Copper-based nanoparticles induce high toxicity in leukemic HL60 cells

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Copper-based nanoparticles induce high toxicity in leukemic HL60 cells

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Ylva Rodhe a, Sara Skoglund b, Inger Odnevall Wallinder b, Zuzana Potácová c, Lennart Möller a,⇑

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a

Department of Biosciences and Nutrition, Karolinska Institutet, SE-141 83 Huddinge, Stockholm, Sweden KTH Royal Institute of Technology, Division of Surface and Corrosion Science, School of Chemical Science and Engineering, SE-100 44 Stockholm, Sweden c Department of Laboratory Medicine, Karolinska Institutet, SE-141 83 Huddinge, Stockholm, Sweden b

a r t i c l e

i n f o

Article history: Received 11 June 2014 Revised 25 May 2015 Accepted 27 May 2015 Available online xxxx Keywords: Nanoparticles Cu CuO Oxidative stress Cytotoxicity Mitochondrial damage

a b s t r a c t From the increasing societal use of nanoparticles (NPs) follows the necessity to understand their potential toxic effects. This requires an in-depth understanding of the relationship between their physicochemical properties and their toxicological behavior. The aim of the present work was to study the toxicity of Cu and CuO NPs toward the leukemic cell line HL60. The toxicity was explored in terms of mitochondrial damage, DNA damage, oxidative DNA damage, cell death and reactive oxygen species (ROS) formation. Particle characteristics and copper release were specifically investigated in order to gain an improved understanding of prevailing toxic mechanisms. The Cu NPs revealed higher toxicity compared with both CuO NPs and dissolved copper (CuCl2), as well as a more rapid copper release compared with CuO NPs. Mitochondrial damage was induced by Cu NPs already after 2 h exposure. Cu NPs induced oxidation at high levels in an acellular ROS assay, and a small increase of intracellular ROS was observed. The increase of DNA damage was limited. CuO NPs did not induce any mitochondrial damage up to 6 h of exposure. No acellular ROS was induced by the CuO NPs, and the levels of intracellular ROS and DNA damage were limited after 2 h exposure. Necrosis was the main type of cell death observed after 18 h exposure to CuO NP and dissolved copper. Ó 2015 Published by Elsevier Ltd.

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1. Introduction

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Nanoparticles (NPs) hold physicochemical characteristics that differ dramatically from larger sized particles, and from the bulk material of the same chemical composition (Borm et al., 2006). Their unique properties make them highly attractive as components in novel and established applications such as medical products, electronic devices, cosmetic products and inks (Dastjerdi and Montazer, 2010; Choi et al., 2012; Saison et al., 2010; Chaloupka et al., 2010). NPs can also unintentionally form during e.g. combustion, production of electronics and during welding processes. (Peters et al., 2006; Zimmer et al., 2002). From the increased usage

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Abbreviations: AAS, atomic absorption spectroscopy; ALS, alkali-labile sites; CI, confidence interval; DCFH-DA, 20 ,70 -dichlorofluorescein-diacetate; DLS, dynamic light scattering; DMEM, Dulbecco’s Minimal Essential Medium; FBS, fetal bovine serum; FPG, Formamido Pyrimidine DNA Glycosylase; IC, inhibitory concentration; NP, nanoparticle; PBS, phosphate buffer solution; PCCS, photon cross correlation spectroscopy; PI, propidium iodide; PS, phosphatidylserine; ROS, reactive oxygen species; RPMI, Roswell Park Memorial Institute cell medium; SBs, strand breaks; TEM, transmission electron microscopy; TMRM, tetramethyl rhodamine methyl ester. ⇑ Corresponding author. E-mail address: [email protected] (L. Möller).

of NPs raises questions on their potential health and environmental effects. Different physicochemical properties of NPs, compared to larger sized particles and their bulk materials, often result in altered toxicological profiles. Even though the molecular understanding behind the toxicity of NPs is not fully understood, it has been shown that the particle size, shape, charge, chemical composition and solubility have impact on the toxicity (Lanone et al., 2009; Piret et al., 2012; Karlsson et al., 2009; Gliga et al., 2014). A larger surface area related to the particle size is a potential cause of higher reactivity and toxicity. The release of metals from the NPs is also of importance and several studies suggest that NP toxicity is induced by the particles as such, whereas other studies derive the toxicity to intra- or extracellular particle dissolution and release of metal ions (Studer et al., 2010; Hanagata et al., 2011). A Trojan-horse type mechanism is commonly used to describe the process where the cell takes up NPs that, for most metallic particles, dissolve to different extent in the acidic pH environment of the lysosomes (pH  4.5), and release metal ions within the cell (Studer et al., 2010; Cronholm et al., 2013). This pathway disables the capacity of the cell to protect itself against high levels of metals. The cell can normally regulate the metal uptake through metal sensing and transport proteins (Sinani et al., 2007). Toxic

