Oral malodorous compound triggers mitochondrial-dependent apoptosis and causes genomic DNA damage in human gingival epithelial cells.
OBJECTIVE
Volatile sulfur compounds are the main compounds causing halitosis. One of these compounds, hydrogen sulfide (H(2)S), which is responsible for physiological halitosis, is reported also to have periodontal pathogenic activities. Hydrogen sulfide has been shown to activate the apoptotic process in different tissues. Apoptosis plays an important role in the development of periodontitis. The aim of this study was to determine whether H(2)S causes apoptosis in human gingival epithelial cells and to examine the cellular signaling pathway initiating the process.


METHODS
Human gingival epithelial cells were incubated with 50 ng/mL H(2)S in air contining 5% CO(2) for 24, 48 or 72 h. To detect apoptosis, the cells were stained with annexin V and 7-amino actinomycin D, and analyzed using flow cytometry. Reactive oxygen species, mitochondrial membrane depolarization and release of cytochrome C into the cytosol were assessed using flow cytometry and enzyme-linked immunosorbent assay. Activity levels for the key apoptotic enzymes caspase-9, -8 and -3 were also determined. Genomic DNA damage was detected using single-cell gel electrophoresis.


RESULTS
Apoptosis was significantly increased to 24.5 +/- 5.7 at 24 h and 41.5 +/- 8.9% at 48 h (p < 0.01). Reactive oxygen species were enhanced and mitochondrial membrane depolarization was collapsed. Cytochrome C release was dramatically increased (0.12 +/- 0.02 vs. 0.02 +/- 0.01 at 24 h and 0.21 +/- 0.02 vs. 0.02 +/- 0.01 ng/mL at 48 h; p < 0.05). Caspase-9 and -3 were strongly activated, while caspase-8 activity remained low. The percentage of DNA strand breaks increased, especially at 48 h.


CONCLUSIONS
Hydrogen sulfide induces apoptosis in human gingival epithelial cells by activating the mitochondrial pathway.
Calenic
B
B
Department of Oral Health, Nippon Dental University, Tokyo, Japan.
Yaegaki
K
K
Murata
T
T
Imai
T
T
Aoyama
I
I
Sato
T
T
Ii
H
H
D017304:Annexin A5; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3 / Q000032:analysis; D053181:Caspase 8 / Q000032:analysis; D053453:Caspase 9 / Q000032:analysis; D002460:Cell Line; D002478:Cells, Cultured; D020552:Comet Assay; D045304:Cytochromes c / Q000032:analysis; D004249:DNA Damage; D003609:Dactinomycin / Q000031:analogs & derivatives; D004847:Epithelial Cells / Q000187:drug effects / Q000648:ultrastructure; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D016678:Genome; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006209:Halitosis / Q000378:metabolism*; D006801:Humans; D006862:Hydrogen Sulfide / Q000009:adverse effects*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000032:analysis; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D055549:Volatile Organic Compounds / Q000009:adverse effects
D017304:Annexin A5; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D055549:Volatile Organic Compounds; D003609:Dactinomycin; C025942:7-aminoactinomycin D; D045304:Cytochromes c; C505530:CASP8 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D006862:Hydrogen Sulfide