations increased fast and remained largely unchanged between 4 and 48 hours after ZnO NP exposure (Fig. 2). We observed higher values of Zn2+ in the nucleus compared to the cytoplasm, indicating enrichment of dissolved Zn2+ ions. Next we analysed cellular viability starting 4 hours up to 48 hours post exposure and observed a significant time dependent decrease of viability at the highest (100 µg/mL) concentration (Fig. 3). Interestingly, we observed at the lower NP-concentrations slightly increased (n.s.) cell counts of viable cells, suggesting stimulated proliferation. Next, we exposed cells again to the highest concentration (100 µg/mL) but changed the culture media (fresh media without NP) after 4, 6, and 8 hours and analysed viability 24 hours later. We found that the particles showed a reduced viability of 20 % in comparison to the untreated controls at any point of time (Fig. 4A). Notably, viability decreased to levels comparable to those observed in cultures without media change, indicating that impairment of cellular viability was induced early prior to the media replacement. To evaluate whether cellular toxicity needs solid ZnO-particles, we incubated the ZnO NP in culture medium for 10 minutes, centrifuged this suspension and used the supernatant for incubation of the cells
J. Name., 2012, 00, 1-3 | 3
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Fig. 3. Analysis of viable cell counts after ZnO NP exposure. High concentrations (100 µg/mL) of NP resulted in a significant time dependent decrease of viable cell counts. Interestingly, the lower NP-concentrations resulted up to 48 hours in slightly (n.s.) increased viability. Impaired viability of ZnO NP treated cells was completely abolished by DTPA (shown for 48 h). *** = p
Nanoscale Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: J. Heim, E. Felder, M. N. Tahir, A. Kaltbeitzel, U. Heinrich, C. Brochhausen, V. Mailänder, W. Tremel and J. Brieger, Nanoscale, 2015, DOI: 10.1039/C5NR01167A.
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors. Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
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The potential toxicity of nanoparticles currently raises public and scientific discussions, and attempts for developing generally accepted handling procedures for nanoparticles are underway. The investigation of the impact of nanoparticles on human health is overdue and reliable test systems accounting for the special properties of nanomaterials must be developed. Nanoparticular zinc oxide (ZnO) may be internalised through the ambient air or topical application of cosmetics, only to name a few, with unpredictable health effects. Therefore, we analysed the determinants of ZnO nanoparticle (NP) genotoxicity. ZnO NP (15-18 nm in diameter) were investigated at concentrations of 0.1, 10, and 100 µg/mL using the cell line A549. Internalised NPs were only infrequently detectable by TEM, but strongly increased Zn 2+ levels in the cytoplasm and even more in the nuclear fraction were measured by atom absorption spectroscopy, indicative for internalised zinc and nuclear accumulation. We observed a time and dosage dependent reduction of cellular viability after ZnO NP exposure. ZnCl 2 exposure of cells induced similar impairments of cellular viability. Complexation of Zn 2+ with diethylene triamine pentaacetic acid (DTPA) resulted in loss of toxicity of NPs, indicating a relevant role of Zn2+ for ZnO NP toxicity. Foci analyses showed the induction of DNA double strand breaks (DSBs) by ZnO NP and increased intracellular reactive oxygen species (ROS) levels. Treatment of the cells with the ROS scavenger N-acetyl-L-cysteine (NAC) resulted in strongly decreased intracellular ROS levels and reduced DNA damages. However, slow increase of ROS after ZnO NP exposure, and reduced but not abolished DSBs after NAC-treatment suggest that Zn2+ may exert genotoxic activities without the necessity of preceding ROS-induction. Our data indicate that ZnO NP toxicity is a result of cellular Zn 2+ intake. Subsequently increased ROS-levels cause DNA damages. However, we found evidence for the assumption that DNA-DSBs could be caused by Zn2+ without the involvement of ROS.
