Mechanism of graphene-induced cytotoxicity: Role of endonucleases.

Graphene, a crystalline allotrope or carbon, presents numerous useful properties; however, its toxicity is yet to be determined. One of the most dramatic and irreversible toxic abilities of carbon nanomaterials is the induction of DNA fragmentation produced by endogenous cellular endonucleases. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells is capable of inducing DNA fragmentation measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. TUNEL (cell death) and endonuclease activity measured using a near infrared fluorescence probe was significantly higher in cells containing graphene aggregates detected by Raman spectroscopy. The elevation of TUNEL coincided with the increased abundance of heme oxygenase 1 (HO-1), heat shock protein 90 (HSP90), active caspase-3 and endonucleases (deoxyribonuclease I [DNase I] and endonuclease G [EndoG]), as measured by quantitative immunocytochemistry. Specific inhibitors for HO-1, HSP90, caspase-3, DNase I and EndoG almost completely blocked the DNA fragmentation induced by graphene exposure. Therefore, graphene induces cell death through oxidative injury, caspase-mediated and caspase-independent pathways; and endonucleases DNase I and EndoG are important for graphene toxicity. Inhibition of these pathways may ameliorate cell injury produced by graphene. Copyright (c) 2017 John Wiley & Sons, Ltd. This study demonstrated that pristine graphene exposed to cultured kidney tubular epithelial cells induced DNA fragmentation measured by deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, which is usually associated with cell death. The elevation of TUNEL coincided with the increased abundances of heme oxygenase-1, HSP90, caspase-3, DNase I and EndoG, and blocked by specific inhibitors to these proteins/enzymes. Therefore, caspase-mediated and caspase-independent pathways, and endonucleases DNase I and EndoG are important for graphene toxicity.