Oral malodorous compounds are periodontally pathogenic and carcinogenic

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1 Japanese Dental Science Review (2008) 44, available at journal homepage: REVIEW ARTICLE Oral malodorous compounds are periodontally pathogenic and carcinogenic Ken Yaegaki * Department of Oral Health, Nippon Dental University, Fujimi, Chiyoda-ku, Tokyo , Japan Received 31 March 2008; received in revised form 17 June 2008; accepted 19 June 2008 KEYWORDS Hydrogen sulfide; Cancer; Methyl mercaptan; Apoptosis; Halitosis Summary Volatile sulfur compounds (VSCs), mainly composed of hydrogen sulfide (H 2 S) and methyl mercaptan (CH 3 SH), cause halitosis. VSCs increase the permeability of a model for gingival crevicular epithelia, causing an increase in the penetration of lipopolysaccharide, as well as prostaglandin, into the tissue. VSCs inhibit the proliferation of human gingival fibroblasts (HGF), human gingival epithelial cells (HGEC) and osteoblasts. H 2 S also causes apoptosis in HGF and HGEC. Furthermore, VSCs increase collagen degradation and reduce collagen synthesis in HGF and suppress wound healing, especially the formation of basal membrane and Type IV collagen synthesis. Moreover, VSCs stimulate interleukin-1 production, resulting in an increase in prostaglandin E and matrix metalloproteinase 1. Thus, VSCs may induce the initial damage to the epithelial barrier in the progression of periodontal disease. VSCs, especially H 2 S, strongly inhibit cytochrome c oxidase, which is a key enzyme for oxidative phosphorylation in the respiratory chain. Therefore, high concentration of VSCs causes lethal toxicities as well as cyanide. On the other hand, very low concentration of H 2 S at lower concentration in periodontal pocket causes genomic DNA damages in both HGF and HGEC. It has been suggested that VSCs may be one of the contributing factors for carcinogenesis because of increasing oxidative stress, and as the Ras/mitogen activated protein kinase signaling pathway, which is constitutively activated in many types of cancer, is enhanced. # 2008 Japanese Association for Dental Science. Published by Elsevier Ireland. All rights reserved. Contents 1. Introduction Toxicity of VSCs in general conditions Carcinogenicity of VSCs Effects of VSCs on apoptosis and cell proliferation Effects of VSCs on the permeability of the mucosa Effects of VSCs on collagen synthesis and degradation Effects of VSCs on immunological activities Effects of VSCs on the cytoskeleton and cell migration * Tel.: ; fax: address: yaegaki-k@tky.ndu.ac.jp /$ see front matter # 2008 Japanese Association for Dental Science. Published by Elsevier Ireland. All rights reserved. doi: /j.jdsr

2 Toxicities of oral malodorous compounds Conclusion Acknowledgements References Introduction An oversupply of dentists has become a big topic in Japanese dental society, where only 16% of the population receive regular dental examinations [1], while 66% of the population in USA visit dentists at least once a year [2]. The shortage of dental practitioners in the USA started several years ago, although the number of practitioners per capita is similar to the Japanese number [3]. People s attitudes and behaviors towards their own oral health are transformed by oral health promotion, and dentistry in developed countries has thus achieved social recognition. Dental research contributes greatly to oral health promotion, but a particular area of research that is generally disregarded by dental researchers the very one that, ironically, rouses much public interest in dentistry is halitosis, which can provide excellent educational material to encourage people to visit dentists regularly. Oral malodor is caused by volatile sulfur compounds (VSCs) such as hydrogen sulfide (H 2 S) and methyl mercaptan (CH 3 SH). Yaegaki and Sanada [4] have proved that H 2 Sisa main cause of physiological halitosis, while CH 3 SH is responsible for oral pathologic halitosis caused by periodontal conditions. The guidelines of halitosis treatment have been ascertained by Miyazaki et al. [5], and halitosis clinics have been well established for the past decade [6]. Consequently, clinicians interest in halitosis has increased; however, it is not yet generally known that VSCs are toxic to human tissues, especially periodontal tissue, as