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mechanisms of metal ions include the formation of hydroxyl radicals, via Fenton-reactions, non-specific binding to protein ligands and a competition with other essential metals resulting in dysfunctional proteins (Stohs and Bagchi, 1995; Asmuss et al., 2000). Oxidative stress and generation of reactive oxygen species (ROS), either directly at the particle surface, via release of redox active ions, or by secondary formation of inflammatory signals, are generally proposed as underlying toxic mechanisms in NP toxicity. Damage to the mitochondria is a source of ROS, and a close relation exists between the induction of mitochondrial damage caused by ROS and an excess production of ROS due to mitochondrial damage. Studies have shown translocations of NPs into mitochondria, as well as mitochondrial damage after exposing cells to NPs (Li et al., 2003; Karlsson et al., 2009). Copper-based NPs are of interest for applications within medical products, electronics and antimicrobial products. However, it has been shown that CuO NPs are more toxic compared with micron-sized CuO particles (Semisch et al., 2014; Sun et al., 2012, 2011). Previous studies have shown that CuO NPs are highly toxic and induce oxidative DNA damage and mitochondrial damage in human lung cells (Karlsson et al., 2009, 2008). Since it has been shown that NPs can translocate into the circulatory system and secondary organs upon inhalation (Oberdorster et al., 2005; Nemmar et al., 2002), the human cell line HL60 was chosen in the present study as an in vitro model for blood cells. Most toxicity studies on copper-based NPs have focused on their effects on epithelial cells, and the studies in HL60 cells are scarce. However, amorphous and crystalline CuS NPs were in one previous study shown to inhibit the proliferation and induce apoptosis in HL60 cells (Guo et al., 2010). The aim of the present work was to study the toxicity of Cu and CuO NPs in HL60 cells and to determine the dominant cellular toxic mechanisms. Toxic mechanisms (mitochondrial damage, DNA damage and ROS formation) and type of cell death induced by Cu and CuO NPs were investigated and compared with parallel findings for dissolved copper (from readily soluble CuCl2). The impact of particle characteristics and release of copper on the observed toxicity was specifically investigated.

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2. Materials and methods

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2.1. Cell culture

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The acute myeloid leukemia cell line HL60 (DSMZ, Braunschweig, Germany) was maintained in RPMI (GIBCOÒ Invitrogen) supplemented with 10% fetal bovine serum (FBS). The cells were grown in a humidified environment with 5% CO2 at 37 °C. Cells were one day prior to exposure seeded in 24-well plates at a concentration of 0.16 million cells/well.

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2.2. Particles

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The Cu NPs originated from Ionic Liquides Technologies (Heilbronn, Germany). CuO NPs and CuCl2 were purchased from Sigma–Aldrich (St. Louis, USA). According to the manufacturers the particle size of the Cu NPs was 50 nm, and the size and surface area of the CuO NPs were 42 nm and 23 m2/g, respectively.

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2.3. Transmission electron microscopy

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Size and shape of the Cu and CuO NPs were analyzed using transmission electron microscopy (TEM). The NPs were dispersed in Milli-Q water (0.1 mg/mL) and droplets were applied on TEM grids and dried for 5 min. Imaging was performed using a Mega View III digital camera from Soft Imaging System, GmbH (Munich,

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Germany) in a Tecnai 10 apparatus (Fei, The Netherlands) at an acceleration voltage of 100 kV.

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2.4. Particle size distribution

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Hydrodynamic size distributions of Cu and CuO NPs in complete cell medium (RPMI + 10% FBS) were assessed using dynamic light scattering (DLS) with photon cross correlation spectroscopy (PCCS, Nanophox, Sympatec GmbH, Clausthal-Zellerfeld, Germany). The measurements were performed at 37 °C, immediately after sonication and after 1 and 2 h. Duplicate samples were measured three times each and the data was integrated to produce a single distribution with the PCCS software (Windox 5). Standard latex samples (20 ± 2 nm, Sympatec GmbH) and blank samples were tested to ensure the accuracy of the measurements.

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2.5. Exposure conditions and cytotoxicity analysis

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Stock solutions of NPs (1 mg/mL) in complete cell medium were vortexed for 20 s and sonicated for 2  20 s. Further dilutions were made in preheated (37 °C) complete medium. The cytotoxicity was assessed using the trypan blue assay. Exposed cells were collected and incubated with trypan blue 0.4% (Sigma–Aldrich) for 3 min (1:1 v/v). The percentage of stained cells (i.e. non-viable cells) was counted using a Bürker chamber and a light microscope (minimum 100 cells per sample). Dose–response relationships for Cu, CuO NPs and dissolved copper (from readily soluble CuCl2) after 18 h exposure, were investigated. Concentrations for which the cell viability was inhibited by 25%, 50% and 75% (IC25, IC50 and IC75) were graphically determined by linear interpolation. Dissolved copper was expressed as equivalent Cu mass in CuCl2 per mL. The IC-values were further used for analysis of cell death, mitochondrial damage, DNA damage and intracellular ROS formation.

