Dose-dependent genotoxicity of copper oxide nanoparticles stimulated by reactive oxygen species in human lung epithelial cells.

Toxicologyhttp://tih.sagepub.com/ and Industrial Health

Dose-dependent genotoxicity of copper oxide nanoparticles stimulated by reactive oxygen species in human lung epithelial cells Mohammad Javed Akhtar, Sudhir Kumar, Hisham A Alhadlaq, Salman A Alrokayan, Khalid M Abu-Salah and Maqusood Ahamed Toxicol Ind Health published online 5 December 2013 DOI: 10.1177/0748233713511512 The online version of this article can be found at: http://tih.sagepub.com/content/early/2013/12/03/0748233713511512

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Article

Dose-dependent genotoxicity of copper oxide nanoparticles stimulated by reactive oxygen species in human lung epithelial cells

Toxicology and Industrial Health 1–13 © The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0748233713511512 tih.sagepub.com

Mohammad Javed Akhtar1, Sudhir Kumar2, Hisham A Alhadlaq1,3, Salman A Alrokayan4, Khalid M Abu-Salah1 and Maqusood Ahamed1 Abstract Copper oxide nanoparticles (CuO NPs) are of great interest in nanoscience and nanotechnology because of their broad industrial and commercial applications. Therefore, toxicity of CuO NPs needs to be thoroughly understood. The aim of this study was to investigate the cytotoxicity, genotoxicity, and oxidative stress induced by CuO NPs in human lung epithelial (A549) cells. CuO NPs were synthesized by solvothermal method and the size of NPs measured under transmission electron microscopy (TEM) was found to be around 23 nm. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) and lactate dehydrogenase (LDH) assays showed that CuO NPs (5–15 mg/ml) exert cytotoxicity in A549 cells in a dose-dependent manner. Comet assay suggested concentration-dependent induction of DNA damage due to the exposure to CuO NPs. The comet tail moment was 27% at 15 mg/ml of CuO NPs, whereas it was 5% in control (p < 0.05). The flow cytometry data revealed that CuO NPs induced micronuclei (MN) in A549 cells dose dependently. The frequency of MN was 25/103 cells at 15 mg/ml of CuO NPs, whereas it was 2/103 cells for control. CuO NPs were also found to induce oxidative stress in a concentration-dependent manner, which was indicated by induction of reactive oxygen species (ROS) and lipid peroxidation along with glutathione depletion. Moreover, MN induction and DNA damage were significantly correlated with ROS (R2 ¼ 0.937 for ROS vs. olive tail moment, and R2 ¼ 0.944 for ROS vs. MN). Taken together, this study suggested that CuO NPs induce genotoxicity in A549 cells, which is likely to be mediated through ROS generation and oxidative stress. Keywords Nanotoxicity, nanoparticle, oxidative stress, apoptosis, DNA damage

Introduction At present, metal oxide nanoparticles (NPs) are used in manufacturing of hundreds of commercial products, and their industrial applications are expected to expand during the next decade. Metal oxide NPs are of great interest in nanotechnology, in part because the family of metal oxides offers intriguing new properties for applications in future technologies. Copper oxide (CuO) NPs are being used in various applications such as antimicrobial preparations, heat transfer fluids, semiconductors, and intrauterine contraceptive devices (Aruoja et al., 2009; Chang et al., 2005; Zhou et al., 2006). Furthermore, CuO NPs, for example, can be used to dope other materials for photocatalysis (Song et al., 2008), can be added to fluids

to create novel thermal properties as the so-called nanofluid (Karthikeyan et al., 2008; Khandekar et al., 2008),

1

King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, Saudi Arabia 2 Department of Zoology, University of Lucknow, Lucknow, India 3 Department of Physics and Astronomy, College of Science, King Saud University, Riyadh, Saudi Arabia 4 Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia Corresponding author: Maqusood Ahamed, King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia. Email: [email protected]


