Abstract 3872: Genotoxicity of zinc oxid nanoparticles and the activation of ATM-Chk2 DNA-damage-response pathway are caused by zinc-ions
Authors: Heim Julia , Tahir Nawaz Muhammad , Kaltbeitzel Anke , Mailänder Volker , Lüddens Hartmut , Tremel Wolfgang , Brieger Jürgen 
Published: August 2015

Abstract
Zinc oxide nanoparticles (ZnO-NP) are used in a variety of applications including cosmetics and pharmaceuticals. The common utilization of ZnO-NPs results in an increasing environmental release with unknown health effects. Recent studies have shown that ZnO-NP exposure cause cellular toxicity including DNA-damages. Discussed mechanisms are interactions of the ZnO-NP with intracellular structures and molecules after internalization, extracellular or intracellular dissociation of the particles, and the generation of ROS. In the present study we evaluated the genotoxic properties of ZnO-NP as well as the cellular DNA damage response in dependence of oxidative distress and ZnO-NP dissociation into Zn2+.
For this purpose, we exposed the human epithelial cell line A549 to defined ZnO-NP (100μg/ml). Genotoxic effects of the particles were evaluated by Comet assay and γH2AX-foci, measuring DNA double-strand breaks (DNA-DSBs). Dissociation of ZnO-NP and the intracellular increase of Zn2+ were performed with spectrofluorimetric and cLSM measurements by the use of FluoZin3. Oxidative distress was determined by the compound dihydrodichlorofluorescein diacetate (H2DCF-DA). The Activation of the DNA damage response pathway was proven by analyses of H2AX-, ATM-, Chk1- and Chk2 phosphorylation, and p21, p53 up-regulation after ZnO-NP exposure by western blot or ELISA. To figure out, if the genotoxic potential of ZnO-NP is dependent of released Zn2+ or the generation of ROS we inhibited oxidative distress by the antioxidant N-acetyl-L-cysteine (NAC) and Zn2+ were extracellular chelated by diethylene triamine pentaacetic acid (DTPA).
We found a fast extracellular dissociation of ZnO-NP and a quick intracellular increase of Zn2+. Furthermore, we ascertained an activation of H2AX induced by ZnO-NP; this phosphorylation is totally inhibited by DTPA. ZnO-NP activated the DSB-damage response indicated by ATM-, Chk2-phosphorylation as well as an up-regulation of p53 and p21. Pretreatment with NAC scavenging ROS leads to a reduced and Zn2+ complexation to no activation of ATM-Chk2-DNA damage pathway. The ATR/Chk1 pathway was not activated by ZnO-NP.
We demonstrated ZnO-NP induced DNA-damages and the activation of the ATM/Chk2 DNA damage response pathway but not the ATR/Chk1 pathway, indicating that ZnO-NP exposure induces DNA-DSBs. Scavanging of ROS leads to a slight reduction of DNA-DSBs but extracellular Zn2+ compelxation to a complete impairment suggesting that the mechanism of ZnO-NP genotoxicity is not mediated via ROS-generation but rather a direct interaction of dissociated Zn-ions with the DNA.
Citation Format: Julia Heim, Nawaz Muhammad Tahir, Anke Kaltbeitzel, Volker Mailänder, Hartmut Lüddens, Wolfgang Tremel, Jürgen Brieger. Genotoxicity of zinc oxid nanoparticles and the activation of ATM-Chk2 DNA-damage-response pathway are caused by zinc-ions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3872. doi:10.1158/1538-7445.AM2015-3872