ZnO nanoparticles induce apoptosis in human dermal fibroblasts via 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, USA b King Abdullah Institute for Nanotechnology, King Saud University, Riyadh 11451, Saudi Arabia article info Article history: Received 29 May 2011 Accepted 20 August 2011 Available online xxxx Keywords: Zinc oxide nanoparticles Apoptosis p53 p38 Human dermal fibroblasts abstract The production of engineered nanoparticles is growing rapidly as the field of nanotechnology continues to 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 the biological effects and risks of ZnO NPs. This study was designed to investigate the apoptosis induction by ZnO NPs via mitogen-activated protein kinase p38 and cell cycle checkpoint protein p53 pathways in 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 lower density and a rounded morphology. Apoptosis induction was confirmed by the annexin V assay and Western blot analysis showed the up-regulation of p53 and phospho-p38 proteins. Furthermore, in ZnO NP exposed cells, p53 protein was phosphorylated at Ser 33 and Ser 46 sites known to be phosphory- lated by p38. Our results suggest that ZnO NPs have the potential to induce apoptosis in human dermal fibroblasts 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 towards biosensing, 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 ZnO NPs, there is lack of information concerning the toxicity of these nanoparticles at the cellular and molecular levels. Previous studies have reported the cytotoxic effects of ZnO in various mammalian cell lines. After a 72 h exposure to 15 lg/ml of 19 nm particles, nearly complete cell death was observed in human mesothelioma and rodent fibroblasts ( Brunner et al., 2006). In neuroblastoma culture, 50 nm ZnO particle exposure at 100 lg/ml resulted in nearly 50% cell death (Jeng and Swanson, 2006). ZnO particles between 20 and 70 nm at 50 lg/ml decreased vascular endothelial cell survivorship by 50% (Gojova et al., 2007). Human neural cells exhibited approximately a 50% decrease in survival after a 48 h exposure to 11 lg/ml ZnO NPs, and human alveolar epithelial cell viability was reduced by half at 24 h by 50 nm ZnO at 4 lg/ml (Lai et al., 2008; Lanone et al., 2009). A reduction in neural stem cell survivorship was observed upon 24 h incubation with 12 lg/ml ZnO NPs (Deng et al., 2009). However, cytotoxic effects were observed in human T cells beginning only at 5 mM (over 400 lg/ml) (Reddy et al., 2007). In addition, cancer T cells were demonstrated to have around 30 times more sensitivity than normal T cells to ZnO NP toxicity (Hanley et al., 2008). We choose human dermal fibroblast cells as a model to assess whether ZnO NPs induce toxicity because dermal fibroblasts are the most abundant cell in the skin tissue and represent the first level of exposure to many toxicants. The present study was designed to investigate the toxicity of ZnO NPs through three approaches. First, cell morphology and survivorship were examined by phase contrast microscopy and the cell viability assay. Second, apoptosis in response to ZnO NP exposure was determined by annexin V staining. Third, Western blot analysis was used to examine the expressions of cell cycle checkpoint protein p53 and p38 mitogen-activated protein kinase (MAPK) after exposure to ZnO NPs. The p53–p38 pathway is a key signal transduction system that mediates several extracellular signals through a cascade of protein phosphorylation. It was shown that p38 MAPK phosphorylates p53 at Ser 33 and Ser 46 and plays a prominent role in an integrated regulation of N-terminal that regulates p53-mediated apoptosis after UV radiation (Bulavin et al., 1999). Therefore, we further explored the interaction 0887-2333/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.tiv.2011.08.011 ⇑ Corresponding author. Tel.: +1 937 229 3429; fax: +1 937 229 2021. E-mail addresses: maqusood@gmail.com (M. Ahamed), yiling.hong@notes. udayton.edu (Y. Hong). Toxicology in Vitro xxx (2011) xxx–xxx Contents lists available at SciVerse ScienceDirect Toxicology in Vitro journal homepage: www.elsevier.com/locate/toxinvit Please cite this article in press as: Meyer, K., et al. ZnO nanoparticles induce apoptosis in human dermal fibroblasts via p53 and p38 pathways. Toxicol. in Vitro (2011), doi:10.1016/j.tiv.2011.08.011