demonstrated by Tonzetich and co-workers [7]. Periodontitis is initiated by the invasion of periodontal pathogens and/or the penetration of microbial products into the gingival tissues, accompanied by a series of inflammatory reactions by host factors. The host factors of periodontitis are clearly identified, but the etiology of periodontitis has not yet been explicitly illustrated, especially at its initial stage [7]. It has been recently elucidated that VSCs reduce the effectiveness of the gingival epithelial tissues that normally play a key role as a barrier against penetration and invasion by detrimental compounds and microorganisms [8 25]. Some epidemiological and clinical studies have demonstrated that H 2 S is related to general conditions. Specifically, a high concentration of H 2 S in the colon has been reported as being one of the most likely causes of colon cancer [26]. We have recently found that H 2 S existing in periodontal pockets inhibits a superoxide scavenger that prevents oxidative damage of the tissues, and thus it causes genomic DNA damage [27]. In this review, the toxic effects of VSCs on human tissues are described. 2. Toxicity of VSCs in general conditions As the toxicity of VSCs, especially H 2 S, is similar to that of hydrogen cyanide, VSCs toxic effects on human tissue are well documented. H 2 S is recognized to be a potent inhibitor of cytochrome c oxidase (COX), which is a key enzyme for oxidative phosphorylation in the respiratory chain in the mitochondria to produce adenosine triphosphate (ATP) [28]. COX inhibition by H 2 S has been observed not only in vivo for some tissues, but also in purified COX from mitochondria [28,29]. The key mechanism by which H 2 S inhibits COX is in its binding to the heme iron of the enzyme, thus completely inhibiting the aerobic metabolism producing ATP [29]. Exposure to H 2 S can also occur in some occupational accidents or by means of environmental pollution. Concentrations over 500 ppm result in immediate death caused by cardiac arrhythmia and respiratory paralysis [30]. Healthy human subjects show blood sulfide ranging from 10 to 100 mmol/l, equivalent to ppm in blood [31], although the free sulfide in the ultrafiltered serum was not detected [32]. As 60 ppb 0.6 ppm of sulfide were demonstrated in the culture medium utilized for experiments that examined the adverse effects of VSCs on human cell cultures, these studies, which were discussed in this paper, could simulate of the conditions of human tissues [8 25]. Total VSCs or H 2 S concentration in mouth air from healthy subjects is reported as a maximum of 2 ppm or 1 ppm, while several times this concentration is found in periodontal patients [4,33,34]. In a group of asthmatics exposed to 2 ppm hydrogen sulfide for 30 min, airway resistance increased, implying bronchial obstruction [35]. Furthermore, a longitudinal study demonstrated the experience of general symptoms in subjects exposed to ambient-air malodorous sulfur compounds (reference: daily mean H 2 S concentration is <7 ppb; medium exposure: 7 21 ppb H 2 S; high exposure: >21 ppb H 2 S). The incidence of eye, respiratory and central nervous symptoms was significantly higher on days of medium and high exposure compared with the reference [36]. Eye injury was also documented; acute exposure to 25 ppb H 2 S is the lowest concentration to irritate the eyes; with chronic exposure, serious eye effects are possible [37]. These results imply that much lower levels of H 2 S than the concentration in mouth air cause exposure-related adverse effects. Therefore, VSCs in mouth air are very likely to cause unpleasant outcomes, but no clinical study has reported the relationship between inhaled VSCs in mouth air and these conditions. On the other hand, the mass absorption of VSCs was found from periodontal pocket into oral tissues [7 9],and it is expected that absorbed VSCs will be carried by blood as well as VSCs produced in the colon. However, VSCs absorbed by the colon carried into the liver where the toxic compounds are detoxified, whereas VSCs absorbed by oral tissues are directly carried to the other organs including heart or brain. Further studies for the toxicities of VSCs produced in the oral cavity would be required. 3. Carcinogenicity of VSCs H 2 S causes genomic DNA damage in human tissues [27]. DNA damage in HGF and human gingival epithelial cells (HGEC) exposed to 100 ng/ml H 2 S were detected by single-cell gel electrophoresis assay (SCGE) (CometAssy TM, Trevigen,