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2.6. Copper release into cell medium

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Atomic absorption spectroscopy (AAS) was used to determine the amount of released copper from Cu and CuO NPs into complete cell medium. Suspensions of Cu and CuO NPs were prepared in duplicate and kept for 0, 2, 4, 6 and 24 h at the same conditions as for the cell experiments. The samples were centrifuged 15 min at 20,800 rcf, and the supernatants were collected and acidified with HNO3 (65%) to pH < 2. Successful separation of supernatant and particles was verified by PCCS. The released amount of copper was analyzed by flame AAS (Perkin Elmer, Analyst 800). Calibration standards at 1, 3 and 10 lg/mL copper were prepared from a 1 mg/mL standard from Perkin Elmer. The detection limit was 6 ng/mL. Blank samples and quality control samples of known concentrations were regularly analyzed for quality control.

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2.7. Cell death analysis

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The FITC Annexin V Apoptosis Detection kit 1 (BD Biosciences, San Jose CA, USA) was used to analyze the type of cell death. Exposed cells were washed in PBS and re-suspended in Annexin V Binding buffer. FITC-Annexin V and PI were added, followed by 15 min incubation. Binding buffer was added prior analysis with FACS-Calibur flow cytometer (Becton Dickinson, Franklin Lakes, USA) on the FL-1 and FL-2 channels. Three cell populations were detected; viable cells (Annexin V negative/PI negative), early apoptotic cells (Annexin V positive/PI negative) and necrotic/late apoptotic cells (Annexin V positive/PI positive). Cisplatin (Sigma– Aldrich) was used as positive control.

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2.8. Mitochondrial damage

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The fluorescent probe tetramethyl rhodamine methyl ester (TMRM, Molecular probesÒ/Invitrogen Ltd) was used to evaluate loss of mitochondrial membrane potential. Cells were incubated with Cu NPs, CuO NPs and dissolved copper (CuCl2) for 2–24 h. At the end of the exposure was TMRM, diluted in DMSO, added at a final concentration of 25 nM for 30 min (final concentration of DMSO was 0.5%). The cells were centrifuged and washed in PBS prior to analysis with FACS-Calibur flow cytometer. The percentage of cells (10,000 cells/sample) with depolarized mitochondrial membranes was assessed using Cell Quest Software (Becton Dickinson).

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2.9. DNA damage and oxidatively damaged DNA

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The comet assay (single cell gel electrophoresis) was used to investigate DNA damage in terms of strand breaks (SBs) and alkali-labile sites (ALS). To assess oxidatively damaged DNA, an additional step of Formamido Pyrimidine DNA Glycosylase (FPG) treatment was performed. Cells were exposed to Cu NPs, CuO NPs and dissolved copper (CuCl2) for 2 h. The comet assay was then performed as previously described (Karlsson et al., 2008). Staining with ethidium bromide and examination using a fluorescence microscope (Olympus BH-2, 20x apochromatic objective, Komet 4.0 software from Kinetic Imaging Ltd) enabled visualization of comets and computerized scoring of the % DNA in tail, to estimate the level of DNA damage. 100 cells were scored per sample. Cells exposed to H2O2 (2 mM, 5 min) or to the photosensitizer Ro 19-8022 (0.4 lM, 5 min irradiation with a 500-W tungsten halogen lamp) were used as positive controls.

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2.10. Acellular and intracellular ROS formation 0

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The 2 ,7 -dichlorofluorescein-diacetate (DCFH-DA) dye was used to assess acellular and intracellular ROS formation. Upon oxidation of DCFH, the fluorescent compound DCF is formed, which can be detected by fluorescence measurements. In the acellular approach, 0.01 M NaOH was added to deacetylate DCFH-DA. The reaction was ended after 30 min by the addition of PBS. Cu NPs, CuO NPs and dissolved copper (CuCl2) were added to final concentrations of 10, 80 and 175 lg/mL. The fluorescence was measured using a microplate reader (Spectra Max Gemini, Molecular Devices, USA) at the excitation wavelength of 485 nm and the emission wavelength of 530 nm. Measurements were performed 5 min after particle addition, repeated every 10 min up to 65 min, and then after 2, 3, 4, 5 and 6 h. In the cellular approach, HL60 cells were exposed to the NPs and the CuCl2, and subsequently centrifuged and washed in PBS. After 30 min incubation with 100 lM DCFH-DA and diluted in Hanks balanced salt solution, the cells were washed and transferred to a 96-well microplate for fluorescence measurements. Micron-sized MnO particles (10 lm, Sigma–Aldrich) were used as positive control.