Introduction Zinc oxide nanoparticles (ZnO NP) are used in a variety of applications including cosmetics and coatings.1-6 The common utilization of ZnO NP results in an increasing environmental release (estimated ~500 tons in 2009 in the U.S.) and may lead to adverse health effects.7 The inflammatory potential of inhaled metal oxides like ZnO is well known since decades as acute metal fume fever syndrome.8 Recent studies demonstrated that ZnO NP induce multiple toxic responses in mammalian cells, bacteria, algae, fish, nematodes and plants.9-19 Several studies have been reported with the aim to decipher the role of solid ZnO NP, dissolved Zn 2+ and triggered intracellular metabolites and pathways. Whereas some authors found an impact of both, solid ZnO NP as well as dissolved Zn ions, for toxic responses of exposed cells12,20 other studies suggested that ZnO NP toxicity is mainly attributed to the intake of the particles by endocytosis, their dissolution and subsequently in mitochondria generated reactive oxygen species (ROS).21 The release of ROS has been reported in cells exposed to ZnO NPs,12,18,22-25 suggesting destructive oxidation of cellular components and structures as possible downstream mechanism of ZnO NP exposure. More recently, DNAdouble strand breaks (DSBs) have been observed after ZnO NP-
This journal is © The Royal Society of Chemistry 2013
exposure, i.e., genotoxicity has turned up as another possible feature of ZnO toxicity.26,27 This appears comprehensible as the observed intracellular high ROS-levels after ZnO NP-exposure could be DNA-damaging. However, beside the most likely involvement of ROS in ZnO NP genotoxicity, other genotoxic mechanisms of ZnO NP or dissolved Zn2+ ions like direct interaction of internalised ZnO NP or Zn2+ with vital intracellular structures remain possible.2125,28,29 In accordance with the assumed role of Zn for DNA damages, Hackenberg et al demonstrated the cellular uptake and nuclear accumulation of BSA-coated and thereby stabilized ZnO NPs in nasal mucosa cells.27 These results of ZnO NP uptake are in agreement with several previous data obtained with different cell lines such as primary human epidermal keratinocytes, acute monocytic leukemia cells and nasal mucosa cells.24,25,28,29 In contrast to this result Valdiglesias et al showed no cellular uptake of ZnO NPs in neuronal cells by flow cytometric analysis.29 The later data support the hypothesis of Koa et al. who suggested an endocytotic ZnO NP uptake and subsequent dissociation into Zn ions in the acidic environment of early endosomes.21 Overall, the data remain controversial. Therefore, we here analysed in detail the DNA-damaging properties of ZnO NP at concentrations
J. Name., 2013, 00, 1-3 | 1
Nanoscale Accepted Manuscript
Published on 16 April 2015. Downloaded by University of Sussex on 21/04/2015 14:21:15.
Julia Heim, a Eva Felder, a Muhammad Nawaz Tahir, b Anke Kaltbeitzel, d Ulf Ruediger Heinrich, a Christoph Brochhausen, c Volker Mailänder, d,e Wolfgang Tremel, b Juergen Briegera*
Nanoscale
occurring in the environment and clarified whether ZnO NP, Zn2+– ions or induced metabolites exert DNA damages. Our study provides insights into the toxic mechanisms of synthesized ZnO NP in a model system of respiratory epithelia. The data suggests that ZnO NP dissolve quickly into Zn2+ prior to internalisation and nuclear accumulation, resulting in reduced cellular viability and DNA damages. Our data suggests direct genotoxic properties of Zn2+ besides those caused by Zn2+ induced ROS.
Published on 16 April 2015. Downloaded by University of Sussex on 21/04/2015 14:21:15.
