Molecular damage and responses of oral keratinocyte to hydrogen peroxide

Background: Hydrogen peroxide (H₂O₂)-based tooth bleaching reagents have recently increased in popularity and controversy. H₂O₂ gel (3%) is used in a Nightguard for vital bleaching; transient tooth sensitivity and oral mucosa irritation have been reported. Genotoxicity and carcinogenicity have also been significant concerns. Methods: We used primary cultured normal human oral keratinocytes (NHOKs) as an in vitro model to investigate the pathological effects to mitochondria functions on human oral keratinocytes exposed to different doses of H₂O₂ for different durations. Results: An MTT assay showed compromised cell viability at a dose over 5 mM. The treatments induced nuclear DNA damage, measured using a single-cell gel electrophoresis assay. A real-time quantitative polymerase chain reaction showed H₂O₂ induced significant increase in mitochondrial 4977-bp deletion. Mitochondrial membrane potential and apoptosis assays suggested that oxidative damage defense mechanisms were activated after prolonged exposure to H₂O₂. Reduced intracellular glutathione was an effective defense against oxidative damage from 5 mM of H₂O₂. Conclusion: Our study suggests the importance for keratinocyte damage of the dose and the duration of the exposure to H₂O₂ in at-home-bleaching. A treatment dose ≥100 mM directly causes severe cytotoxicity with as little as 15 min of exposure.