RESUMO
The toxicity of hydrogen sulfide (H(2)S), an oral malodorous compound, is well reported. We have recently established an experimental model of hepatic differentiation from human tooth-pulp stem cells (HTPC) using serum-free medium. The objective of the present study is to determine the effect of H(2)S on hepatic differentiation. The CD117 positive cell fraction was obtained from deciduous HTPC using magnetic cell sorting. After 3-4 passages, cells were grown in Dulbecco's modified Eagle's medium supplemented with insulin-transferrin-selenium-x (ITS-x), embryotrophic factor (ETF) and hepatocyte growth factor (HGF) for hepatic commitment (five days). For hepatic differentiation the cells were cultured in Iscove's modified Dulbecco's medium supplemented with ITS-x, ETF, oncostatin, HGF and dexamethasone for 15 days in air containing 5% CO(2), with or without H(2)S at 0.05 ng ml(-1). Cells were assayed for the expression of hepatic markers α-fetoprotein, albumin or carbamoyl phosphate synthetase, and urea concentrations and glycogen synthesis were also determined. The panel of hepatic markers was expressed more in the test groups exposed to H(2)S than in the control groups. Urea and glycogen production were also increased, especially glycogen which was approximately five times greater compared to the control (p < 0.01). We concluded that H(2)S at physiological concentrations increased the ability of HTPC to undergo hepatogenic differentiation.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Polpa Dentária/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Sulfeto de Hidrogênio/farmacologia , Células-Tronco/efeitos dos fármacos , Dente Decíduo/citologia , Biomarcadores , Polpa Dentária/citologia , Imunofluorescência , Hepatócitos/citologia , Humanos , Imuno-Histoquímica , Células-Tronco/citologia , Dente Decíduo/efeitos dos fármacosRESUMO
Oral malodor is caused by volatile sulfur compounds (VSCs) composed mainly of hydrogen sulfide (H(2)S) and methyl mercaptan. In particular, H(2)S is an important compound, since it is a major component of physiologic halitosis. The toxicity of VSCs is similar to that of hydrogen cyanide, and is well investigated. The role of VSCs in reducing collagen in human gingival fibroblasts is one of the main sources of their toxicity to human oral tissues. It has been reported recently that H(2)S may cause apoptosis in several periodontal tissues. In human gingival fibroblasts, H(2)S inhibits not only cytochrome c oxidase activity but also superoxide dismutase activity. The levels of reactive oxygen species are markedly increased, which causes the release of cytochrome c into the cytoplasm, resulting in caspase-9 activation; finally, the executor caspase, caspase-3, is activated. This pathway is commonly observed in cells from all periodontal tissues. Moreover, p53, an apoptotic factor, and phosphorlylated p53, which is the activated form, are increased by H(2)S in keratinocyte stem cells and osteoblasts. H(2)S also increases the expression of Bax, a primary response gene playing an important role in p53-mediated apoptosis, but maintains a lower expression of Bcl-2, an anti-apoptotic factor, in osteoblasts. It is concluded that the Bax apoptotic pathway and the mitochondrial pathway are activated by H(2)S.
Assuntos
Apoptose/fisiologia , Fibroblastos/patologia , Gengiva/metabolismo , Halitose/metabolismo , Boca/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Caspases/metabolismo , Humanos , Sulfeto de Hidrogênio/metabolismo , Queratinócitos/metabolismo , Periodonto/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
INTRODUCTION: Untreated dental caries will eventually lead to pulpal inflammation. Although much is known regarding the interaction of microbial antigens and the immunologic defense systems of pulp, many aspects of the pathogenesis of pulpitis are not fully understood. The relationship between human pulp stem cells (HPSCs) and the pathogenesis of pulpitis remains among the poorly understood areas. Many of the invading bacteria are known to produce considerable amounts of hydrogen sulfide (H(2)S), which causes apoptosis in some tissues. The aims of this study were to determine whether H(2)S causes apoptosis in HPSCs and to examine its signaling pathway. METHODS: Stem cells were isolated from human dental pulp and incubated with 50 ng/mL H(2)S for 48 hours. To detect apoptosis, the cells were analyzed by using flow cytometry. The mitochondrial signaling pathway was examined by determining mitochondrial membrane depolarization. Activation of the key apoptotic enzymes caspase-9, caspase-8, and caspase-3 was assessed by using enzyme-linked immunosorbent assay. Release of cytochrome C from mitochondria was also determined. RESULTS: The number of apoptotic cells increased significantly with H(2)S treatment from 1.6% to 16.3% (P < .01). Significant increases were also measured in the amounts of caspase-9 and caspase-3 and in cytochrome C release (all P < .01) and in mitochondrial membrane depolarization (P < .05), whereas caspase-8 activity was not found. CONCLUSIONS: H(2)S causes apoptosis in HPSCs by activating the mitochondrial pathway. It is suggested that H(2)S might be one of the factors modifying the pathogenesis of pulpitis by causing loss of viability of HPSCs through apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Polpa Dentária/citologia , Sulfeto de Hidrogênio/efeitos adversos , Células-Tronco/efeitos dos fármacos , Benzimidazóis , Carbocianinas , Caspase 3/análise , Caspase 8/análise , Caspase 9/análise , Contagem de Células , Técnicas de Cultura de Células , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Corantes , Citocromos c/análise , Polpa Dentária/efeitos dos fármacos , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Imuno-Histoquímica , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
We report a clinical trial of the effects of test tablets containing bovine lactoferrin and lactoperoxidase on oral malodor and salivary bacteria. Fifteen subjects with volatile sulfur compounds (VSCs) in mouth air above the olfactory threshold (H(2)S >1.5 or CH(3)SH >0.5 ng/10 ml) as detected by gas chromatography were enrolled in the trial. Either a test or a placebo tablet was ingested twice at 1-h intervals in two crossover phases. Mouth air was monitored for VSC levels at the baseline before ingestion of a tablet, 10 min after the first ingestion, 1 h (just before the second ingestion), and 2 h after the first ingestion. Whole saliva was analyzed at the baseline and at 2 h for bacterial numbers. At 10 min, the level of CH(3)SH was significantly lower in the test group (median [interquartile range] = 0.28 [0.00-0.68] ng/10 ml) compared to that in the placebo group (0.73 [0.47-1.00] ng/10 ml; P = 0.011). The median concentration of CH(3)SH in the test group was below the olfactory threshold after 10 min until 2 h, whereas the level in the placebo group was above the threshold during the experimental period. No difference in the numbers of salivary bacteria was detected by culturing or quantitative PCR, but terminal restriction fragment length polymorphism detected one fragment with a significantly lower copy number at 2 h in the test group (mean ± standard error, 4.89 ± 0.11 log(10) copies/10 µl) compared to that in the placebo group (5.38 ± 0.15 log(10) copies/10 µl; P = 0.033). These results indicate a suppressive effect of the test composition on oral malodor and suggest an influence on oral bacteria.