Continuous presence of H2O2 induces mitochondrial-mediated, MAPK- and caspase-independent growth inhibition and cytotoxicity in human gingival fibroblasts

The continuous generation of reactive oxygen species (ROS) is one of the most important events that occur during periodontal inflammation. Hydrogen peroxide (H2O2) is widely used in dental clinics. Many investigators have tried to elucidate the exact effect of H2O2 on human gingival fibroblasts (HGFs). These studies have shown that H2O2 induces growth inhibition and apoptosis in cells. However, the mechanisms involved in H2O2-induced cell death in HGFs are not completely understood. In this study, we examine how continuously generated H2O2 affects the viability and proliferation of HGFs using glucose oxidase (GO). We also explored the mechanisms by which the continuous presence of H2O2 induces cell death. GO treatment not only inhibited HGF growth and proliferation, but it also induced cell death in HGFs without typical apoptotic features such as nuclear DNA laddering. This GO-mediated cytotoxicity was proportional to the levels of intracellular ROS that were generated, rather than proportional to changes of cellular antioxidant activities. GO treatment also resulted in the loss of mitochondrial membrane potential and the relocation of mitochondrial apoptogenic factors. There was also an acute and severe depletion of cellular ATP levels. However, none of the pharmacological inhibitors specific for mitogen-activated protein kinases (MAPKs) or pancaspase prevented GO-induced cell death. Treatment with either catalase or acteoside significantly attenuated the GO-mediated cytotoxicity in the HGFs, thereby suggesting a protective effect of antioxidants against ROS-mediated gingival damage. Here we demonstrate that continuously generated H2O2 not only inhibits the viability and proliferation of HGFs, but also causes pyknotic/necrotic cell death through mitochondrial stress-mediated, MAPK- and caspase-independent pathways.

Highlights► This study examined the effects of glucose oxidase (GO) on human gingival fibroblasts (HGFs). ► Continuously generated H2O2 by GO caused oxidative cell death without typical apoptotic features. ► Continuous presence of H2O2 induced MMP loss and mitochondrial alteration of apoptogenic factors. ► Continuous H2O2 exposure also led to the marked depletion of intracellular ATP level in HGFs. ► Further, MAPK or caspase inhibitors did not prevent H2O2-mediated toxicity in HGFs.