Toxicology in Vitro 25 (2011) 1721–1726

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Toxicology in Vitro

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ZnO nanoparticles induce apoptosis in human dermal fibroblastsvia p53 and p38 pathways

Kyle Meyer a, Pavan Rajanahalli a, Maqusood Ahamed b, John J. Rowe a, Yiling Hong a,⇑a Department of Biology and Center for Tissue Regeneration and Engineering, University of Dayton, Dayton, OH 45469, USAb King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 May 2011Accepted 20 August 2011Available online 26 August 2011

Keywords:Zinc oxide nanoparticlesApoptosisp53p38Human dermal fibroblasts

0887-2333/$ - see front matter Published by Elsevierdoi:10.1016/j.tiv.2011.08.011

⇑ Corresponding author. Tel.: +1 937 229 3429; faxE-mail addresses: maqusood@gmail.com (M. A

udayton.edu (Y. Hong).

The production of engineered nanoparticles is growing rapidly as the field of nanotechnology continuesto expand. Zinc oxide nanoparticles (ZnO NPs) are used in various applications, including catalysis, elec-tronics, biosensors, medicine, paints, sunscreens and cosmetics, thus it is important to understand thebiological effects and risks of ZnO NPs. This study was designed to investigate the apoptosis inductionby ZnO NPs via mitogen-activated protein kinase p38 and cell cycle checkpoint protein p53 pathwaysin human dermal fibroblasts. MTT-based cell viability assay showed a significant decrease in cell survi-vorship after ZnO NP exposure, and phase contrast images revealed that ZnO NP treated cells had lowerdensity and a rounded morphology. Apoptosis induction was confirmed by the annexin V assay andWestern blot analysis showed the up-regulation of p53 and phospho-p38 proteins. Furthermore, inZnO NP exposed cells, p53 protein was phosphorylated at Ser33 and Ser46 sites known to be phosphory-lated by p38. Our results suggest that ZnO NPs have the potential to induce apoptosis in human dermalfibroblasts via p53–p38 pathways.

Published by Elsevier Ltd.

1. Introduction

Zinc oxide nanoparticles (ZnO NPs) are used in various products,including cosmetics and sunscreens due to UV-filtering properties(NCPI, 2010). Recent interest in ZnO NPs has been directed towardsbiosensing, cell imaging, photodynamic therapy and nanomedicine(Brayner et al., 2006; Hanley et al., 2008; Premanathan et al., 2011;Ahamed et al., 2011). Despite the widespread application of ZnONPs, there is lack of information concerning the toxicity of thesenanoparticles at the cellular and molecular levels.

Previous studies have reported the cytotoxic effects of ZnO invarious mammalian cell lines. After a 72 h exposure to 15 lg/mlof 19 nm particles, nearly complete cell death was observed inhuman mesothelioma and rodent fibroblasts (Brunner et al.,2006). In neuroblastoma culture, 50 nm ZnO particle exposure at100 lg/ml resulted in nearly 50% cell death (Jeng and Swanson,2006). ZnO particles between 20 and 70 nm at 50 lg/ml decreasedvascular endothelial cell survivorship by 50% (Gojova et al., 2007).Human neural cells exhibited approximately a 50% decrease insurvival after a 48 h exposure to 11 lg/ml ZnO NPs, and humanalveolar epithelial cell viability was reduced by half at 24 h by

Ltd.

: +1 937 229 2021.hamed), yiling.hong@notes.

50 nm ZnO at 4 lg/ml (Lai et al., 2008; Lanone et al., 2009). Areduction in neural stem cell survivorship was observed upon24 h incubation with 12 lg/ml ZnO NPs (Deng et al., 2009).However, cytotoxic effects were observed in human T cellsbeginning only at 5 mM (over 400 lg/ml) (Reddy et al., 2007). Inaddition, cancer T cells were demonstrated to have around 30times more sensitivity than normal T cells to ZnO NP toxicity(Hanley et al., 2008).