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2.11. Statistical analysis

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All experiments were performed at least three independent times unless else wise stated, and the generated data is presented as mean values and standard deviations. The data was analyzed using unpaired student’s t-test and ANOVA followed by Tukey’s HSD test for data sets with equal variances, or Games–Howell for unequal variances. Two-way ANOVA was conducted to examine time and concentration dependency for the mitochondrial damage in Fig. 6. Normal distribution of data was examined with Shapiro– Wilks test, and equality in variances was examined with both

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Levene’s test and Hartley’s test. If necessary, data sets were transformed prior the statistical analysis to improve the normal distribution and equality in variances. The SPSS software version 20.0 (IBM Corporation, NY, USA) was used to perform the statistical tests. GraphPad Prism 6 (GraphPad Software, Inc, CA, USA) was used to calculate IC50-values and 95% confidence intervals using non-linear interpolations.

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3. Results

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3.1. Primary particle size

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Estimates of the primary particle size and shape of the Cu and CuO NPs were made using TEM (Fig. 1). Both NPs revealed relatively spherical particles, typically sized from 20 to 50 nm, and from 15 to 100 nm in diameter for Cu and CuO, respectively. Particle agglomeration in the solvent (in this case MilliQ water) restrained in both cases the possibility to accurately determine the size of individual particles.

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3.2. Particle size distribution in solution

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Density distributions (volume) of Cu and CuO NPs were measured immediately after sonication (0 h), and after 1 and 2 h using PCCS (Fig. 2A and C). Agglomeration occurred in both cases upon dispersion. Only the size distribution exceeding 20 nm was considered in the evaluation to minimize the risk to overestimate the amount of small particles due to rotational diffusion that can take place for non-spherical particles and give rise to peaks at particle sizes <10 nm (Khlebtsov and Khlebtsov, 2011). The possible presence of smaller particles can though not be excluded. The Cu NPs formed larger agglomerates with time shifting from an initial average volume size of 350–600 nm after 1 h to several mm after 2 h. The size distribution also became wider with time. CuO NPs revealed a monodisperse, and in time stable, size distribution with an average volume size of approximately 300 nm. An important factor when describing the stability of particles in solution is the scattered light intensity, which increases non-linearly with increasing particle size. The intensity of the scattered light of the CuO NP suspension (Fig. 2D) did not change significantly over time, whereas a considerable reduction was evident for the Cu NPs (Fig. 2B). This reduction was interpreted as an effect of particle dissolution (see Section 3.4) and of sedimentation, following the formation of large agglomerates with time.

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3.3. Cytotoxicity and dose–response relationships in HL60 cells

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Dose–response relationships were investigated for Cu NPs, CuO NPs and dissolved copper (CuCl2) in HL60 cells after 18 h exposure (Fig. 3). In the figure, the x-axis refers to the actual copper concentration in the CuO NPs and CuCl2. The cytotoxicity of Cu NPs increased already at low concentrations and showed a dose-dependent manner (p = 0.003). The CuO NPs and dissolved copper (from CuCl2) also induced cytotoxicity in a dosedependent manner (p < 0.001 for both). The particle concentrations, and copper concentration in CuCl2, that decreased the viability by 25%, 50% and 75% (IC25, IC50 and IC75) after 18 h exposure, were graphically calculated by linear interpolation and are presented in Table 1. These concentrations were further used for analysis of cell death, mitochondrial damage, DNA damage and intracellular ROS formation. In addition, IC50 and 95% confidence intervals (CI) were calculated using non-linear interpolations to 15 lg/mL (95% CI 14–17; R2 0.89) for Cu NPs, 85 lg/mL (95% CI 78–93; R2 0.98) for CuO NPs and 45 lg/mL (95% CI 42–47; R2 0.99) copper concentration for CuCl2.

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Fig. 1. Particle size and shape of Cu and CuO NPs determined by TEM (transmission electron microscopy) image analysis after particle suspension and sonication in Milli-Q water (0.1 mg/mL). The particle sizes varied between 20 and 50 nm for the Cu NPs and between 15 and 100 nm for the CuO NPs.

Fig. 2. Particle size distribution measurements in solution measured by PCCS (photon cross correlation spectroscopy). Results are based on (A and C) the volume densities of Cu and CuO NPs (80 lg/mL) after 0, 1 and 2 h in complete cell medium (RPMI), and (B and D) corresponding intensities of the scattered light for each time point. The results are presented as mean values and deviations based on duplicate samples, each measured three times.