3 102 K. Yaegaki Gaithersburg, MD, USA). The basis of SCGE is the migration of DNA in an agarose gel under electrophoretic conditions. Intact DNA migrates more slowly and remains within the nucleoid, whereas damaged DNA migrates faster. When viewed under a microscope, a cell has the appearance of a comet, with a head (the nuclear region) and a tail containing DNA fragments. Evaluation of the DNA comet tail shape allows for assessment of DNA damage. Therefore, the DNA damage caused by H 2 S was determined by assessing tail length (=whole length of comet length of head), %DNA in the tail (=tail area/(tail area + head area) 100), and tail moment (=tail length %DNA in the tail). In HGF, a significant difference was found between the test groups in tail length and tail moment ( p < , respectively) as well as in DNA in the tail ( p < 0.05). (Fig. 1). Similar results were also demonstrated in HGEC (data are not shown). Matias et al. [38] have shown genotoxicity of H 2 S in several kinds of cell lines using SCGE, and have suggested that H 2 S-induced DNA damage might be mutagenic or carcinogenic, or play a role in chromosomal instability formation. Yang and Wong [39] have indicated that the effects of H 2 S on cell function have two aspects, apoptosis and cell-proliferation, indicating its carcinogenicity. In comparison to the sulfur concentration in the periodontal pocket, much higher concentration of H 2 S, over 1000 ppm greater than the lethal dose exists in the colon [40 42]. Colonic epithelium cope with this toxicity by methylation and de-methylation [42,43]. The detoxification activity of H 2 S in the colonic mucosa is also much higher than in other tissues, therefore the colonic mucosa protects itself from severe injuries such as ulcerative colitis [42 46]. Ulcerative colitis is recognized as a pre-cancerous condition, and this condition is linked to increased fecal production of VSCs as potential causative agents [47,48]. Thiols react with sulfhydryl-containing compounds to form persulfides, and then alter cell metabolism including VSCs detoxification; consequently a defect in VSCs metabolism may allow accumulation of VSCs, with resultant tissue damage [42]. Incubation of colonic mucosa with H 2 Sproduced Figure 2 Possible mechanisms of carcinogenicity of H 2 S [26,45 50]. significant damage including apoptosis, loss of goblet cells, and superficial ulcerations [49]. H 2 S concentration in feces from subjects at high risk of sigmoid colon cancer was significantly higher than in the control group [41]. Proliferation of colonic mucous cells was accelerated by H 2 S, and controlled by the Ras family that consists of Ha-ras, Ki-ras, and N-ras genes [50,51]. In fact, activated Ki-ras was frequently found in human colorectal cancers [51]. As Ras activates mitogen activated protein kinase (MAPK) which affects its downstream pathways, Ras oncogene is implicated in colorectal carcinogenesis [52]. Possible mechanisms have been illustrated for the subcellular explanation of hydrogen sulfide s carcinogenicity (Fig. 2) [26,45 50]. Deplancke and Gaskins have found that H 2 Scausesareduced redox environment in intestinal cells, which triggers the Ras/MAPK pathway (Fig. 3) [26]. Similar carcinogenicity in oral tissues should be investigated in further studies. Superoxide dismutase (SOD) is a critical enzyme responsible for the elimination of superoxide radicals. Accumula- Figure 1 Quantitation of DNA strand breaks caused by H 2 S incubation [27]. The cells were incubated with either 5% CO 2 in air with H 2 S (100 ng/ml) or 5% CO 2 in air alone for 72 h. DNA strand breaks were detected using the CometAssay TM. Data were obtained from six independent experiments, 75 nuclei per experiment. Asterisk indicates a statistically significant difference between the test treated with H 2 S and the control ( p < ). Figure 3 Hypothesis of carcinogenesis mechanism by high concentration of H 2 S through Ras/MAPK pathway, modified from Deplancke and Gaskins [26]. Dependent on genetic background, this event may result in hyperproliferation, ulcerative colitis and colorectal cancer [26]. (Reproduced with the permission of Dr. Rex H. Gaskins.)