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Toxicology and Industrial Health

can aid in the synthesis of fullerenes (Liu et al., 2008), and can be used in energetic materials such as explosives and propellants (Shende et al., 2008). The suitability of NPs in biological and, specifically, biomedical applications must be supported by rigorous studies of their potential toxicity (Singh and Nalwa, 2007; Ye and Mahato, 2008). There is also potential for multiple adverse interactions such as oxidative stress and inflammatory responses (Gwinn and Vallyathan, 2006). Such cellular processes may lead to cell death via cell necrosis or apoptosis. Thus, in the development of suitable NPs for use as drug or biomolecules delivery platforms, induction of potential NPs in adverse cellular reactions must be considered (Kagan et al., 2005). Exposure to NPs typically occurs in the epithelia of the lung, skin, or gastrointestinal tract. The present study uses A549 cells, in vitro model of human lung epithelia, to analyze cytotoxicity, genotoxicity, and oxidative stress-induced by CuO NPs exposed. We determined the cytotoxicity by (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) MTT and lactate dehydrogenase (LDH) leakage in response to different concentrations of CuO NPs in A549 cells. Genotoxicity induced by CuO NPs in A549 cells was determined by Comet assay, a technique that detects single- and double-strand DNA breaks in individual nuclei by measuring the migration of denatured DNA fragments through an agarose gel (McKelvey-Martin et al., 1993; Plewa et al., 2002). In addition to comet assay, the micronuclei (MN) induction was also determined by flow cytometry that has the capability of analyzing thousands of events rapidly in three dimensions, leading to a reduction in false negative errors, a powerful technique over conventional measurement, which is usually conducted microscopically and is limited to 2D (Dertinger et al., 2002; Smolewksi et al., 2001). Parameters of oxidative stress, in addition to cytogenotoxicity, have also been determined in the present study. Oxidative stress biomarkers including reduced glutathione (GSH) as a cellular antioxidant, reactive oxygen species (ROS) generation as a collective marker of hydrogen peroxide (H2O2), superoxide anion (O2), hydroxyl radical (HO•), and thiobarbituric acid reactive substances (TBARS) as an indicator of membrane lipid peroxidation (LPO) were determined.

Material and methods Chemicals and reagents Fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM), Hank’s balanced salt

solution (HBSS), penicillin–streptomycin, and trypsin were bought from Invitrogen Co. (California, USA). GSH, MTT, 5,5-dithio-bis-(2-nitrobenzoic acid), 2,7-dichlorofluorescin diacetate (DCFH-DA), pyruvic acid, propidium iodide (PI), O-phthalaldehyde (OPT), 1,1,3,3-tetraethoxypropane, thiobarbituric acid (TBA), sodium dodecyl sulfate (SDS), and copper chloride (CuCl2) were purchased from SigmaAldrich (Missouri, USA). All other chemicals used were of the highest purity available from commercial sources.

Synthesis of CuO NPs CuO NPs were synthesized by solvo-thermal method as given subsequently. Briefly, 3 mM of CuCl23H2O and 6 mM of 1, 10-phenanthroline were dissolved into the mixed distilled water and ethanol. Then 2 M sodium hydroxide (NaOH) aqueous solution was added under magnetic stirring. This alkaline solution was transferred into a Teflon-lined autoclave with about 80% capacity. The autoclave was then sealed and maintained at 160 C for 24 h. After cooling to room temperature, the black precipitates were filtered, washed with distilled water and absolute ethanol several times. The resulting product was then dried at 60 C for 6 h to get the dry nanopowder of CuO.

