Takatoshi Murata

Oral malodorous compound triggers mitochondrial-dependent apoptosis and causes genomic DNA damage in human gingival epithelial cells

BACKGROUND AND OBJECTIVEnVolatile 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.nnnMATERIAL AND METHODSnHuman 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.nnnRESULTSnApoptosis 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.nnnCONCLUSIONnHydrogen sulfide induces apoptosis in human gingival epithelial cells by activating the mitochondrial pathway.

Oral Malodorous Compound Inhibits Osteoblast Proliferation

BACKGROUNDnOral malodorous compounds including hydrogen sulfide (H2S) are causative agents of periodontitis because the toxicities are similar to that of cyanate. Previous studies demonstrated that volatile sulfur compounds (VSCs) were highly toxic to periodontal tissues, causing a large reduction in the amount of collagen in human gingival fibroblasts and extracellular matrix as well as, for example, apoptosis, immunologic responses, and matrix metalloproteinase production. The objective of this study was to determine the effect of H2S on the proliferation of osteoblasts and a signaling transduction pathway through the mitogen-activated protein kinase (MAPK).nnnMETHODSnNormal human osteoblasts (NHOst) and murine osteoblasts (cell line MC3T3-E1) were incubated with H2S. Cell proliferation was assessed by measuring [3H]thymidine incorporation. The effects of H2S on the signal transduction pathways, the MAPK cascade, that control cell proliferation were evaluated in NHOst by determining extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation with a Western blot analysis.nnnRESULTSnAfter incubating NHOst with H2S for 24 hours, [3H]thymidine incorporation into the DNA significantly decreased dose-dependently with H(2)S. At a concentration of 100 ng/ml H2S, [3H]thymidine incorporation decreased 79% compared to the control. Similar results were obtained from MC3T3-E1. The phosphorylation of ERK1/2 and p38 was increased by H2S at 10 minutes after starting the treatment and then decreased time dependently. The activation of ERK1/2 and p38 induced by H2S was inhibited by the specific inhibitor of MAPK/ERK kinase ([MEK]; U0126) or p38 (SB203580).nnnCONCLUSIONnH2S inhibited the proliferation of human osteoblastic cells through the MAPK pathway.

Hydrogen sulfide induces apoptosis in epithelial cells derived from human gingiva.

Hydrogen sulfide (H(2)S) is not only one of the main causes of halitosis but is also an agent of toxicity against periodontal cells and tissues in biofilm-related periodontal diseases. Also, apoptosis of gingival epithelial cells may play an important role in the onset and progress of periodontitis. We examined the effect of H(2)S on the induction of apoptosis, using human gingival fibroblasts (HGF) and keratinocyte-like Ca9-22 cells derived from human gingiva. The cells were incubated with H(2)S (100 ng ml(-1)) for 24, 48 or 72 h by adding H(2)S to air containing 5% CO(2), supplied constantly to the culture environment during incubation. The incidence of apoptosis caused by H(2)S was determined with Annexin V staining by flow cytometry. The proportion of apoptotic cells was significantly increased by exposure to H(2)S for 48 h in comparison with the control in both Ca9-22 cells and HGF. A concentration of 100 ng ml(-1) H(2)S in air is possible in the gingival sulcus. This study indicates that apoptosis in gingival epithelial cells and HGF by H(2)S may occur in the oral cavity, which may cause a periodontal condition.

Inhibitory effects of MoAbs against a surface protein antigen in real-time adherence in vitro and recolonization in vivo of Streptococcus mutans.

A surface protein antigen (PAc) of Streptococcus mutans, particularly the A‐region of the molecule, has been reported to interact with salivary components on the tooth surface. It might be a candidate antigen inducing the production of antibodies against the adherence of S. mutans to the tooth surface. We investigated the effects of monoclonal antibodies (MoAbs) obtained by immunization of synthetic PAc peptides that completely correspond to the amino acid sequence of part of the A‐region. These MoAbs recognize several core B‐cell epitopes in the sequence. Two (KH5 and SH2) of these antibodies reacted with both S. mutans and Streptococcus sobrinus, but not with Streptococcus sanguis, Streptococcus salivarius, Porphyromonas gingivalis or Lactobacillus casei. They clearly inhibited the real‐time adherence of S. mutans to salivary components in a biosensor. KH5, which showed a real‐time inhibition (71%), also significantly prevented the recolonization of S. mutans on the tooth surface in rats. These results suggested that the core B‐cell epitope (‐Y‐‐‐L‐‐Y‐‐‐‐) recognized by KH5 was the essential sequence in the antigenic epitopes of PAc protein recognized specifically by the inhibitory antibody. Therefore, the amino acid residues were found to be important in the initial attachment of S. mutans to the tooth surface. These results provide for the mechanism of PAc molecule in the initial attachment of S. mutans on the tooth surface and more effective designs for the removal of S. mutans and S. sobrinus from the oral cavity.

