Molecular damage and responses of oral keratinocyte to hydrogen peroxide

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.

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.

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.

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.