4 Toxicities of oral malodorous compounds 103 tion of reactive oxygen species (ROS) results in cellular oxidative stress; if the stress is not corrected in time, it can lead to the damage of important biomolecules such as membrane lipids, proteins and DNA. Prolonged accumulation of free radicals may cause irreversible cellular injury. SOD is the key enzyme in the first metabolic step of superoxide elimination. Deficiency in SOD or inhibition of its activity may cause severe accumulation of O 2 or H 2 O 2 in cells. We recently found that very low concentrations of H 2 S, much lower than those found in human periodontal pockets, inhibit SOD activity in HGF, as well as Cu, Zn SOD and Mn SOD activities [27]. We also determined that H 2 S elevates the level of ROS, which may cause oxidative stress that is associated with cancer or aging [27]. 4. Effects of VSCs on apoptosis and cell proliferation VSCs induce cell death in gingival tissue; a large number of dead or impaired cells were found in an epithelial model of gingival crevices after exposure to VSCs [9]. VSCs also delay wound healing. CH 3 SH has been found to delay wound healing in rats; furthermore, in a wound treated with CH 3 SH in an epithelial model of gingival crevices, no formation of Type IV collagen was found [19]. The effects of VSCs on the proliferation of both fibroblasts and epithelial cells have been studied. It was found that VSCs adversely affect only certain types of fibroblasts, which may indicate that there is a selection process for particular fibroblast cell populations [14]. The proliferation of both HGF and HGEC was inhibited by VSCs [19,20,53]. We have recently found that H 2 S caused apoptosis in HGF (Fig. 4) [27]. Apoptosis in HGEC was also caused by H 2 S; moreover, H 2 S induced cell-cycle arrest via the expression of p21 in HGEC [53,54]. The upper stream of the signaling pathway involving Caspase 3 may be activated, because H 2 S significantly elevates the level of the enzyme [27]. It has also been suggested that H 2 S propels the cell toward apoptotic death triggered initially by stabilization of p53 and that subsequently involves a cascade of downstream products [55]. In the early stages of periodontitis, the number of apoptotic cells among periodontal ligament fibroblasts increases significantly [56]. Apoptosis has also been found in these tissues in human aggressive or severe periodontitis [57 60]. Periodontal pathogenic microorganisms play etiological roles in the apoptotic process of periodontal tissues [58,60]. Lipoprotein of Bacteroids forsythus also induced apoptosis in HGF and human gingival keratinocytes [61]. A signaling pathway of apoptosis caused by lipoprotein was mediated by Toll-like receptor 2 [62]. In this process, NF-kappa B was partly activated via the PI3 kinase/akt pathway in HGF after Porphyromonas gingivalis infection [60]. At the later stages of infection, the antiapoptotic genes were largely shut down and the pro-apoptotic genes were activated [63]. Therefore, apoptosis occurring in periodontal tissues is considered to be one of the etiologic processes of periodontitis. To investigate the effect of oral malodorous compounds on the proliferation of osteoblasts and on a signaling transduction pathway through the MAPK cascade, normal human osteoblasts (NHOst) and murine osteoblasts (MC3T3-E1), were cultured in the presence of H 2 S. Cell proliferations of both NHOst and MC3T3-E1were significantly suppressed by H 2 S, furthermore, the phosphorylation of ERK1/2 and p38 increased as the H 2 S concentration increased (data are not shown). 5. Effects of VSCs on the permeability of the mucosa Basal membrane of epithelium is the barrier against the penetration of tumor cells into subcutaneous tissue. In periodontal tissue, the onset of periodontal disease follows initial damage to the gingival sulcular epithelial barrier [7]. VSCs increased permeability in porcine sublingual nonkeratinized mucosa, a model for crevicular epithelia (Fig. 5) [8]. A possible mechanism is that VSCs induce de-aggregation of proteoglycans, which are elements of the extracellular matrix of connective tissue, because sulfide cleaves disulfide bonds [8]. The effect of VSCs on the permeability of lipopolysaccharide (LPS) in tissues was studied. There was a large variation in the ability of LPS to diffuse through the tissues Figure 4 Ratio of early apoptosis induced by H 2 S [27]. The cells were incubated with either 5% CO 2 in air with H 2 S (100 ng/ml) or 5% CO 2 in air alone for the times indicated. Cells were stained with annexin V and propidium iodide. Apoptotic cells were detected by flow cytometry. Asterisk indicates a statistically significant difference between treatment with H 2 S and the control ( p < 0.05). Figure 5 Percentage increase in permeability of oral mucosa subjected for various periods of time to H 2 SorCH 3 SH [5]. (a) 15 ng H 2 SorCH 3 SH per ml 95% air/5% CO 2, (b) 95% air/5% CO 2.