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3.4. Release of copper in cell medium

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Time-dependent (0, 2, 4, 6 and 24 h) released amounts of copper from Cu and CuO NPs into complete cell medium were analyzed by AAS. Fig. 4A and B shows the percentage of released copper of the total amount of copper added in the Cu and CuO NPs, respectively. Release of copper was rapidly taking place (10–20% of particle mass) for the Cu NPs directly after sonication and reached relatively constant and similar levels for the different concentrations already after 2 h (around 60%), a level remaining also after 24 h. The release of copper from the CuO NPs was initially slower compared with Cu NPs, with released fractions increasing from approximately 5% after sonication to 28% after 2 h, and 68% after 24 h. The percentage of released copper after 24 h was slightly higher for CuO NPs (68%) compared with Cu

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NPs (60%). The released copper fraction was relatively independent on particle concentration for both NPs. The total amounts of released copper from the IC-values and from particle concentration 80 lg/mL are shown in Table 2. The released amounts from CuO NPs were considerably higher compared with from Cu NPs due to significantly higher loadings. Notable is that the levels of released amounts from the IC-values of CuO NPs are similar to the IC-values for CuCl2 in Table 1.

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3.5. Cell death analysis

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Cell death analysis using Annexin V–PI staining showed a small reduction in cell viability after 18 h exposure to Cu NPs (Fig. 5). There was a small but significant increase of necrotic/late apoptotic cells after exposure to 15 lg/mL for 18 h (p = 0.002). Exposure to

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Fig. 3. Cytotoxic effects in HL60 cells, measured by the trypan blue exclusion assay after 18 h exposure to Cu NPs, CuO NPs and dissolved copper (from readily soluble CuCl2). The x-axis refers to the actual copper concentration in CuO NPs and CuCl2. The results are presented as mean values and standard deviations of three independent experiments.

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CuO NPs resulted in a significantly reduced cell viability with most cells being necrotic/late stage apoptotic cells. There was a small but still significant increase of early apoptotic cells after exposure to both 60 and 90 lg/mL CuO NPs (p = 0.004 and p = 0.01). The cell viability significantly decreased after exposure to 32 and 58 lg/mL (p = 0.027 and p = 0.028) dissolved copper (CuCl2), predominantly as necrotic cell death. The percentage of early apoptotic cells after exposure to 45 and 58 lg/mL dissolved copper (CuCl2) was low but significantly higher compared with the controls (p = 0.011 and p = 0.037). Noteworthy, the estimated viability using Annexin V–PI staining was similar to the viability measured by the trypan blue assay for the CuO NPs. In contrast, the assessed viability after exposure to Cu NPs and CuCl2 was higher as measured by the Annexin V–PI assay compared to the trypan blue assay.

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3.6. Mitochondrial damage

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Two-way ANOVA was conducted to investigate the effect of particle concentration and exposure time on the mitochondrial damage (Fig. 6). For Cu NPs there was a significant effect of both concentration (p < 0.001) and time (p = 0.016), and a significant interaction between concentration and time (p = 0.004). The level of mitochondrial damage increased significantly already after 2 h exposure for the highest Cu NP concentration (p = 0.009, IC75). A relatively high mitochondrial damage was observed for all three concentrations after 24 h. Significant effects of both concentration and time (p < 0.001) were observed for both CuO NPs and dissolved copper (CuCl2), as well as a significant interaction between particle concentration and time (p < 0.001). No increase in mitochondrial damage was observed up to 6 h for either CuO NPs or dissolved copper (CuCl2). All concentrations revealed mitochondrial damage (p < 0.001) after 24 h.

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Table 1 Concentrations (lg/mL) where the cell viability was inhibited by 25%, 50% and 75% in HL60 cells after 18 h exposure, estimated graphically by linear interpolation. Measured by trypan blue exclusion assay. For dissolved copper (CuCl2), the concentration is expressed as equivalent Cu mass.

Cu NP CuO NP CuCl2

IC25 (lg/mL)

IC50 (lg/mL)

IC75 (lg/mL)

12 60 32

15 90 45

17 120 58

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3.7. DNA damage and oxidatively damaged DNA

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A tendency for increased DNA damage was evident after 2 h exposure, but none of the exposed concentrations showed any significant difference compared with the control, neither in terms of SBs and ALS (Supplementary Fig. S1A), nor in oxidatively damaged DNA (Supplementary Fig. S1B). Cell viability examined after 2 h exposure did not fall below 90%. The positive control H2O2 induced 38% SBs and ALS, and Ro exposure induced 40% oxidatively damaged DNA. Representative images of the comets are given in Supplementary Fig. S1C.

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3.8. Acellular and intracellular ROS formation

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The acellular assay showed that the Cu NPs induced oxidation of DCFH at high levels in a dose- and time-dependent manner (Fig. 7), whereas the oxidative potential of the CuO NPs did not differ significantly from the control up to 65 min followed by only a slight increase. Dissolved copper (CuCl2) followed the same trend with no significant differences from the control up to 65 min, followed by increased levels of DCFH oxidation in a dose and time-dependent manner, however to a lower extent than Cu NPs. The intracellular ROS formation after 2 h exposure is shown in Supplementary Fig. S2. All exposures, especially the Cu NP exposure, resulted in slightly increased levels of intracellular ROS. However, it was only the IC75 of Cu NPs (17 lg/mL) that showed a statistically significant increase compared with the control (p = 0.01). Similar or even lower levels of intracellular ROS were observed after 4 and 24 h exposure (data not shown). Lower levels for exposures exceeding 2 h are likely due to reduced cell viability.