Characterization of CuO NPs Morphology of CuO NPs were evaluated by field emission transmission electron microscopy (FETEM, JEM-2100F, JEOL Inc., Japan) at an accelerating voltage of 200 kV. In brief, dry powder of CuO NPs was suspended in deionized water at a concentration of 1 mg/ml and then sonicated using a sonicator bath at room temperature for 15 min at 40 W to form a homogeneous suspension. For size measurement, sonicated 1 mg/ml CuO NP stock solution was then diluted to a 50–100 mg/ml working solutions. Then a drop of CuO NP suspension was placed onto a carbon-coated copper grid, air-dried, and observed with FETEM. Dynamic light scattering (DLS) used for the characterization of hydrodynamic size and zeta potential of CuO NPs in cell culture medium were performed on Malvern Instruments (Zetasizer Nano-ZS, UK) as described by Murdock et al. (2008). Briefly, dry powder of CuO NPs was suspended in cell culture medium at a concentration of 15 mg/ml for 24 h. Then suspension of CuO NPs was sonicated using a sonicator bath


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at room temperature for 15 min at 40 W and performed the measurements.

Cell culture and CuO NPs exposure Cells were used between passages 10–20. Cells were cultured in DMEM/F12 medium supplemented with 10% FBS and 100 U/ml penicillin–streptomycin at 5% CO2 and 37 C. At 85% confluence, cells were harvested using 0.25% trypsin and were subcultured into 25 cm2 flasks, 6-well plates, or 96-well plates according to the selection of experiments. Cells were allowed to attach to the surface for 24 h prior to treatment. CuO NPs were suspended in cell culture medium and diluted to appropriate concentrations (5, 10, and 15 mg/ml). The appropriate dilutions of CuO NPs were then sonicated using a sonicator bath at room temperature for 10 min at 40 W to avoid NP agglomeration prior to administration to the cells. Selection of dosage range (5–15 mg/ml) and exposure time of CuO NPs was based on a preliminary doseresponse study (data not shown).

When plasma membrane integrity is disrupted, LDH leaks into culture media and its extracellular level is elevated. LDH assay was carried out with the method described earlier (Welder et al., 1991; Wroblewski and LaDue, 1955). In brief, 10,000 cells per well were seeded in 96-well plate and exposed to CuO NPs at the concentrations of 5, 10, and 15 mg/ml for 24 h. At the end of exposure, 96-well plate was centrifuged at 2300g for 10 min to get the cell culture media. Then, a 100 ml of culture media transferred to new fresh tube containing 100 ml of sodium pyruvate (2.5 mg/ml phosphate buffer) and 100 ml of reduced nicotinamide adenine dinucleotide (NADH) (2.5 mg/ml phosphate buffer) in a total volume of 3.0 ml (0.1 M potassium phosphate buffer, pH 7.4). The rate of NADH oxidation was determined by following the decrease in absorbance at 340 nm for 3 min at 30 s interval using a spectrophotometer (ThermoSpectronic, New York, USA). The amount of LDH released is represented as LDH activity (IU/l) in culture media.

Cell viability assay

Comet assay

Viability of cells after exposure to CuO NPs was assessed by MTT assay as described by Mossman (1983) with some specific modifications (Akhtar et al., 2010). The MTT assay assesses the mitochondrial function by measuring ability of viable cells to reduce MTT into blue-formazon product. Briefly, 1000 cells/well were seeded in 96-well plates and exposed to CuO NPs at the concentrations of 5, 10, and 15 mg/ml for 24 h. At the end of exposure, medium was removed from each well to avoid interference of NPs and replaced with new medium containing MTT solution in an amount equal to 10% of culture volume and incubated for 3 h at 37 C until a purple colored formazan product developed. The resulting formazan product was dissolved in acidified isopropanol. Further, the 96-well plate was centrifuged at 2300g for 5 min so that the remaining NPs present in the solution settle. Then, a 100 ml supernatant was transferred to other fresh wells of 96-well plate and absorbance was measured at 570 nm by using a microplate reader (Synergy-HT, BioTek, Virginia, USA).

