Effects of Hydrogen Sulfide on Cell Proliferation in Epithelial Cells

Objectives: Oral malodor is caused mainly by volatile sulphur compounds (VSC) such as hydrogen sulfide (H₂S) and methyl mercaptan (CH₃SH). These compounds might contribute to the initiation and progression of periodontal disease. However, little is known about the physiological effects of VSC in oral cavity. H₂S has been recently reported to inhibit cell proliferation in vascular smooth muscle cells, but not in oral epithelial cells. The aim of this study was to evaluate the effects of H₂S on cell proliferation and cell cycle in oral epithelial cells. Methods: Human gingival carcinoma cell line (Ca9-22) was used in this study. Cells were cultured in conditions of 5%CO₂/95%air with or without H₂S (5, 10 and 20ng/ml) for 24 hours. Cell viability was determined by trypan blue dye exclusion test. DNA synthesis was measured using BrdU ELISA kit (Roche Diagnostics). Cell cycle was analyzed by flow cytometry. The expression of cyclin-dependent kinase inhibitor (p21^Cip1) was evaluated by Western immunoblotting. To determine statistical significance, t test was used. Results: Exposure to 5, 10 and 20ng/ml H₂S did not induce cell death in Ca9-22. Exposure to 5, 10 and 20ng/ml of H₂S decreased DNA synthesis significantly (p<0.005). Flow cytometric analysis for DNA content showed that exposure to 5, 10 and 20ng/ml of H₂S decreased in the proportion of cells in G₂/M phase. Western immnoblotting showed that p21^Cip1 was strongly expressed by the exposure to 20ng/ml H₂S. Conclusion: These results indicated that H₂S inhibited cell proliferation and induced G₀/G₁ phase arrest in Ca9-22. This cell cycle arrest was induced by the elevation of p21^Cip1 level.