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4. Discussion

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The aim of the present work was to study the toxicity of Cu and CuO NPs, and to determine their cellular toxic mechanisms in the leukemic cell line HL60. Particle characteristics and the release of copper from the NPs were specifically investigated, as it is known that physicochemical properties of NPs influence their interactions with biological systems. In the present study, the particle size, extent of agglomeration and degree of copper release from Cu and CuO NPs in cell medium were investigated. The particle size and extent of agglomeration were determined using both TEM and PCCS measurements. Observed size distributions in solution were different from the TEM findings, primarily due to that TEM visualizes the particle size at high vacuum conditions after being dispersed in MilliQ water, and PCCS measures its changes in hydrodynamic size in cell medium. The solvent for TEM grid preparations has previously been shown to influence the ex situ size estimation (Skoglund et al., 2013). PCCS findings showed that particle agglomeration as well as particle dissolution and sedimentation took place for the Cu NPs with time. For the CuO NPs, no significant changes in the size of the rapidly formed agglomerates were observed. However, this does not necessarily mean that the particles remained unchanged in the solution. The Cu NPs revealed a rapid release of copper and reached relatively constant levels already after 2 h, while the release of copper from the CuO NPs was slower. The observed toxicity induced by Cu and CuO NPs in HL60 cells was compared with parallel studies using readily soluble CuCl2, assuming its complete dissolution. Unpublished findings by the authors do reveal that complexation between dissolved copper ions and cell medium components will rapidly take place and change the speciation of copper in solution, hence reducing the free ion concentration with time. These effects are not considered

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Fig. 4. Release of copper into complete cell medium (RPMI + 10% FBS) from (A) Cu NPs and (B) CuO NPs, measured by AAS. The results are expressed as the percentage released copper in solution per amount of total copper mass in added Cu and CuO NPs, respectively. The results are presented as mean values and deviations based on duplicate samples for each time period, each measured three times.

Table 2 Released concentrations (lg/mL) of copper from Cu and CuO NPs after 24 h at corresponding IC-values and 80 lg/mL. Data are presented as mean values and standard deviations of two independent experiments.

Cu NP CuO NP

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IC25 (lg/mL)

IC50 (lg/mL)

IC75 (lg/mL)

80 (lg/mL)

8 ± 0.4 32 ± 1.4

9 ± 0.2 47 ± 0.01

10 ± 0.5 61 ± 2.3

49 ± 1.0 43 ± 0.18

in this study as also complexation will occur for released copper from NPs of both Cu and CuO. The dose–response relationships in HL60 cells showed that the Cu NPs (IC50 = 15 lg/mL) were most toxic, followed by dissolved copper (IC50 = 45 lg/mL) and the CuO NPs (IC50 = 90 lg/mL). The high toxicity observed for copper-based NPs is in agreement with previous in vitro studies reporting cytotoxic effects in other cell lines (Karlsson et al., 2008; Semisch et al., 2014; Kasemets et al., 2009). However, the lower toxicity of CuO NPs compared with dissolved copper toward HL60 cells differ from previous findings in A549 cells where CuO NPs were far more toxic compared with dissolved coppers (Karlsson et al., 2008; Moschini et al., 2013). A few other studies have reported viability inhibition concentrations of Cu and CuO NPs. Lanone et al. reported TC50 values (toxic concentrations inducing 50% cell mortality) ranging from 5.3 to 6.6 lg/mL for Cu NPs (90 nm in diameter) and from 3 to 7.3 lg/mL for CuO NPs (30 nm) in THP-1 cells after 24 h exposure measured by MTT (Lanone et al., 2009). Wang et al. reported an IC50-value of 15 lg/mL CuO NPs (20–40 nm) for A549 cells exposed for 24 h, evaluated by means of the Cell Counting Kit-8 as cell viability assay (Wang et al., 2012). By comparing these data with findings of this study it can be concluded that HL60 cells are less sensitive to copper-based NPs, especially with regard to the CuO NPs. Toxicity of NPs has also previously been shown to vary dependent on the studied cell line (Kroll et al., 2011). Varying mechanisms for cellular uptake and intracellular fate of particles may explain observed differences in toxicity (Zhao et al., 2011). In addition, differences in experimental set-ups have impact on the toxic response. The presence of serum in the culture medium highly influences the toxicity of NPs, likely by the formation of a corona of proteins at the particle surface with effects on the released amount of copper (Midander et al., 2009; Cronholm et al., 2011). The presence of serum (and other solution species) and prevailing particle and surface characteristics, influenced by the particle dispersion methodology, govern the actual dose of particles and reactions at the surface, in solution and with the cell membrane. The delivered dose, which is dependent on the material density, has been shown to largely vary between different nanomaterials for given particle concentrations (Cohen et al., 2014; DeLoid et al.,

