Hydrogen sulphide increases hepatic differentiation of human tooth pulp stem cells compared with human bone marrow stem cells.

AIM: To determine the differences in stem cell properties, in hepatic differentiation and in the effects of hydrogen sulphide (H2 S) on hepatic differentiation between human bone marrow stem cells (hBMC) and stem cells from human exfoliated primary tooth pulp (SHED). METHODOLOGY: CD117(+) cells were magnetically separated and subjected to hepatic differentiation. CD117(+) cell lineages were characterized for transcription factors indicative of stem cells by qRT-PCR. For the last 9 days of the differentiation, the test cells were exposed to 0.1 ng mL(-1) H2 S. Immunocytochemistry and flow cytometry of albumin, alpha-fetoprotein and carbamoyl phosphate synthetase were carried out after differentiation. Urea concentration and glycogen synthesis were also determined. RESULTS: Genes expressed in SHED were also expressed in BMC. No difference in expression level of hepatic markers was shown by immunofluorescence. SHED showed more positive cells than hBMC (P < 0.01). H2 S increased the number of positive cells in both cultures (P < 0.01). Urea concentration and glycogen synthesis increased significantly after H2 S exposure (P < 0.001 and P < 0.05, respectively). Real-time PCR data were analysed by RT(2) profiler RT-PCR Array Data Analysis version 3.5 (Qiagen), and ELISA data were analysed by Bonferroni's multiple comparison using Windows spss version 16 (SPSS Inc, Chicago, IL, USA). Bonferroni's multiple comparison test was also carried out after angle transformation for the percentage data of flow cytometer using Windows spss(®) version 16 (SPSS Inc). Statistical significance was accepted at P < 0.05. CONCLUSIONS: Stem cells from human exfoliated primary tooth pulp and BMC have similar properties. The level of hepatic differentiation in SHED compared with BMC was the same or higher. H2 S increased the level of hepatic differentiation.

Halitosis management by the general dental practitioner--results of an international consensus workshop.

Clinical investigations on patients suffering from halitosis clearly reveal that in the vast majority of cases the source for an offensive breath odor can be found within the oral cavity (90%). Based on these studies, the main sources for intra-oral halitosis where tongue coating, gingivitis/periodontitis or a combination of the two. Thus, it is perfectly logical that general dental practitioners (GDPs) should be able to manage intra-oral halitosis under the conditions found in a normal dental practice. However, GDPs who are interested in diagnosing and treating halitosis are challenged to incorporate scientifically based strategies for use in their clinics. Therefore, the present paper summarizes the results of a consensus workshop of international authorities held with the aim to reach a consensus on general guidelines on how to assess and diagnose patients' breath odor concerns and general guidelines on regimens for the treatment of halitosis.

p53-Pathway activity and apoptosis in hydrogen sulfide-exposed stem cells separated from human gingival epithelium.

BACKGROUND AND OBJECTIVE: Hydrogen sulfide ( H2S ) is a volatile sulfur compound responsible for physiological halitosis. H2S was also reported as having periodontal pathologic activities. Gingival crevicular epithelium is the first barrier against periodontal pathogens and their products; oral keratinocyte stem cells OKSCs play key roles in maintaining this barrier. The p53 pathway is responsible for regulating key biological events. Increased apoptosis and cell-cycle arrest of DNA repair can affect keratinocyte stem cells, having a direct impact on the architecture of the oral epithelial tissue. However, the link between H2S , p53 activity and OKSCs has not yet been fully explored. The main objective of the present study was to explore the implications of the p53 pathway in OKSCs following exposure to H2S. MATERIAL AND METHODS: OKSCs were isolated from human gingival epithelium and incubated with physiological levels of H2S for 24 and 48 h. Apoptosis and the mitochondrial membrane potential were detected using flow cytometry. Cytochrome c, total p53, phosphorylated p53 and caspase activity were assessed using specific ELISAs. p53 Pathway gene activity was assayed using quantitative RT-PCR. RESULTS: The levels of apoptosis were significantly increased following incubation in the presence of H2S, especially after 48 h (36.95 ± 1.91% vs. 4.77 ± 0.74%). Caspases 9 and 3 were activated, whereas caspase-8 activity remained low. Total p53 activity and particularly phosphorylated p53 at serine 46, were significantly enhanced compared with controls (47.11 ± 9.84 units/mL vs. 1.5 ± 0 units/mL and 32.22 ± 10.23 units/mL vs. 0.15 ± 0 units/mL, respectively, at 48 h). Among p53 pathway genes, apoptosis-related genes [i.e. phosphatase and tensin homolog ( PTEN ), B-cell CLL/lymphoma 2 ( BCL2), sirtuin 3 ( SIRT3) and caspases]) were dramatically increased when compared with controls. Moreover, cell-cycle progression genes [i.e. E2F transcription factor (E2F) family and histone deacetylase ( HDAC )] and DNA-repair genes [i.e. growth arrest and DNA-damage-inducible, gamma ( GADD45G ) family and serine/threonine-protein kinase Chk2 ( CHEK2)] were also increased. CONCLUSION: Following incubation with H2 S , OKSCs express multiple p53-associated genes, including programmed cell death, cell-cycle control and DNA-repair genes.

Oral malodorous compound causes caspase-8 and -9 mediated programmed cell death in osteoblasts.

