Oral Malodorous Compound Increases Oxidative Stress in Human Gingival Fibroblasts

Objective: Hydrogen sulfide (H₂S) is oral malodorous compounds. H₂S has been elucidated to involve periodontal pathogenicities. Furthermore, we have previously reported that H₂S induces apoptosis in human gingival fibroblasts(HGF), which may have a role in periodontal pathogenesis and/or carcinogenesis. In this study, we therefore investigated whether H₂S increases oxidative stress which initiates apoptosis in HGF. Methods: The apoptosis caused by H₂S (100ng/ml air) in HGF was determined with Cell Death Detection ELISA plus^TM (Roche, Canada). Intracellular reactive oxygen species (ROS) was detected using MitoSOX^TM Red (Invitrogen, CA) by a flow cytometry. The cell viability and H₂S cytotoxicity were counted with using trypan blue staining (Invitrogen, CA) and a Cytotoxicity Detection kit (Roche). The effect of H₂S on SOD activity was determined using the SOD Assay kit-WST (Dojindo, Japan). Results: DNA fragmentation in cytoplasm was increased by H₂S for 48 and 72 hours incubations (79 ± 4 mU vs. 65 ± 3 mU and 108 ± 24 mU vs. 64 ± 3 mU, p<0.001, respectively ). Significant difference of the cell viability was not observed between test and control groups. The cytotoxicities of H₂S were less than 10% at each incubation time. MitoSOX™ positive cells were significantly increased comparison with controls (5.6 ± 2.6% vs. 39.8 ± 21.5% at 48-hour incubation: p<0.001, and 3.5 ± 2.7% vs. 29.2 ± 9.5% at 72 hour incubation: p<0.001). The activity of Cu,Zn-SOD was found to decrease by 80% (P<0.001) when exposed to H₂S and that of HGF-SOD activity decreased by 42% (P<0.001). Conclusion: H₂S induced apoptosis in HGF, but did not cause significant numbers of necrosis. H₂S also increased ROS in HGF, specially in mitochondria, since SOD was strongly inhibited by H₂S. It was postulated oxidative stress in mitochondria may activate a mitochondrial apoptotic pathways.