Experimental Synthesis of ZnO nanoparticles The ZnO nanoparticles were synthesized following a procedure reported by Cheng et al. with slight modifications.30 Zn(Ac)2 x 2H2O (1.09 g) was dissolved in 10 mL of methanol. (5+15) mL of the tetra methyl ammonium hydroxide (25% w/w in high-purity H2O and tetramethyl ammonium hydroxide 25% w/w in methanol) were added under stirring at room temperature. The solution was transferred to a Teflon-lined stainless steel autoclave and heated at 50 °C for 24 h. The white precipitate was collected and purified by washing with water and dried in air at room temperature. Prior to use in cell culture experiments particles were weighted and solubilized in high-purity H2O as described under cell studies. Sample characterization ζ-potential Measurements. The measurement of the ζ-potential of the ZnO particles was performed using a Zetasizer Nano ZS from Malvern Instruments using a disposable capillary cell. Typically, 1 mL aliquots of each sample were injected into the capillary cell and 5−10 measurements per sample were performed at 25°C. Electron microscopy. The size and morphology of the ZnO NP were investigated using transmission electron microscopy (TEM, Philips EM 420, Philips International B.V., Amsterdam, Netherlands; instrument with an acceleration voltage of 120 kV). Samples for transmission electron microscopy were prepared by placing a drop of dilute nanoparticle solution in hexane on a carbon coated copper grid. Low-resolution TEM images and ED patterns were recorded on a Philips EM420 microscope (Firma) operating at an acceleration voltage of 120kV. X-ray diffraction. X-ray powder diffraction measurements were performed on a Bruker D8 Advance powder diffractometer operating with Mo-Kα radiation and a Sol-X energy-dispersive detector. Samples were prepared as loose powder on nearly background free Sisingle crystal plates. Background, lattice parameters, crystallite sizes, scale factors and the partial occupation factors of Zn and O were refined. Cell studies Nanoparticle treatment conditions. ZnO NPs were synthesized with a defined size of 15-18 nm. ZnO NP or ZnCl2 (Sigma-Aldrich, St. Louis, Missouri, USA) were weighted on the day of experiment and suspended in high-purity H2O (Cayman Chemical Company, Ann Arbor, Michigan, USA) at a concentration of 1 mg/mL. The ZnO NP dispersion or ZnCl2 solution were sonicated using a Sonorex Super RK 510 H (BANDELIN electronics, Berlin, Germany) for 5 min at 32 W amplitude. The cells were treated immediately in a final concentration of 0.1 µg/mL, 10 µg/mL or 100 µg/mL for 1, 15,
2 | J. Name., 2012, 00, 1-3
Page 2 of 7
Journal Name 30 min, and 1, 4, 24, and 48 h. Controls were cells cultured in standard medium without nanoparticle treatment. Concentrations were chosen according to values measured in sun protection (up to 100mg/ml and drinking water 10 µg/ml.44,45 Preparation of ZnO NP free supernatant. ZnO NP, in a final concentration of 100 µg/mL were added for 10 min to 8 mL cell culture medium and stirred at RT. Afterwards the suspension was centrifuged at 16,000 g for 10 min. The supernatant was used for cell treatments. Cell culture conditions. We used the alveolar epithelial-like type-II cell line A549 (ATCC, Manassas, VA, USA), maintained in DMEM/Ham’s F12 (Sigma-Aldrich), supplemented with 5 % fetal calf serum (FCS, Sigma-Aldrich) and 1 % Penicillin-Streptomycin (Penicillin 100 U/mL and Streptomycin 100 µg/mL; Sigma-Aldrich) at 37 °C in 5 % CO2. Cell preparation for TEM-imaging. For the ultra structural analysis of ZnO NP uptake, cell pellets of ZnO NP (100 µg/mL; exposure time 15 min, 30 min and 1 h) treated A549 cells were applied. They were washed with PBS and centrifuged at 300 g for 5 min. This step was repeated three times. Afterwards, cell pellets were incubated for 15 min in fixation buffer (4 % paraformaldehyde, 0.1 % glutardialdehyde, and 0.2 % picric acid, in 0.1 mol Sørensen buffer). Pellets were washed two times again for 5 min in 0.1 M Sørensen buffer. Pellets were dehydrated in a concentration series of EtOH (70, 80, 90 and 100 %), for 5 min each. Afterwards, they were embedded for 10 min in a 1:1 suspension of 100 % EtOH and LR-White™ (London Resin Company Ltd, London, United Kingdom) and further two times for 30 min in LR-White™. After embedding the pellets were polymerised over night at 58 °C. Ultrathin slides were prepared (80100 nm) and the intracellular deposition of NPs was evaluated by electron microscopy (LEO 906: Zeiss, Oberkochen, Germany). Measurement of Zn2+ concentrations in the cytoplasmatic and nuclear fractions. For atomic absorption spectroscopy (AAS) measurement of Zn, 600,000 cells were incubated with 100 µg/mL ZnO NP for 4, 24 and 48 h and counted (CASY TT®, Roche, Mannheim, Germany) prior to fractionation. Cells were washed with ice cold PBS and centrifuged for 10 min at 400 g. The supernatants were removed and each pellet was resuspended in 250 µL of hypotonic buffer (20 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2; pH 7) including 10 µL DTT (0.5 mM) and incubated for 15 min on ice. Afterwards, 12.5 µL NP-40 detergent was added and mixed for 1 min. The homogenate was centrifuged at 4 °C at 1000 g for 10 min for separation of the cytoplasmatic fraction (supernatant) and the nuclear fraction (pellet). The nuclear fraction was resuspended in 30 µL of cell extraction buffer (10 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na4P2O7, 2 mM Na3VO4, 1 % Triton X-100, 10 % Glycerol, 0.1 % SDS, 0.5 % Na-deoxycholate) and incubated for 30 min on ice. Nuclear fractions were sonicated for 5 min and further centrifuged by 4 °C at 16,000 g for 30 min. Finally, plasma and nuclear fractions were filled up with aqua dest. to a total volume of 4 mL. Zn2+-concentrations in the cytoplasmatic and nuclear fractions were measured by atomic absorption spectrometry (AAS). The samples were prepared by dissolving the aliquots in 2N HNO3 solution. The measured Zn-concentrations were allocated with the total volume of 4 mL giving the total Zn content and than divided through viable cell counts measured before giving absolute values of Zn/- nucleus and cytoplasm of a single cell.