5 104 K. Yaegaki Table 1 Increase in diffusion of [ 14 C]-LPS through mucosa pretreated with CH 3 SH 9 Average dpm of [ 14 C]-LPS Diffused through mucosa Unit 1 Unit 2 Test Control Test Control Each value represents an average of three separate experiments. Each unit originated from different sources. Test tissues were exposed to 10 ng CH 3 SH/ml air/co 2 for 3 h. Then, they were overlayed with 14 C-LPS for 3 h. Table 2 Effects of VSC on permeability of mucosa to PGE 2 [8,9] System %Change in permeability Control a 7.7 CH 3 SH-treated b c CH 3 SH/ZnCl a Mucosa was exposed to 95% air/5% CO 2 atmosphere for 60 min. b Mucosa was exposed for 60 min to 15 ng CH 3 SH/ml 95% air/5% CO 2 atmosphere. c Following 60 min to 15 ng CH3SH/ml 95% air/5% CO2 atmosphere, mucosa was treated for 15 min with 0.22% ZnCl 2 solution. due to the different thicknesses of mucosa and/or different tissue sources. 9) Taking this variation into account, the pretreatment of mucosa with CH 3 SH increased its permeability to LPS 2 3-fold (Table 1) [9]. The CH 3 SH-exposed epithelial surface of mucosa showed a markedly rough surface caused by cell exfoliation and dissolution of the intercellular matrix [9]. P. gingivalis, which has been implicated in the etiology of periodontal disease, produces a large amount of CH 3 SH [10]. P. gingivalis also produces vesicles involving proteolytic activities [9]. It has been demonstrated that CH 3 SH enhances the activity of vesicles in digesting Type IV and Type I collagen, as collagenase activity is thiol-dependent [9]. The permeability of tissues to prostaglandin (PG) E 2 increased by 66% after exposure to CH 3 SH (Table 2) [9]. It is suggested that PGE 2 that is diffused into the tissues may have a role in the suppression of collagen synthesis, or may augment the production of endotoxin-induced collagenase [9]. Accordingly, an increment in the permeability of the sulcular tissues induces damage to the epithelial barrier in the process of periodontal disease. Zinc ions are considered to be one of the most effective deodorants for oral malodor. A zinc-containing mouthwash reduces VSCs concentration by more than 90% for 3 h; zinc inhibits cysteine proteinase activity, which plays a key role in VSCs production [11]. Interestingly, the treatment of mucosa with 0.22% zinc chloride nullified the effect of VSCs and restored tissue permeability to its previous state (Table 2) [9]. Zinc also inhibited the destruction of Type I collagen by both CH 3 SH and P. gingivalis vesicles [9]. Hence, zinc ions appear to act not only as a deodorant but also as a protective agent for the maintenance of the permeability barrier. in gingival fibroblasts exposed to CH 3 SH was suppressed by 44.1% [14]. Further studies also indicated a reduction in proline transport in the range of 40 50% in cultures exposed to CH 3 SH [13,14]. The conversion of proline to hydroxyproline was measured to assess the effect of CH 3 SH on the intra- and extracellular metabolism of collagen in human gingival fibroblast cultures, utilizing the methods of Yaegaki et al. [17]. After a 30-min pulse, CH 3 SH had suppressed collagen synthesis by approximately 40% and increased the intracellular degradation of newly synthesized collagen markedly (Figs. 6 and 7) [16]. An increase in the intracellular degradation of collagen was observed in both 30-min and 12-h pulsed cultures (Fig. 7) [16]. Extracellular degradation of collagen in 12-h pulsed cultures was also increased over 13-fold (Fig. 7) [16]. The CH 3 SH-exposed cultures had a 70% reduction in collagen, resulting from the combined effects of suppressed synthesis and increased degradation [14]. CH 3 SH induced reductions in both a1 and a2 chains of Type I collagen and 6. Effects of VSCs on collagen synthesis and degradation VSCs have a detrimental effect on the integrity of collagen. The H 2 S and CH 3 SH thiol groups combine with Type I collagen, and H 2 S is incorporated directly into the peptide chains [12,13]. Intermolecular cross-linkages of Type I acid-soluble collagen are cleaved by VSCs, and it reverts to a more soluble molecule [14,15]. It is concluded that exposure to CH 3 SH causes an alteration in collagen metabolism. VSCs can modulate the metabolism of gingival fibroblasts. It has been found that the protein content of CH 3 SH-exposed cell cultures was decreased by approximately 25 35% [14,16]. This deleterious effect was irreversible in test cultures subsequently incubated in a VSCs-free environment [14]. DNA synthesis Figure 6 Methyl mercaptan-induced suppression of collagen synthesis by human gingival fibroblast cell cultures [16]. (a) Results are derived from triplicate samples with approximately 150,000 dpm total in controls. Data indicate percent reduction in radioactivity of CH 3 SH-exposed cultures when compared with corresponding controls exposed to air/co 2. (b) All [ 14 C]-OH-Pro activity was located within the cells during the 30 min pulse period. The reduction, differences between test and control, were analyzed by Student s t-test, and significant differences at p < were found except the media of 30 min incubation study.