We choose human dermal fibroblast cells as a model to assesswhether ZnO NPs induce toxicity because dermal fibroblasts arethe most abundant cell in the skin tissue and represent the firstlevel of exposure to many toxicants. The present study wasdesigned to investigate the toxicity of ZnO NPs through threeapproaches. First, cell morphology and survivorship wereexamined by phase contrast microscopy and the cell viabilityassay. Second, apoptosis in response to ZnO NP exposure wasdetermined by annexin V staining. Third, Western blot analysiswas used to examine the expressions of cell cycle checkpointprotein p53 and p38 mitogen-activated protein kinase (MAPK)after exposure to ZnO NPs. The p53–p38 pathway is a key signaltransduction system that mediates several extracellular signalsthrough a cascade of protein phosphorylation. It was shown thatp38 MAPK phosphorylates p53 at Ser33 and Ser46 and plays aprominent role in an integrated regulation of N-terminal thatregulates p53-mediated apoptosis after UV radiation (Bulavinet al., 1999). Therefore, we further explored the interaction

1722 K. Meyer et al. / Toxicology in Vitro 25 (2011) 1721–1726

between p38 and p53 by examining levels of p53 phosphorylatedat Ser33 and Ser46 in response to ZnO NPs. This study providesmolecular evidence for the ZnO NP-induced apoptosis in humandermal fibroblast cells.

2. Materials and methods

2.1. Zinc oxide nanoparticles

ZnO NP dry powder was purchased from Nanostructure &Amorphous Materials Inc., Houston, TX (www.nanoamor.com).The characteristics provided by the supplier are as follows:20 nm average particle size, 99.5% purity, 50 m2/g specific surfacearea, milky white color, nearly spherical morphology, 0.3–0.45g/cm3 bulk density, and 5.606 g/cm3 true density.

2.2. Characterization of zinc oxide nanoparticles

We also characterized the shape and size of ZnO NPs usingtransmission electron microscopy (TEM) on a Hitachi H-7600tungsten-tip instrument at an accelerating voltage of 100 kV.Dry powder of ZnO NPs was suspended in deionized water at1 mg/ml and then sonicated using a Branson-1510 sonicator bathat room temperature for 15 min at 35–40 W to aid in mixing andforming a homogeneous dispersion. For size measurements,sonicated 1 mg/ml ZnO NP stock solution was then diluted to a50 lg/ml working solution. ZnO NPs were examined after NPssuspensions were deposited in carbon film-coated Cu grids. Theadvance microscopy techniques (AMT) software for the digitalTEM camera was calibrated for size measurements of the nano-particles. Information on mean size and SD was calculated usingthe point-to-point method as described elsewhere (Murdocket al., 2008).

2.3. Cell culture and exposure to zinc oxide nanoparticles

Human dermal fibroblasts were ordered from Promo-cell (Cat#C-12302). Human dermal fibroblast cells were cultured at 37 �Cand 5% CO2 in high glucose Dulbecco’s Modified Eagle Medium(DMEM, Gibco# 11965) containing 10% fetal bovine serum, 0.5%penicillin–streptomycin (Gibco# 15140), 1% glutamine (Gibco#35050), and 1% non-essential amino acids (Gibco# 11140). Cellswere passaged using 0.25% trypsin (Gibco# 25200). At 85% conflu-ence, cells were harvested using 0.25% trypsin and were sub-cultured into 6-well plates or 96-well plates. Cells were incubatedfor 24 h prior to treatment. ZnO NPs were suspended in cell culturemedium and diluted to the target concentrations. The dilutions ofZnO NPs were then sonicated using a sonicator bath at room tem-perature for 10 min at 40 W to avoid agglomeration prior toadministration to the cells.

2.4. Cell viability

Cell viability was determined by MTT assay (CGD-1 kit, Sigma–Aldrich). Human dermal fibroblast cells (1 � 104 cells/well) wereseeded into a 96-well plate (PerkinElmer SpectraPlate). After24 h, the media were replaced with 100 ll of media prepared withZnO NPs (2.5, 10, 25, 50, and 100 lg/ml) and incubated for 24 h.After the exposure, the medium was removed from each welland replaced with a new medium containing MTT solution in anamount equal to 10% of culture volume and incubated for 3 h at37 �C until a purple colored formazan product developed. Theresulting formazan product was dissolved in acidified isopropanol,and the absorbance was measured at 570 and 690 nm by using amicroplate reader (Synegy-HT Biotek). Background absorbance at690 nm was subtracted from absorbance at 570 nm.

2.5. Cell morphological examination

Cell morphology of human dermal fibroblasts was examinedfollowing exposure to different concentrations ZnO NPs for 4 and24 h using a phase contrast inverted microscope (Nikon TS100).