Extracts of Actinobacillus actinomycetemcomitans Induce Apoptotic Cell Death in Human Osteoblastic MG63 Cells

Whether an extracellular component of periodontal-disease-causing bacteria induces apoptotic cell death in bone-related cells is unknown. To study the effects on osteoblasts of extracts obtained from sonicated Actinobacillus actinomycetemcomitans and Prevotella intermedia, we cultured human osteoblastic cell lines MG63 and Saos-2 cells and mouse osteoblastic cell line MC3T3-E1 cells in the presence of such extracts. The addition of the extracts from Actinobacillus actinomycetemcomitans induced cell death in MG63 cells in a dose- and time-dependent fashion over the concentration range of 0.1 to 10 μg/mL. By contrast, the extracts from Prevotella intermedia did not induce cell death in these cells, even in the presence of 10 μg/mL protein. By using the Hoechst 33342 staining technique, we observed marked nuclear condensation and fragmentation of chromatin in MG63 cells treated with the extracts of Actinobacillus actinomycetemcomitans. DNA ladder formation, a hallmark of apoptosis, also was detected in MG63 cells treated with extracts from Actinobacillus actinomycetemcomitans. In MG63 cells, DNA ladder formation was dose-dependent, with a maximal effect at a concentration of 10 μg/mL, and time-dependent, from 12 to 48 hrs. However, the extracts from Prevotella intermedia did not induce DNA fragmentation in MG63, Saos-2, or MC3T3-E1 cells. The extracts from Actinobacillus actinomycetemcomitans did not induce cell death and DNA fragmentation in Saos-2 and MC3T3-E1 cells. Sonicated extracts of Actinobacillus actinomycetemcomitans that had been treated with heat and trypsin did not induce DNA ladder formation in MG63 cells, suggesting that the apoptosis-inducing factors are proteinaceous. Cycloheximide prevented the Actinobacillus actinomycetemcomitans-induced DNA ladder formation in MG63 cells in a dose-dependent fashion, suggesting that new gene transcription and protein synthesis are regulated for Actinobacillus actinomycetemcomitans-induced apoptosis in MG63 cells. Our results indicate that apoptosis in alveolar bone cells induced by Actinobacillus actinomycetemcomitans plays an important role in periodontal diseases.

Effects of a composition containing lactoferrin and lactoperoxidase on oral malodor and salivary bacteria: a randomized, double-blind, crossover, placebo-controlled clinical trial

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 (H2S >1.5 or CH3SH >0.5xa0ng/10xa0ml) 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, 10xa0min after the first ingestion, 1xa0h (just before the second ingestion), and 2xa0h after the first ingestion. Whole saliva was analyzed at the baseline and at 2xa0h for bacterial numbers. At 10xa0min, the level of CH3SH was significantly lower in the test group (median [interquartile range]u2009=u20090.28 [0.00–0.68]u2009ng/10xa0ml) compared to that in the placebo group (0.73 [0.47–1.00]u2009ng/10xa0ml; Pu2009=u20090.011). The median concentration of CH3SH in the test group was below the olfactory threshold after 10xa0min until 2xa0h, 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 2xa0h in the test group (mean ± standard error, 4.89u2009±u20090.11 log10 copies/10xa0µl) compared to that in the placebo group (5.38u2009±u20090.15 log10 copies/10xa0µl; Pu2009=u20090.033). These results indicate a suppressive effect of the test composition on oral malodor and suggest an influence on oral bacteria.

Oral malodorous compound activates mitochondrial pathway inducing apoptosis in human gingival fibroblasts.

Hydrogen sulfide (H2S) is a main cause of physiologic halitosis. H2S induces apoptosis in human gingival cells, which may play an important role in periodontal pathology. Recently, it has been reported that H2S induced apoptosis and DNA damage in human gingival fibroblasts (HGFs) by increasing the levels of reactive oxygen species. However, the mechanisms of H2S-induced apoptosis have not been clarified in HGFs. The objective of this study was to determine the apoptotic pathway activated by H2S in HGFs. The HGFs were exposed to 50xa0ng/mL H2S, resulting in 18xa0ng/mL in the culture medium, which is lower than the concentration in periodontal pockets. The number of apoptotic cells after 24 and 48xa0h incubation was significantly higher than that in the control cultures (pu2009<u20090.05). Mitochondrial membrane depolarization and the release of cytochrome c, and caspase-3, and caspase-9 were also significantly increased after both 24- and 48-h incubation (pu2009<u20090.05), whereas caspase-8, a key enzyme in the receptor ligand-mediated pathway causing apoptosis, was not activated. The present study shows that H2S triggered the mitochondrial pathway causing apoptosis in HGFs but did not activate the receptor ligand-mediated pathway.

Transblot identification of avidin-interacting proteins in rat salivary glands.

Blotting of rat parotid gland proteins separated by SDS‐PAGE and transferred to Immobilon transfer membranes revealed that avidin‐peroxidase conjugate interacted with bands having estimated molecular weights of 72, 74, and 120 kDa. Even at the lowest concentration of avidin‐peroxidase used in the general ABC method (1:2000 dilution), three bands were clearly discernible. The staining reaction of parotid gland proteins was eliminated on preincubating the proteins with native avidin. The staining reaction was markedly reduced with the proteins obtained from submandibular/sublingual glands.