Comet assay was performed as described by Singh et al. (1988) with some specific modifications (Ali et al., 2010). In brief, 80,000 cells/well were seeded in a 12-well plate. After 24 h of seeding, cells were treated with different concentrations of CuO NPs (5–15 mg/ml) for 24 h. At the end of exposure, cells were trypsinized and resuspended in DMEM supplemented with 10% FBS and the cell suspension was centrifuged at 2300g for 5 min at 4 C. The cell pellet was finally suspended in ice-chilled phosphate buffer saline for comet assay. Then, 15 ml of cell suspension (approximately 20,000 cells) were mixed with 85 ml of low melting point agarose (0.5%) and layered on one end of a frosted plain glass slide, precoated with a layer of 200 ml normal agarose (1%). Thereafter, it was covered with a third layer of 100 ml low melting point agarose (0.5%). After solidification of the gel, the slides were immersed in a freshly prepared lysing solution (2.5 M sodium chloride (NaCl), 100 mM disodium ethylenediaminetatraacetate (Na2EDTA), and 10 mM Tris pH 10 with 10% dimethyl sulfoxide (DMSO) and 1% Triton X-100) overnight at 4 C. The slides were then placed in a horizontal gel electrophoresis unit. Fresh cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, and 0.2% DMSO, pH 13.5) was poured into the chamber and left for 20 min at 4 C

Lactate dehydrogenase leakage assay Lactate dehydrogenase (LDH) is an enzyme widely present in cytosol, which converts lactate to pyruvate.


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for DNA unwinding and conversion of alkali-labile sites to single-strand breaks. Electrophoresis was carried out using the same solution at 4 C for 20 min at 15 V. The slides were neutralized gently with 0.4 M tris buffer at pH 7.5 and stained with 75 ml ethidium bromide (20 mg/ml). The slides were stored at 4 C in a humidified slide box until scoring. Slides were scored at a final magnification of 400 using an image analysis Komet 5.5 (Kinetic Imaging Limited, Liverpool, UK) attached to a fluorescent microscope (LeicaOptiphase, Germany) equipped with appropriate filters. The undamaged cell resembles an intact nucleus without a tail and a damaged cell has the appearance of a comet. The comet parameter was used to measure the mean tail moment of the cells. Images from 200 random cells (50 cells from each of 4 replicate slides) were analyzed for each experiment.

Flow cytometric analysis of MN induction MN formation due to exposure to CuO NPs was examined by flow cytometer as described by Nusse et al. (1994). Briefly, cells were exposed to CuO NPs at the concentrations of 5, 10, and 15 mg/ml for 24 h. After exposure, the cells were washed with cold phosphate buffer saline. Further cell suspension was centrifuged for 5 min at 500g and supernatant was removed, the cell pellet was suspended in solution I (10 mM NaCl, 3.4 mM sodium citrate, 25 mg/ml PI, 0.01 mg RNase from bovine pancreas, and 0.3 ml/ml triton-X). After 1 h at room temperature, an equal volume of solution II (78.1 mM citric acid, 40 mg/ml PI, and 0.25 M sucrose) was added. After 15 min, the suspension was filtered through a 53-mm nylon mesh and stored on ice until analyzed on Flow cytometer (Becton-Dickinson LSR II, California, USA) using ‘Cell Quest’ 3.3 analysis software.

Intracellular ROS measurement The production of intracellular ROS was measured using DCFH-DA (Wang and Joseph, 1999) with some specific modifications (Akhtar et al., 2010). The DCFH-DA passively enters the cell where it reacts with ROS to form the highly fluorescent compound dichlorofluorescein (DCF). Briefly, 10 mM DCFHDA stock solution (in methanol) were diluted in culture medium without serum or other additive to yield a 100 mM working solution. After exposure to CuO NPs, the cells were washed twice with HBSS. Then the cells were incubated in 1 ml working solution of DCFH-DA at 37 C for 30 min. Cells were lysed in

alkaline solution and centrifuged at 2300g for 10 min to avoid interference of NPs and cell debris. A 200 ml supernatant were transferred to 96-well plate and fluorescence was measured at 485 nm excitation and 520 nm emission using a microplate reader (Synergy-HT, BioTek). The intensity of untreated control well was assumed to be 100% and data are represented in percentage of control.