2.6. Apoptosis detection by annexin V staining

We examined apoptosis induction in human dermal fibroblastsexposed to 50 lg/ml ZnO NPs for 24 h using annexin V staining.After exposure, cells were fixed with Formalde-Fresh (formalde-hyde 4% w/v, methanol 1% w/v), permeabilized in PBS containing1% NP40, and blocked with 10% horse serum for 1 h. The coverslipswith the cells were stained with annexin V (Abcam# 14196) andfluorescent-labeled secondary antibody Alexa Fluor 594 (1:400,Invitrogen# A31632). Images were acquired with a Fluoview laserscanning confocal microscope (10� magnification).

2.7. Western blot

For Western blot analysis cells were cultured in 6-well plates andexposed to different concentrations of ZnO NPs for 4 and 24 h. At theend of the exposure, cells were lyzed with RIPA buffer (50 mMTris–HCl, pH 8.0, 150 mM NaCl, 1% NPs-40, 1% sodium deoxycholate,and 0.1% SDS) containing protease inhibitors. Cell lysates wereseparated on a 12% SDS–PAGE and transferred to a polyvinylidenefluoride membrane (Millipore# IPVH00010). After blocking themembrane for 1 h in TBST (20 mM Tris–HCl, pH 7.6, 136 mM NaCl,and 0.1% Tween-20) containing 3% nonfat milk, membranes wereincubated at 4 �C overnight with primary antibody diluted in 3%milk–TBST solution. Following incubation, the membranes werewashed with TBST and incubated at room temperature for 1 h withhorseradish peroxidase conjugated secondary antibody. Blots weredeveloped using Amersham ECL detection reagents (RPN2106) andKodak BioMax Light Film (Cat# 868 9358). The following primaryand secondary antibodies were used: anti-p53 (1:2000, CellSignaling# 2524), anti-p53 (1:400, Calbiochem OP03), anti-phospho-p38 MAPK (1:1000, Cell Signaling# 9215), anti-phospho-p53 (Ser33) (1:1000, Cell Signaling# 2526), anti-phospho-p53(Ser46) (1:1000, Cell Signaling# 2521), anti-b-actin (1:4000, SigmaAldrich A1978), goat anti-mouse IgG-HRP (1:2000, Santa Cruzsc-2005), and goat anti-rabbit IgG-HRP (1:5000, Santa Cruz sc-2004).

2.8. Statistical analysis

Statistical significance for Student’s t-test was set at p < 0.05.

3. Results

3.1. Zinc oxide nanoparticle characterization

Fig. 1A shows an example TEM image of ZnO NPs. Informationon mean size and SD was obtained by measuring over 100 nano-particles in random fields of view. The mean ± SD of ZnO NPswas 23.47 ± 5.54 nm. Fig. 1B represents the frequency of size distri-bution of ZnO NPs.

3.2. Effect of zinc oxide nanoparticles on cell viability

Human dermal fibroblast cells were exposed to ZnO NPs atconcentrations of 2.5, 10, 25, 50, and 100 lg/ml for 24 h, and cellviability was determined using the MTT assay (Fig. 2). ZnO NPsat the concentration of 2.5 lg/ml did not produce significantreduction in cell viability. Cells exposed to 10 lg/ml ZnO NPsshowed 20% reduction in cell survivorship (p < 0.05), while greater

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Fig. 1. Transmission electron microscopy characterization of ZnO NPs. (A) indicates that the majority of ZnO NPs were spherical in shape. Information on mean size and SDwas obtained by measuring over 100 nanoparticles. The mean ± SD was 23.47 ± 5.54 nm. (B) represents the frequency of size distribution.

Fig. 2. Cell viability (MTT assay) of human dermal fibroblasts exposed to differentconcentrations of ZnO NPs for 24 h. Data presented are mean ± SD of triplicatevalues. Statistically significant differences were assessed as compared to thecontrols. ⁄p < 0.05, ⁄⁄p < 0.001.

Fig. 3. Morphology of human dermal fibroblasts exposed to ZnO NPs. (A) and (D) are c(F) 100 lg/ml for 24 h.