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Nanoscale
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Journal Name Viability measurement with CASY TT®. Viability was measured by the use of the CASY TT® (Roche Diagnostics GmbH, Penzberg, Germany), an electric field multi-channel cell counting system. CASY TT® measures cell number, cell size and cell viability and is based on a pulse surface analysis. The cells were sucked with a constant speed into a capillary between to electrodes. The magnetoresistance of the capillary equates to the cell volume. The cell membrane of a dead or injured cell is more permeable than those of an intact cell and causes less change in magneto-resistance. The cells were treated with three different concentrations of ZnO NPs (0.1; 10; 100 µg/mL) or the supernatants of ZnO NPs. After exposure the cells were washed with PBS and were covered with fresh medium (5 % FCS, 2 % P/S). The cellular viability of the cells was determined after 4, 24, and 48 h of ZnO NP exposure. After incubation cells were trypsinized and centrifuged at 450 g for 8 min. The cells were resuspended with 750 µL (4 h, 24 h) or 200 µL (48 h) cell culture medium depending on the exposure time. Afterwards, 4 tubes with 10 mL of CASY-ton were supplied with 200 µL (50:1) cell suspension of each time point and were measured by CASY TT®. Chelating studies. Diethylene triamine pentaacetic acid (DTPA) was used as extracellular chelator of dissociated Zn2+. Cells were seeded in a 6-well-plate (control, 100 µg/mL ZnO NP, 100 µg/mL ZnO NP + DTPA) and cultured for 24 h. On the following day the medium of the controls was replaced by fresh medium. The second well was treated with 100 µg/mL ZnO NP, prepared as described before and the third well was treated with (100 µg/mL ZnO NP + 0.06 mM DTPA, rate 2:1). After 48 h cells were measured by CASY TT®. ROS-measurement and NAC-treatment. The generation of ROS at different time points after ZnO NP treatment (100 µg/mL) was assessed in A549 cells by using 2,7-dichlorofluorescin diacetate (H2DFCDA, Invitrogen, Oregon, USA). The non-fluorescent compound H2DCF-DA is de-esterified and oxidized in presence of ROS to fluorescent DCF (2´,7´- dichlorofluorscein). ROS generation was studied by fluorometric analysis. Cells (200,000 per well) were seeded in 6-well culture plates and allowed to adhere for 24 h. On the following day the culture medium was replaced with fresh culture medium containing 5 mM NAC (Sigma-Aldrich). After 24 h of NAC treatment, the cells were incubated with H2DCF-DA (10 mM) for 30 min at 37 °C. The reaction mixture was removed and the cells washed with 2 mL PBS each well (Sigma-Aldrich). Afterwards, cells were covered with 2 mL culture medium containing ZnO NPs (100 µg/mL). Fluorescence intensities were measured after 1 min, 15 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h using a fluorescent microplate reader (Labsystems, Ramsey, Minnesota, USA) at an excitation wavelength of 485 nm and emission wavelength of 528 nm, and values were expressed as percentage of fluorescence intensity relative to the controls. γH2A.X immunofluorescence assay. For the evaluation of γH2A.X immunofluorescence assay after ZnO NP and ZnCl2 treatment, 200,000 cells were cultivated on coverslips (IDL GmbH & Co. KG, Nidderau, Germany) in 6 well plates (Thermo Scientific, Waltham, Massachusetts, USA). We investigated three different concentrations of ZnO NP or ZnCl2 (0.1, 10, 100 µg/mL). After 1 min, 15 min, 1 h, 4 h, and 24 h cells were fixed (15 min with 4 % paraformaldehyde and 10 min methanol) and washed with PBS (Sigma-Aldrich). Nonspecific staining was blocked by incubation with 5 % BSA/PBS, 0.3 % Triton X-100 for 60 min. The cells were incubated with the pri-
This journal is © The Royal Society of Chemistry 2012
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DOI: 10.1039/C5NR01167A