LPO assay LPO was assessed by the TBARS assay, which detects mainly malondialdehyde (MDA), an end product of the peroxidation of polyunsaturated fatty acids and related esters. TBARS was measured by slight modification of the method of Ohkawa et al. (1979). Sub-confluent cells were scraped in 75 cm2 flasks, washed two times in isotonic trace element-free Tris-HCl buffer (400 mM, pH 7.3). A 200 ml aliquot of cell suspension were subsequently mixed with 800 ml of LPO assay cocktail containing 0.4% (w/v) TBA, 0.5% (w/v) SDS, 5% (v/v) acetic acid, pH 3.5, and incubated for 60 min at 95 C. The sample was cooled using tap water and centrifuged at 2300g for 5 min. The absorbance of the supernatants was read at 532 nm against a standard curve prepared using the MDA standard (10 mM 1,1,3,3tetramethoxypropane in 20 mM Tris-HCl, pH 7.4). Results were calculated as nmol of TBARS/mg protein.

Intracellular GSH assay Intracellular GSH was quantified using the method of Hissin and Hilf (1976). Briefly, cells were cultured in 75 cm2 culture flask and exposed to CuO NPs at the concentrations of 5, 10, and 15 mg/ml for 24 h. After the exposure, the cells were lysed in 20 mM Tris (pH 7.0) and the centrifuged at 2300g for 15 min at 4 C. Further, protein of the supernatant was precipitated using 1% perchloric acid and again centrifuged at 2300g for 10 min at 4 C to get the supernatant. Then 20 ml of supernatant were mixed with 160 ml of 0.1 M potassium phosphate-5 mM EDTA buffer (pH 8.3), and 20 ml OPT (1 mg/ml in methanol) in a black 96-well plate. After 2 h of incubation at room temperature in the dark, fluorescence was measured at the emission wavelength of 460 nm and excitation wavelength of 350 nm. The amount of GSH was expressed as nmol GSH/mg protein.


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Cell viability (% of control)

(a) 120 100 *

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Figure 1. Characterization of copper oxide nanoparticles. (a) Transmission electron microscopic image and (b) hydrodynamic size and zeta potential determined by dynamic light scattering.

LDH leakage (IU/L)

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Estimation of protein Protein concentration was measured by the Bradford method (Bradford, 1976) using Bradford reagent (Sigma-Aldrich, Missouri, USA) and bovine serum albumin as the standard.

Statistics Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test. Significance was ascribed at p < 0.05. All analyses were conducted using the Prism software package (GraphPad Software, Version 5.0, GraphPad Software Inc., California, USA).

Results Shape and size of CuO NPs Figure 1(a) shows the typical TEM image of the CuO NPs. This picture shows that the majority of the particles were in spherical shape with smooth surfaces. TEM average diameter was calculated by measuring over 100 particles in random fields of TEM view. The average TEM diameter of CuO NPs was approximately 23 nm. The average hydrodynamic diameter and zeta potential of the CuO NPs suspension in

5 µg/ml

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15 µg/ml

Figure 2. Copper oxide nanoparticles induced cytotoxicity in A549 cells. (a) MTT assay and (b) LDH assay. Data represented are mean + standard deviation of three identical experiments made in three replicates. *Statistically significant difference as compared to the controls (p < 0.05 for each). MTT: 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; LDH: lactate dehydrogenase.

culture medium determined by DLS were found to be 87 nm and 25 mV, respectively (Figure 1(b)). The higher size of CuO NPs in aqueous suspension than the TEM size might be due to the tendency of particles to aggregate in aqueous state. This finding is supported by other investigators (Bai et al., 2009) and has been briefly discussed in our previous publication (Ahamed et al., 2010).