Fig. 5. Cell death analysis using Annexin V–PI staining and flow cytometry in HL60 cells after 18 h exposure to (A) Cu NPs, (B) CuO NPs and (C) dissolved copper (from CuCl2) at particle concentrations corresponding to the IC25, IC50 and IC75-values. The results are presented as mean values and standard deviations based on three independent experiments. Significant differences compared to the control; ⁄, ⁄⁄ and ⁄⁄⁄ represent; p < 0.05, p < 0.01 and p < 0.001, respectively.

2014). Direct comparison between literature findings is hence not straightforward. Previous findings have shown that NP dissolution is of large importance for their concomitant toxicity (Wang et al., 2012; Piret et al., 2012; Semisch et al., 2014). Cu and CuO NPs revealed

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Fig. 6. Mitochondrial depolarization in HL60 cells after 2, 4, 6 and 24 h exposure to (A) Cu NPs, (B) CuO NPs and (C) dissolved copper (from CuCl2) at particle concentrations corresponding to the IC25, IC50 and IC75-values obtained after 18 h exposure. The results are expressed as the mean values of the percentage of cells with depolarized mitochondria and standard deviations of three independent experiments. ⁄, ⁄⁄ and ⁄⁄⁄ represent significant differences to control with p < 0.05, p < 0.01 and p < 0.001, respectively.

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different dissolution profiles with time, factors that may explain the large difference in their toxicity. Cu NPs consist of a metallic copper core covered by a thin, naturally formed surface oxide of Cu2O and CuO that acts as a physical barrier, whereas CuO is a bulk chemical compound without any surface oxide barrier (Midander et al., 2009; Karlsson et al., 2013; Shi et al., 2012). The dissolution process is hence governed by different mechanisms, chemical and electrochemically induced for Cu NPs and chemical for CuO NPs. Karlsson et al. have shown that Cu NPs caused significantly higher damage to the cell membrane (measured by trypan blue staining) compared with CuO NPs (Karlsson et al., 2013). It was suggested that differences in the chemical reactions taking place at the particle surface, in solution and in contact with the cell surface, including ROS formation could call for the diversity in toxic response. As

Fig. 7. Acellular ROS formation after addition of (A) Cu NPs, (B) CuO NPs and (C) dissolved copper (from CuCl2) to DCFH-solution. The results are presented as mean values and standard deviations of the fluorescent units measured after three independent experiments. Data was monitored every 10 min up to 65 min, and after 2, 3, 4, 5 and 6 h.

explained earlier by Vanwinkle et al. oxidation of Cu NPs can increase the level of H2O2 and oxidative stress, particularly on the cell surface due to higher levels of O2 in the cell membrane (Vanwinkle et al., 2009; Shi et al., 2012). Li et al. observed toxicity of Cu NPs and discussed the influence of released copper ions and their different valence states on the generation of ROS. Cu(I) ions were shown to be rapidly released from the Cu NPs and a

