EVALUATION OF REACTIVE OXYGEN SPECIES GENERATION AND DNA METHYLATION CHANGE IN LINE-1 AND ALU INDUCED BY NANOPARTICLES IN HEK293 AND HACAT CELLS

Abstract

Engineered nanoparticles (ENPs) are one of the most nanomaterials that wildly used in various fields including biomedical applications. However, the adverse effects of ENPs on health risk still need to be concerned. ENPs have been reported to be a matter that cause cellular damage through either direct or indirect, cellular oxidative stress is one of the most nanotoxicity have been found. Reactive oxygen species (ROS) is a cause of cellular oxidative stress that leads to intracellular macro molecules damages and may impact on DNA methylation changes. In this study we aim to investigate the effect of ROS induced by ENPs on DNA methylation that is one important of epigenetic mechanisms. Human embryonic kidney (Hek 293) and human keratinocyte (HaCaT) cells were used as model to expose with three different types of ENPs, gold nanoparticles (AuNPs), Silica nanoparticles (SiNPs) and Chitosan nanoparticles (CSNPs). First, we evaluated cytotoxicity of cells by measuring viability, morphology and ROS levels. Global DNA methylation levels were measured by 5-methylcytosine immunocytochemistry staining, and we also investigated DNA methylation levels of retrotransposable elements, LINE-1 and Alu by using combined bisulfite restriction analysis technique (COBRA). We found ROS level was increased in SiNPs exposed HaCaT cells only. DNA hypomethylation of global and Alu elements was showed in cells were exposed with SiNPs and CSNPs in HaCaT cells only. LINE-1 did not change in both of Hek293 and HaCaT cells. Furthermore, the inversion of Alu DNA methylation level in HaCaT cells exposed with SiNPs and CSNPs was found in antioxidant (N-acetyl cysteine) pretreated cells. Our study demonstrated the new insight that DNA methylation of Alu elements represents the global DNA methylation of cell exposed with ENPs. In this study, the alteration of DNA methylation level in ENPs exposed cells is ROS independent and specific to cell types.