Dose-dependent cytotoxicity of CuO NPs Cytotoxicity of CuO NPs against A549 cells was measured by MTT and LDH assays. MTT results show that cell viability was decreased by CuO NPs and the degree of reduction was dose dependent. Cell viability was decreased to 81, 56, and 31% for the concentrations of 5, 10, and 15 mg/ml, respectively (p < 0.05) (Figure 2(a)). Similarly, LDH leakage, an indicator


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Figure 3. Morphology of A549 cells exposed to different concentrations of copper oxide nanoparticles for 24 h. (a) control, (b) 5 mg/ml, (c) 10 mg/ml, and (d) 15 mg/ml.

of membrane damage was also induced by CuO NPs in A549 cells in a dose-dependent manner. LDH level in cytosol was found to be 1.5-, 2-, and 2.8-fold higher for the concentrations of 5, 10, and 15 mg/ml, respectively (p < 0.05) (Figure 2(b)).

Effect of CuO NPs on cell morphology Cell morphology was assessed following exposure to different concentrations of CuO NPs using a Leica phase contrast microscope. Figure 3 shows the comparative morphologies of control and CuO NPs exposed A549 cells. A significant lowering of cell density and rounding of cells were observed in dosedependent manner supporting the cell viability data.

Dose-dependent genotoxicity of CuO NPs One of the signs of genotoxicity is the induction of DNA damage that can be determined by comet

assay, a widely used method for the detection as well as measurement of DNA strand breaks (Anderson and Plewa, 1998; Singh et al., 1988). Comet assay was carried out in A549 cells against CuO NPs at the concentrations of 5, 10, and 15 mg/ml for 24 h. A dose-dependent increase in DNA damage was observed in CuO NP-treated cells evident by an increase in olive tail moment (arbitrary unit). However, no significant DNA damage was observed in the untreated control cells (Figure 4(a) and (b)). MN induction by flow cytometry further confirmed genotoxic potential of CuO NPs in A549 cells. A significant (p < 0.05) increase in the frequency of MN was observed in A549 cells with increasing concentration of CuO NPs. CuO NP exposure (5, 10, and 15 mg/ml for 24 h) led to induction of MN frequency in A549 cells up to 9, 14, and 25, respectively, whereas in untreated control the frequency was 4 (Figure 5(a) and (b)).


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Olive tail moment (arbitrary unit)

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Figure 4. Copper oxide nanoparticles induced DNA damage in A549 cells. (a) Bar diagram of percentage of tail DNA of treated and control cells. Data represented are mean + SD of three identical experiments made in three replicate. *Significant difference as compared to the controls (p < 0.05 for each). (b) Representative photographs of DNA damage (% tail DNA) of A549 cells treated with copper oxide nanoparticles at the concentration of 15 mg/ml for 24 h along with control cells.

Dose-dependent induction of oxidative stress by CuO NPs It has been suggested that ROS generation and oxidative stress mediate the genotoxicity of NPs (Ahamed et al., 2011; Akhtar et al., 2010, 2012; Nel et al., 2006). Therefore, the ability of CuO NPs to induce oxidative stress in A549 cells was assessed by measuring ROS, LPO, and GSH levels. As expected, we observed that CuO NPs have the potential to induce oxidant generation (ROS and LOP) and reduce antioxidant level (GSH) in A549 cells in a dosedependent manner (Figure 6(a), (b), and (c)). We further examined the role of ROS in cytotoxicity and genotoxicity of CuO NPs using correlation curves. Statistical analysis showed that significant negative correlations observed between ROS-olive tail moment (R2 ¼ 0.937), ROS-MN (R2 ¼ 0.944), and ROS-MTT (R2 ¼ 0.974) (Figure 7(a), (b), and (c)).

