Oral malodorous compound causes oxidative stress and p53-mediated programmed cell death in keratinocyte stem cells.
BACKGROUND
It is well known that hydrogen sulfide (H(2)S), the main substance causing physiologic halitosis, is also involved in the etiology of periodontitis. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; keratinocyte stem cells play key roles in maintaining this barrier. An increased apoptotic process can affect keratinocyte stem cells, having a direct impact on oral epithelial tissue architecture. Our objective is to determine whether H(2)S induces apoptosis in human keratinocyte stem cells.


METHODS
Apoptosis levels; p53 activity; reactive oxygen species; mitochondrial membrane depolarization; cytochrome C release; and caspase-9, -8, and -3 were assessed using enzyme-linked immunosorbent assay and flow cytometry. Genomic DNA damage was examined using single-cell gel electrophoresis. Real-time polymerase chain reaction was used for Bax detection.


RESULTS
The percentage of apoptotic cells was significantly increased (20.5% +/- 1.6% versus 4.5% +/- 1.1% at 24 hours and 37.8% +/- 5.4% versus 4.8% +/- 0.9% at 48 hours; P <0.05, respectively; n = 5). Mitochondrial membrane potential was collapsed and reactive oxygen species levels were significantly increased compared to their control groups. At each time point the amount of released cytochrome C into the cytosol was significantly increased. Caspase-9 and -3 activities were significantly increased (P <0.05), whereas caspase-8 remained inactive. After both 24 and 48 hours, total and phosphorylated p53 levels were significantly increased.


CONCLUSIONS
We conclude that H(2)S can induce apoptosis in human keratinocyte stem cells, a key component of the epithelial barrier, following DNA damage and p53 activation.
Calenic
Bogdan
B
Department of Oral Health, Nippon Dental University, Tokyo, Japan.
Yaegaki
Ken
K
Kozhuharova
Ana
A
Imai
Toshio
T
D053687:Adult Stem Cells / Q000187:drug effects*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D020169:Caspases / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D020552:Comet Assay; D045304:Cytochromes c / Q000032:analysis / Q000378:metabolism; D004249:DNA Damage; D005881:Gingiva / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D006862:Hydrogen Sulfide / Q000494:pharmacology*; D015603:Keratinocytes / Q000187:drug effects*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000032:analysis / Q000378:metabolism
D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D020169:Caspases; D006862:Hydrogen Sulfide