ARTICLE mary antibody, anti-phospho-Histone H2A.X (Ser139, 1:1000, Cell Signalling, Danvers, USA) diluted 1:500 in PBS with 0.3 % Triton X-100 at RT. Cells were washed two times with PBS (SigmaAldrich). The second antibody (Alexa Fluor 488 goat anti-mouse IgG diluted 1:500 in 5 % BSA/PBS with 0.3 % Triton X-100) was applied for one hour at RT. Than cells were washed two times in PBS, incubated for 2 min in TBS with 400 mM NaCl, and washed again once in PBS. The cover slips were mounted on slides using Vectashield® Hard+Set™ Mounting Medium with DAPI. All slides were examined using an inverted microscope (Nikon ECLIPSE TE2000-U). For quantitative analysis foci were counted using the software Image J 1.47f (NIH, Bethesda, USA). Each sample was analysed with at least 60 cells. Statistics. To analyse treatment responses, obtained data were compared to the controls using ANOVA followed by Tukey post tests. For comparison of single treatments, one way paired t-tests were used. Shown are the means +/- SEM of at least three independent experiments.
Results ZnO particle characterization. Transmission electronic micrographs (TEM) show the presence of spherical non-aggregated uniform particles homogeneously dispersed in water. Fig. 1 shows a representative TEM image. The size distribution as obtained from TEM shows that the average size value of the NPs is centered at 1518 nm (Fig. S1a, Supporting Information). The crystallinity and phase identity of the nanoparticles was confirmed using X-ray diffraction (XRD). Representative XRD patterns of as synthesized ZnO NP are presented in Fig. S1b (Supporting Information). All reflections could be indexed to the ZnO structure (wurtzite) with the lattice parameters a = 3.24 Å and c = 5.20 Å and space group (SG) P63mc (No. 186). Evaluation of ZnO toxicity determinants. TEM showed in most cells the absence of solid ZnO NP. Occasionally, agglomerated ZnO NPs were detectable in endosomes and single NPs in the cytoplasm, and the nucleus after NP-exposure (Fig. 1). The intracellular measurement of Zn2+ by AAS indicated Zn-internalisation as well. Intracellular Zn-concentrations increased fast and remained largely unchanged between 4 and 48 hours after ZnO NP exposure (Fig. 2). We observed higher values of Zn2+ in the nucleus compared to the cytoplasm, indicating enrichment of dissolved Zn2+ ions. Next we analysed cellular viability starting 4 hours up to 48 hours post exposure and observed a significant time dependent decrease of viability at the highest (100 µg/mL) concentration (Fig. 3). Interestingly, we observed at the lower NP-concentrations slightly increased (n.s.) cell counts of viable cells, suggesting stimulated proliferation. Next, we exposed cells again to the highest concentration (100 µg/mL) but changed the culture media (fresh media without NP) after 4, 6, and 8 hours and analysed viability 24 hours later. We found that the particles showed a reduced viability of 20 % in comparison to the untreated controls at any point of time (Fig. 4A). Notably, viability decreased to levels comparable to those observed in cultures without media change, indicating that impairment of cellular viability was induced early prior to the media replacement. To evaluate whether cellular toxicity needs solid ZnO-particles, we incubated the ZnO NP in culture medium for 10 minutes, centrifuged this suspension and used the supernatant for incubation of the cells
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DOI: 10.1039/C5NR01167A
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Fig. 3. Analysis of viable cell counts after ZnO NP exposure. High concentrations (100 µg/mL) of NP resulted in a significant time dependent decrease of viable cell counts. Interestingly, the lower NP-concentrations resulted up to 48 hours in slightly (n.s.) increased viability. Impaired viability of ZnO NP treated cells was completely abolished by DTPA (shown for 48 h). *** = p