Discussion This study reported the genotoxic potential of CuO NPs determined by Comet assay and flow cytometry. Several studies have shown a positive correlation among oxidative stress, genotoxicity, and apoptosis (Calviello et al., 2006; Chen et al., 2005). Generation of oxidative stress may also cause DNA damage, which may lead to genotoxicity and cancer (Bump and Malakar, 1998). Results of the present study demonstrate that CuO NP exposure to A549 cells can induce cytotoxicity, genotoxicity, and oxidative stress in a dose-dependent manner. Our study suggests the involvement of oxidative stress in the mechanism of CuO NPs-induced genotoxicity. We found that CuO NPs significantly induced genotoxic effect in human lung cells. Currently, NP genotoxicity testing is based on in vitro methods established for hazard characterization of chemicals (Fubini et al., 2010; Landsiedel et al., 2010). Comet


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Micronuclei induction (MN/1000 cells)

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Figure 5. Flow cytometric analysis of copper oxide nanoparticles induced micronuclei induction in A549 cells. (a) Bar diagram of micronuclei formation in treated and control cells. Data represented are mean + standard deviation of three identical experiments made in three replicates. *Significant difference as compared to the controls (p < 0.05 for each). (b) Representative image of micronuclei induction in A549 cells treated with copper oxide nanoparticles at the concentration of 15 mg/ml for 24 h along with control cells. Gate R1 showing the population of micronuclei.

assay is one of the important and well-applied in vitro methods in genotoxicology and DNA damage studies. It is an in situ method in which embedded cell on agarose base is lysed and electrophoresed on neutral or alkaline conditions (Collins, 2004). The results obtained by the comet indicated that CuO NPs were able to cause a significant increase in tail moment in A549 cells when exposed for 24 h at the concentrations of 15 mg/ml. In addition to comet assay, the MN induction measurement is an established cytogenetic assay that can detect acentric fragments and lagging chromosomes induced by clastogens and aneugens (Mavournin et al., 1990). Manual measurement of MN induction is usually conducted microscopically and is limited to two dimensions and, hence, some MN are not counted due to a lack of visibility. The MN induction measurement by flow cytometry, however, is a powerful technique that has the capability of analyzing thousands of events rapidly in three dimensions leading to a reduction in false negatives errors. Progress

has been made to automate the scoring of MN by flow cytometry (Dertinger et al., 2002; Roman et al., 1998; Smolewksi et al., 2001). The results obtained by the MN assay indicated that CuO NPs were able to cause a significant increase in MN induction in A549 cells when exposed for 24 h at the concentrations of 15 mg/ml. Oxidative stress has been suggested to play an important role in the mechanism of toxicity of a number of compounds either by the production of ROS or by depleting cellular antioxidant capacity. Cellular integrity is affected by oxidative stress when the production of ROS overwhelms antioxidant defense mechanism (Halliwell and Gutteridge, 2007). ROS are oxygen-containing molecules, such as H2O2, O2, and HO•, that have a greater chemical activity than molecular oxygen. There are many evidences showing that NPs increase ROS production and can cause cell death in different types of cultured cells (Akhtar et al., 2010; Park et al., 2008; Peters et al., 2007; Wang et al., 2009). Recently, we have reported oxidative stress as a major mechanism of toxicity


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DCF-fluorescence (% of control)

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Figure 6. Copper oxide nanoparticles induced oxidative stress in A549 cells. (a) Reactive oxygen species level, (b) malondialdehyde level, and (c) glutathione level. Data represented are mean + standard deviation of three identical experiments made in three replicates. *Statistically significant difference as compared to the controls (p < 0.05 for each).