K. Meyer et al. / Toxicology in Vitro 25 (2011) 1721–1726 1723

than 85% reduction in cell viability was observed at higher concen-trations (25–100 lg/ml, p < 0.001) (Fig. 2).

3.3. Effect of zinc oxide nanoparticles on cell morphology

The morphology of human dermal fibroblast cells was examinedafter ZnO NP exposure using phase contrast microscopy. No visualdifferences compared to the unexposed control were observed incells exposed for 4 and 24 h to 2.5, 5, and 10 lg/ml ZnO NPs (datanot shown). A significant lowering of cell density and rounding ofcells were apparent at 50 and 100 lg/ml ZnO NPs after 4 h exposure(Fig. 3B and C) and were present at a greater extent after 24 hexposure (Fig. 3E and F).

3.4. Apoptosis induction by zinc oxide nanoparticles

Phase contrast studies revealed that ZnO NP-exposed cells losttheir normal morphology and were detached from the surface. To

ontrol cells. (B) 50 lg/ml for 4 h, (C) 100 lg/ml for 4 h, (E) 50 lg/ml for 24 h and

50 μg/ml - 24 hours

Annexin V Phase contrast Merge

Control

Fig. 4. Annexin V staining of cells 24 h after treatment with 50 lg/ml ZnO NPs. Upper panel is untreated human dermal fibroblast cells stained for annexin V. Lower panel isZnO NP-treated human dermal fibroblast cells stained for annexin V.

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Fig. 5. Effect of ZnO NPs on the expression level of p53 protein in human dermal fibroblast samples: (A) Lanes 1–4 are 4 h after exposure to (1) control, (2) 10 lg/ml,(3) 50 lg/ml and (4) 100 lg/ml. (B) Lanes 5–8 are 24 h after exposure to (5) control, (6) 10 lg/ml, (7) 50 lg/ml and (8) 100 lg/ml. b-Actin was used as loading control.Statistically significant differences were assessed as compared to the controls. ⁄p < 0.05.

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confirm that cell death was due to apoptosis, we stained forannexin V in human dermal fibroblasts treated with 50 lg/mlZnO NPs for 24 h. Results showed that ZnO NPs induced apoptosisin human dermal fibroblast cells (Fig. 4).

3.5. Zinc oxide nanoparticles induced p53 upregulation andphosphorylation of p53 at Ser33 and Ser46

To understand the molecular mechanisms of apoptosis inducedby ZnO NPs, differential expression of tumor suppressor proteinp53 in response to varying ZnO NP concentrations was analyzedin human dermal fibroblast cells by Western blot analysis. A signifi-cantly increased expression of p53 was observed at 4 h after expo-sure to 10, 50, and 100 lg/ml ZnO NPs (Fig. 5A, lanes 1–4). Almostthe same level of p53 expression was observed after 24 h exposure,suggesting that the expression of p53 protein was the highestwithin 4 h (Fig. 5B, lanes 5–8).

Activation of p53 is modulated by protein phosphorylation. Thelevels of phospho-p53 (Ser33 and Ser46) were analyzed in HF cells.Significantly increased phosphorylation of p53 at Ser33 wasobserved for 4 and 24 h exposure for 10 and 50 lg/ml (Fig. 6A

and B). A similar pattern of expression was observed in Ser46 phos-phorylation (Fig. 7A and B).

3.6. Zinc oxide nanoparticles induced p38 MAP kinase upregulation

p38 is a stress-activated MAP kinase that becomes activatedupon phosphorylation and is involved in apoptosis initiation(Ichijo, 1999). Western blot analysis indicated an increase inphospho-p38 levels at 50 and 100 lg/ml concentrations in humandermal fibroblast cells (Fig. 8A). At 24 h exposure, the phospho-p38expression was still greater than that of the untreated control cells(Fig. 8B). Together, these results indicated that ZnO NP exposureinduced p38 MAP kinase activation and phosphorylation of p53at Ser33 and Ser46, which might play an important role in regulatingp53-mediated apoptosis.

4. Discussion

In spite of many advantages in using nanotechnology, studiesindicate that nanoparticles may cause hazardous effects becauseof their unique physicochemical properties. Although the beneficial

Phospho-p53 Ser 46

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Fig. 7. Effect of ZnO NPs on the expression level of phospho-p53 (Ser46) in human dermal fibroblast cells. (A) Lanes 1–4 are 4 h after exposure to (1) control, (2) 10 lg/ml,(3) 50 lg/ml and (4) 100 lg/ml. (B) Lanes 5–8 are 24 h after exposure to (5) control, (6) 10 lg/ml, (7) 50 lg/ml and (8) 100 lg/ml. Statistically significant differences wereassessed as compared to the controls. ⁄p < 0.05.

