Hydrogen Peroxide Induces Apoptosis in Human Dental Pulp Cells via Caspase-9 Dependent Pathway

Introduction

Reactive oxygen species are a group of metabolic intermediates produced during oxidative metabolism in eukaryotic cells. They include superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radical (·OH), and ¹O₂. Of these intermediates, H₂O₂ is the most stable. Dental pulp cells can be invaded by tooth bleaching, laser radiation, and dental materials. This can influence the intracellular level of reactive oxygen species. Apoptosis, which is the best-known form of programmed cell death, is pivotal to tissue development and regeneration. Little information is available regarding the relationship between H₂O₂ and apoptosis of human dental pulp cells (hDPCs). The purpose of this study was to investigate whether H₂O₂ can induce apoptosis in hDPCs and its signaling way.

Methods

HDPCs were obtained by using a modified tissue explant technique in vitro and cultured at 37°C, 20% O₂ (5% CO₂, 95% air) in Dulbecco modified Eagle medium. Cell viability was investigated by methyl-thiazol-tetrazolium assay. Cell apoptosis was detected by using the annexin V–fluorescein isothiocyanate/propidium iodide apoptosis assay and flow cytometry. Expression of activated caspase-3, cleaved caspase-9, and β-actin was analyzed by using Western blot.

Results

Cell viability of hDPCs decreased more in treated groups than in the control group from days 1 to 7. The relative number of apoptotic cells and the expression of activated caspase-3 and cleaved caspase-9 were much higher in groups exposed to 20 and 50 μmol/L H₂O₂.

Conclusions

These results imply that low concentrations of H₂O₂ are cytotoxic to hDPCs and induce apoptosis in hDPCs in a caspase-9–dependent way.