6 Toxicities of oral malodorous compounds Effects of VSCs on immunological activities Figure 7 Increased intracellular and extracellular degradation in CH 3 SH-exposed fibroblast cell cultures [16]. (a) Results are derived from triplicate samples with approximately 150,000 dpm total in controls. Data compare percent reduction in radioactivity of CH 3 SH-exposed cultures with corresponding controls exposed to air/co 2. (b) All [ 14 C]-OH-Pro activity was shown to be retained in the cells during the 30 min pulse period. Differences between test and control were analyzed by Student s t- test, and significant differences at p < were found except the media of 30 min incubation study. in Type III procollagen. It was suggested that mrna synthesis for Type III procollagen was suppressed in cultures exposed to CH 3 SH because of probably decreased transcription of mrna [16]. Collagen loss in periodontally involved tissue causes periodontal manifestations. Adverse effects of VSCs on collagen metabolism of HGF may therefore have an important role in periodontal etiology. VSCs concentration in mouth air increases with the severity of the periodontal condition. High concentrations of VSCs in mouth air were found in periodontal subjects [64]. The CH 3 SH concentration or CH 3 SH/H 2 S ratio is significantly higher in patients with periodontal disease than in controls (Fig. 8) [4]. This is because there is an elevated concentration of methionine in the gingival sulcular fluid as well as a higher level of CH 3 SH production by periodontal pathogens [64,65]. One would therefore expect CH 3 SH rather than H 2 S to be more toxic in a progressed pathological condition within the pocket. The degenerative changes in periodontal tissue are attributable to a combination of the presence of microorganisms and of the host s immune factors [66]. In addition to the direct effects of microorganisms, microbiota damage the host cells indirectly by activating the host s immune response. Matrix metalloproteinases (MMPs) are involved in the progress of periodontitis. One of the most characteristic changes exerted by MMP is the degradation of Type I collagen, which is the principal component of the extracellular matrix. This destruction is due to increased levels of collagenases, whose activities are increased through activation of the immuneresponse system [66]. LPS stimulates mononuclear cells to produce increased amounts of interleukin-1 (IL-1), which induces the production of MMP. In periodontitis, IL-1 is found at physiologically significant concentrations in gingival crevicular fluid and in gingival tissue [67,68]. Moreover, the production level of IL-1 in gingival crevicular fluid is correlated with the severity of the disease [69]. Thus, higher concentration of IL-1 increases the production of camp, PGE 2, collagenase and plasminogen activator; and PGE 2 mediated with camp promotes bone resorption and stimulates MMP activity (Fig. 9) [18,70,71]. Mononuclear cell cultures were stimulated with LPS in thepresenceorabsenceofch 3 SH, and the IL-1-dependent cellular proliferation was determined. The supernatants derived from cells exposed to CH 3 SH induced a twofold higher stimulation index than supernatants of control cell systems [18]. The amount of IL-1 increased more in the presence of LPS and CH 3 SH than with LPS alone [18]. CH 3 SH alone was also found to increase the production of IL-1 [18]. It has been shown that CH 3 SH stimulates PGE production by gingival fibroblasts. The effect of CH 3 SH was scarcely noticeable during initial exposure to CH 3 SH, but a longer exposure significantly increased PGE production, both in thepresenceandabsenceofil-1[18]. The effect of CH 3 SH on camp production was also ascertained. CH 3 SH-supplemented cultures increased the amount of camp as well as cultures supplemented with IL-1, LPS or IL-1/LPS. CH 3 SH aloneincreasedtheproductionofcollagenase.incombina- Figure 8 Periodontitis and CH 3 SH/H 2 S ratio in mouth air [6]. 3 mm; n = 9, 4 mm; n = 6, 5 mm; n =8,6mm; n =8. Figure 9 [70,71]. The relationship between IL-1 and periodontitis