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Fig. 6. Effect of ZnO NPs on the expression level of phosphor-p53 (Ser33) in human dermal fibroblast cells. (A) Lanes 1–4 are 4 h after exposure to (1) control, (2) 10 lg/ml,(3) 50 lg/ml and (4) 100 lg/ml. (B) Lanes 5–8 are 24 h after exposure to (5) control, (6) 10 lg/ml, (7) 50 lg/ml and (8) 100 lg/ml. Statistically significant differences wereassessed as compared to the controls. ⁄p < 0.05.

K. Meyer et al. / Toxicology in Vitro 25 (2011) 1721–1726 1725

effects of ZnO NPs have attracted considerable attention in termsof nanomedicine (Brayner et al., 2006; Hanley et al., 2008;Premanathan et al., 2011; Ahamed et al., 2011), potential biologicaland environmental hazards should also be considered. We foundthat ZnO NP exposure significantly reduced cell viability of humandermal fibroblasts starting at approximately 10 lg/ml. Cell viabilitydata were further supported by the morphology studies. Thelowering of cell density and the rounding observed suggest that50 and 100 lg/ml nanoparticle concentrations induced substantialcell death. Our cytotoxicity results were similar to those of otherstudies (Deng et al., 2009; Lai et al., 2008; Lanone et al., 2009; Zhaoet al., 2009).

It has been well documented that extensive DNA damage trig-gers apoptosis (Sherr, 2004). We examined ZnO NP induced apop-tosis in human dermal fibroblasts using the annexin V assay. Theresults indicated that ZnO NPs significantly induced cell death/

apoptosis in human dermal fibroblasts. Data from MTT assay,which indirectly measure cell viability by assessing mitochondrialfunction (Ahamed et al., 2008), corroborated the annexin V resultsby showing a reduction in cell survivorship after ZnO treatment.

ZnO NPs induced upregulation of two important molecularmarkers, p53 and phospho-p38. p53 is often regarded as ‘‘theguardian of the genome’’ due to its integral role in preserving theintegrity of the genome through prevention of mutations (Farneboet al., 2010). Upon the cell encountering an insult, p53 arrests thecell cycle, allowing DNA repair enzymes to act, and if the damage isbeyond repair, initiates apoptosis to prevent the mutation frombeing passed on through subsequent cell divisions. Along withthe increased p53 levels, the upregulation of phosphor-p38 indi-cates that ZnO NP exposure resulted in cellular stress. Furthermore,there is substantial interaction between p53 and p38, as demon-strated by a study reporting that during cellular stress induced

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Fig. 8. Effect of ZnO NPs on the expression level of p38 protein in human dermal fibroblast samples: (A) Lanes 1–4 are 4 h after exposure to (1) control, (2) 10 lg/ml,(3) 50 lg/ml and (4) 100 lg/ml. (B) Lanes 5–8 are 24 h after exposure to (5) control, (6) 10 lg/ml, (7) 50 lg/ml and (8) 100 lg/ml. b-Actin was used as loading control.Statistically significant differences were assessed as compared to the controls. ⁄p < 0.05.

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by UV-irradiation p38 phosphorylates p53 at two sites, stabilizingp53 and enhancing the apoptosis response (Bulavin et al., 1999).Our Western blot analysis indicated that phosphorylation at bothof these sites (Ser33 and Ser46) increased within 4 h exposure toZnO NPs.

In summary, our data demonstrate that ZnO NPs have thepotential to induce apoptosis in human dermal fibroblasts and thisprocess is mediated through p53–p38 pathways. These in vitroresults suggest that widespread application of ZnO NPs should becarefully assessed at the in vivo level for potential adverse biologi-cal effects.

Conflict of interest

None declared.

Acknowledgments

Funding for this research was provided by the National ScienceFoundation Grant CBET-0833953 and the University of DaytonResearch Council Seed Grant. Nanoparticle characterization wasperformed by courtesy of Timothy Gorey, University of Dayton.

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