induced by CuO NPs in mouse fibroblast cells (Balb 3T3) (Akhtar et al., 2012), whereas Sun et al. (2012) have reported autophagy as a major mechanism of cell death in A549 cells. Adding to this conflict, however, Song et al (2012) have reported oxidative stress-induced MN induction and DNA damage as the major mechanisms of cell death by several metal NPs including NPs of CuO. This discrepancy prompted us to investigate CuO NPs-induced MN induction and DNA damage, within our limit, in A549 cells along with other parameters of oxidative stress. In the present study, CuO NPs induced significantly higher MN induction and DNA damage, which correlated well with ROS generation in a concentration-dependent manner in A549 cells. Certain other nanomaterials, such as C60, silica, and talc NPs mediate cytotoxicity primarily through LPO, another marker of oxidative stress (Ahmad et al., 2012; Isakovic et al., 2006; Lin et al., 2006; Sayes et al., 2005). Recently, Scarfı` et al. (2009) have reported that plasma membrane contact with quartz, a kind of crystalline silica, is sufficient to trigger membrane LPO, tumor necrosis factor (TNF)-a release, and cell death in mouse macrophages (RAW-264.7). Our present data also

suggest that contact of CuO NPs with cell membranes of A549 cells initiate in the generation of ROS and, in turn, LPO in a concentration-dependent manner. Antioxidant GSH is the most abundant nonproteinous tripeptide containing a sulfhydryl group in virtually all cells and plays a significant role in many biological processes. It also constitutes the first line of the cellular defense mechanism against oxidative injury and is the major intracellular redox buffer in ubiquitous cell types (Meister, 1989). GSH acts as a co-substrate in the GSH peroxidase-catalyzed reduction of H2O2 or LPO leading to its depletion. Previous studies demonstrated that ROS generation following GSH depletion caused mitochondrial damage (Martensson et al., 1989; Meister, 1995), which has been implicated in apoptosis (Green and Reed, 1998). Therefore, increases or decreases in these responses can be interpreted as evidence for oxidative stress as the cell compensates either for increased stress by upregulating the production of antioxidants or the exhaustion of cellular stores of antioxidants by oxidation from reactive nitrogen species (RNS) or ROS. There was a significant depletion of GSH induced due to CuO NP exposure in a concentration-dependent manner when compared with untreated A549.


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25 20 15 10 y = 0.2489x – 20.519 R² = 0.9731

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120 100 80 60 40 y = – 0.7929x + 183.21 R² = 0.9741

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Figure 7. Correlation of reactive oxygen species generation with genotoxicity and cytotoxicity parameters in A549 cells after 24-h exposure to 0, 5, 10, and 15 mg/ml of copper oxide nanoparticles. (a) Significant positive correlation between reactive oxygen species generation and DNA damage, (b) significant positive correlation between reactive oxygen species generation and micronuclei formation, and (c) significant inverse correlation between reactive oxygen species generation and cell viability reduction.

We found that CuO NPs significantly induced genotoxic effect in human lung cells. It has been demonstrated that Cu ions were released from the surface of CuO NPs when suspended in aqueous state (Midander et al., 2009). However, we have not examined the degree of ionization of CuO NPs in aqueous suspension and their biological effects. However, Fahmy and Cormier (2009) studied the cytotoxic response of CuO NPs in airway epithelial cells and found that the release of Cu ions in the cell culture media did not significantly contribute to the cytotoxic response and the oxidative damage of CuO NPs. Further, Griffitt et al. (2007) have also demonstrated that dissolved portion of Cu ions from NPs is insufficient to produce mortality in Zebrafish exposed to CuO NPs. Our study along with other reports, thus, suggests that cytotoxicity and oxidative stress are associated with NPs of CuO per se rather than the released Cu ions. Taken together, we have reported the potential of CuO NPs to cause genotoxicity in human lung epithelial cells (A549), which may be mediated through the

induction of oxidative stress as well as compromising cellular antioxidant potential. This work suggests that the commercial and industrial application of CuO NPs should be carefully evaluated as to their potential hazardous effects to human health. Conflict of interest The authors declared no conflicts of interest.

Funding The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no. RGP-VPP-308.

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