7 106 K. Yaegaki tion with IL-1, LPS, or IL-1/LPS, CH 3 SH enhanced collagenase production significantly in compared with these compounds alone. As cathepsin B requires thiol, it can be speculated that CH 3 SH also enhances the activity of this enzyme [18]. The above findings demonstrate that CH 3 SH is effective in augmenting the LPS activation of IL-1 production by mononuclear cells, and that CH 3 SH acts both directly and via IL-1 on HGF by enhancing the secretion of PGE, camp and collagenase. It is also suggested that CH 3 SH plays a part in the tissue destruction of periodontitis both by an indirect effect through mononuclear cells and by a direct effect on fibroblasts as described above. Leukocytes migrate into the periodontal pocket and produce active oxygen, which is very toxic to human gingival tissues. The effect of active oxygen production in human polymorphonuclear leukocytes has been determined by measuring chemiluminescence [19]. With Fusobacterium stimulation, production of active oxygen was dramatically increased by the presence of CH 3 SH [19]. 8. Effects of VSCs on the cytoskeleton and cell migration The cytoskeleton consists of microfilaments (actin microfilaments), microtubules and intermediate filaments. The cytoskeleton regulates cell shape, mitosis, motility, integrity, endocytosis and intracellular transport. The tight junctions of cells allow compartments with different solute composition to be separate, and this controlled permeability of intracellular space is important not only in preventing microbial invasion but also in cell functions. The permeability is controlled by both internal and extracellular factors, and one of the most important internal factors is the cytoskeleton. Actin filaments circumscribe the cell and fine filaments extend into the tight junction [72]. Within the tight junction, the actin filaments bind directly to the scaffolding proteins, bonding to extracellular compounds through the membrane [73]. The effects of CH 3 SH on the distribution of the cytoskeleton have been investigated [20]. The cells exposed to CH 3 SH were found to be rounder, less spread and less elongated than unexposed cells. Actin microfilament bundles in CH 3 SH-treated cells were found predominantly in the margins of the cell, as opposed to those in control cells that were found in uniform distribution over the entire cell. Microtubules in control cells were uniformly distributed, whereas those in the CH 3 SH-treated cells were found only in the central portion of the cell [20]. VSCs also inhibited the migration of periodontal ligament cells, possibly due to these changes in the cytoskeleton and also to the reduction in fibronectin [22 25]. The signaling cascade is important in regulating the tight junction barrier through the cytoskeleton. Actin microfilaments are regulated through Ras/Rho family GTPases [74]. Because Ras signal transduction is altered by VSCs, it has been suggested that the effect of VSCs on the signal cascade might lead to changes in cytoskeletons as described above [74]. Furthermore, it has been reported that CH 3 SH lowers the resting cytosolic ph, which may affect cell functions that are associated with the cytoskeleton [23,74]. 9. Conclusion It is suggested that VSCs are not only periodontal causative agents but also affect general conditions. In periodontal etiology, VSCs may play an important role in the initial stage of the pathology. Acknowledgements The author thanks Dr. Wei Qian, University of British Columbia, Dr. Yasuhiro Ikeda, Health Science University of Hokkaido, Dr. Tomoko Tanaka and Dr. Takatoshi Murata, Nippon Dental University for their assistance in preparing the manuscript, also Dr. Rex H. Gaskins, University of Illinois, for his advice regarding the carcinogenicity of H 2 S. 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