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Cu(I)-mediated generation of H2O2 was identified as a major contributor to the observed toxicity (Li et al., 2013). Cu NPs were in the present study able to oxidize DCFH to a considerable higher extent when compared with CuO NPs in the acellular setting. The difference is likely due to electrochemical dissolution and release of both Cu(I) and Cu(II) from the Cu NPs that lead to the generation of H2O2 and further redox reactions. The oxidative effect of CuO NPs was minor and increased only after 2 h of exposure. Similar findings were observed for dissolved copper (CuCl2) with no oxidation during the first 2 h, but significant DCFH oxidation for the following exposure period up to 6 h, indicative of oxidative processes in the solution. The lack of effects induced by dissolved copper during short time periods (<2 h) are in agreement with literature findings that generally report short term (15–60 min) ROS measurements (Shi et al., 2012). However, significant oxidation of DCFH was evident after prolonged exposure, an effect that may be associated with auto-oxidation of the dye or Fenton-reactions induced by specific complexes formed with time between copper ions and components of the cell medium. High levels of oxidative processes in the presence of Cu NPs could explain the higher cytotoxicity in the cells and further add evidence to the general theory of oxidative stress as a major mechanism behind toxicity of copper-based NPs. Indeed, oxidative DNA damage and intracellular ROS showed a tendency for higher levels of intracellular oxidation, especially for the Cu NPs. However, it was only the highest concentration of the Cu NPs that induced significantly higher intracellular ROS compared to the control after 2 h exposure. Longer exposures resulted in reduced levels of intracellular ROS, probably due to reduced cell viability. There was also a tendency for increased DNA damage and oxidative DNA damage, as measured by the comet assay, however, these changes were non-significant. It is possible that different antioxidative mechanisms within the cell can counteract the oxidative assault to some extent, or that longer exposure times with lower NP concentrations that do not reduce the cell viability, is required to observe any significant effects. Several studies have reported reduced mitochondrial membrane potential after exposure to NPs (Wang et al., 2012; Karlsson et al., 2009). In the present study mitochondrial depolarization was studied from 2 up to 24 h of exposure. Notable is that the Cu NPs induce mitochondrial damage in a pattern different from CuO NPs and dissolved copper (CuCl2). The Cu NPs induced damage in an early stage of exposure (2 h), whereas the CuO NPs and dissolved copper (CuCl2) did not show any mitochondrial toxicity up to 6 h of exposure. The results differ from the study by Wang et al. who reported mitochondrial depolarization already after 2 h in A549 cells (15 and 50 mg/L). Moreover, small, but non-significant increase of DNA damage levels was observed in the present study. Other studies report genotoxic effects of CuO NPs (Wang et al., 2012; Karlsson et al., 2008). However, different cell types and times of exposure were investigated, variables that may have impact on the levels of DNA damage. Several methodological issues need to be considered when investigating toxicity of NPs. One issue is the presence of serum in the cell culture medium. Proteins (and possibly other molecules) in the serum will adsorb to the particle surface and affect the particle size of the NPs, the extent of agglomeration and release/dissolution of copper into solution. In the present study, serum was added in the same amount for all conditions from which follows that the protein corona and solution effects would take place for all exposure conditions. However, its influence on ROS-generation, possibly taking place at the surface of the Cu NPs may be different. Recent studies have further shown interference of NPs with commonly used viability assays based on spectrophotometric analysis (Holder et al., 2012; Kroll et al., 2012). Cytotoxicity was in this study assessed using trypan blue staining

to diminish potential interferences as this assay is based on the ability of viable cells to exclude the trypan blue dye. Cells are counted manually using a light optical microscope to eliminate such interferences. However, there was a notable discrepancy when comparing the cell viability assessed by the trypan blue assay and the Annexin V–PI assay. For CuO NPs, the results from the two methods correlated well. However, for both dissolved copper (CuCl2) and the Cu NPs, the assessed viabilities were higher when using the Annexin V–PI assay compared with using the trypan blue assay. Overestimation of the cell viability with trypan blue is more probable since the trypan blue molecule is larger and more easily excluded by the cell than the PI molecule. The higher degree of agglomeration and sedimentation rate of the Cu NPs could possible hinder the Annexin V/PI binding.

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5. Conclusions

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The present study showed a high cytotoxicity of Cu and CuO NPs in the HL60 cells. The Cu NPs revealed the highest toxicity, at a significantly lower copper loading compared to the CuO NPs. The metal release analysis showed that copper release from Cu NPs was more rapid compared with the release from the CuO NPs. The observed toxicity was also higher for Cu NPs compared with dissolved copper (CuCl2), which implies both a particle effect and an effect induced by released/dissolved copper. The Cu NPs generated high levels of ROS in an acellular setting, possibly explaining the high cytotoxic effects. Mitochondrial damage was observed already after 2 h. Intracellular ROS formation was increased and a tendency for increased levels of DNA damage and oxidative DNA damage was observed after 2 h of exposure to Cu NPs. CuO NPs were less toxic toward the HL60 cells compared with both the Cu NPs and dissolved copper (CuCl2). The released amounts of copper from the CuO NPs at the IC-values were similar to the IC-values of dissolved copper (CuCl2). The toxic mechanisms of CuO NPs and dissolved copper were similar to each other with regard to the mitochondrial damage, which was observed after a longer exposure time for both CuO NPs and dissolved copper (CuCl2) compared with Cu NPs. Neither DNA damage nor oxidative DNA damage was observed after 2 h exposure. Necrosis was the main type of cell death observed for the CuO NPs and dissolved copper (CuCl2). Findings of this study indicate a lower toxicity of CuO NPs in HL60 cells, when compared with literature findings for other cell lines.

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Conflict of interest statement

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The authors declare no conflicts of interest.

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Acknowledgments

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The authors thank Environmental Cancer Risk, Nutrition and Individual Susceptibility (ECNIS), a Network of Excellence operating in the context of the 6th EU Framework Programme for Research and Development (FP6), for financial support. The photosensitiser Ro 19-8022 was a kind gift from F. Hoffmann-La Roche, Basel, Switzerland. The authors at KTH are grateful for financial support from the Swedish Research Council (VR).

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Appendix A. Supplementary material

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tiv.2015.05.020.

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