BACKGROUND AND OBJECTIVE: Hydrogen sulfide (H(2) S) is one of two volatile sulfur compounds that are known to be the main cause of oral malodor; the other is methyl mercaptan. Other known volatiles existing in mouth air do not contribute significantly to oral malodor originating in the oral cavity. Hydrogen sulfide is also known to be an etiological factor in periodontal disease. However, the effects of H(2) S on alveolar bone remain unclear. The objectives of this study were to determine the apoptotic effects of H(2) S on osteoblasts and to verify the apoptotic molecular pathways. MATERIAL AND METHODS: A clonal murine calvaria cell line was incubated with 50 ng/mL of H(2) S. To detect apoptosis, the cells were analysed by flow cytometry and ELISA. Mitochondrial membrane depolarization was assessed using flow cytometry as well. ELISA was used to evaluate the release of cytochrome c into the cytosol and to assess Fas ligand, p53, tumor necrosis factor α, interleukin IL1-α IL-ß, IL-2, IL-4, IL-10, interferon-γ, granulocyte-colony stimulating factor and granulocyte-macrophage colony stimulating factor. Caspase-3, -8 and -9 activities were estimated. Expression of BAX and Bcl-2 was assessed by real-time quantitative RT-PCR. DNA fragmentation was detected by single-cell gel electrophoresis. Fas receptors were evaluated by western blotting. RESULTS: After H(2) S incubation, apoptotic levels increased significantly in a time-dependent manner. Mitochondrial membrane depolarization, the release of cytochrome c, p53 and caspase-3, -8 and -9 and DNA fragmentation were all significantly greater. BAX gene activity was upregulated, whereas Bcl-2 remained low. Fas ligand/Fas receptor, tumor necrosis factor α and other cytokines were not increased to a significant degree. CONCLUSION: At less-than-pathological concentrations in gingival crevicular fluid, H(2) S induces apoptosis in osteoblasts. The molecular mechanisms underlying the apoptotic process include p53, a mitochondrial pathway and caspase-8 activation.

Characterization of oral keratinocyte stem cells and prospects of its differentiation to oral epithelial equivalents.

OBJECTIVE: Although oral keratinocyte stem cells play a key role in tissue homeostasis, wound healing, and neoplasia, they remain difficult to identify and characterize. The specific aim of the present study is to characterize an oral keratinocyte stem-cell population separated using a magnetic technique. MATERIAL AND METHODS: Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using a proliferation-related marker, CD71 and α6ß4 integrin. The expression of different stem cell markers: CD44H, Nestin, Nanog, Oct 3÷4, CD117 was checked by immunofluorescence. The ability of α6ß4pos CD71neg fraction to form oral epithelial equivalents was also assayed. RESULTS: Three different oral keratinocyte subpopulations were obtained following magnetic separation: α6ß4pos CD71neg, α6ß4pos CD71pos and α6ß4neg. Our α6ß4pos CD71neg stem cell fraction was positive for Oct 3÷4, CD44H and cytokeratin 19 while Nanog, Nestin and CD117 expression was absent. At the same time, the other two cell fractions α6ß4pos CD71pos and α6ß4neg were negative for all stem cell markers. Also, α6ß4pos CD71neg fraction was able to regenerate a well stratified and organized oral epithelial equivalent. The distribution of cytokeratin 19 and involucrin in the oral epithelial equivalent reflected the in vivo situation in oral gingival epithelium. CONCLUSIONS: The human gingival α6ß4pos CD71neg fraction was strongly positive for a panel of stem cell markers and could form oral epithelial equivalent. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.

Magnetic separation and characterization of keratinocyte stem cells from human gingiva.

BACKGROUND AND OBJECTIVE: Although keratinocyte stem cells play a key role in tissue homeostasis, wound healing and neoplasia, they remain difficult to identify and characterize. The purpose of this study was to isolate and characterize an oral keratinocyte stem-cell population. MATERIAL AND METHODS: Oral human keratinocytes obtained from keratinized oral mucosa were magnetically separated using α(6) ß(4) integrin and a proliferation-related marker, CD71. The isolated cell fractions were analyzed for cell size, cell cycle stage (using flow cytometry) and colony-forming ability. The expression of stem cell markers p63 and cytokeratin 19 and of differentiation markers cytokeratin 10 and involucrin was checked using immunocytochemical analysis. RESULTS: The stem cell CD71(neg) fraction had the smallest cell size compared with CD71(pos) and fractions [780.7 ± 141.5 (pixels), 1422.9 ± 264.6 (pixels) and 3844.4 ± 220.1 (pixels) respectively, p < 0.01; analysis of variance (ANOVA)]. Also, the CD71(neg) subpopulation consistently had the highest colony-forming ability among the three cell fractions (126.2 ± 21.7 vs. 32.8 ± 4.5 vs. 12.4 ± 2.1 compared with CD71(pos) and subpopulations, respectively, p < 0.01; ANOVA). Moreover, the CD71(neg) fraction contained more quiescent cells and fewer actively cycling cells than the CD71(pos) cell fraction. The candidate stem cells were positive for cytokeratin 19 and p63 keratinocyte stem cell markers, while differentiation markers such as cytokeratin 10 or involucrin were absent. CONCLUSION: The human gingival CD71(neg) cell fraction, separated by a magnetic system, demonstrated several characteristics of gingival keratinocyte stem cells. It is also suggested that a magnetic system may be an important tool in acquiring oral keratinocyte stem cells for research.

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

BACKGROUND AND OBJECTIVE: Volatile 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. MATERIAL AND METHODS: Human 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. RESULTS: Apoptosis 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. CONCLUSION: Hydrogen sulfide induces apoptosis in human gingival epithelial cells by activating the mitochondrial pathway.

Oral malodorous compound causes apoptosis and genomic DNA damage in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Volatile sulfur compounds are the main cause of halitosis. Hydrogen sulfide is one of these volatile sulfur compounds and the principal malodorous compound in physiological halitosis. Periodontally pathogenic activities of hydrogen sulfide have been previously reported. Hydrogen sulfide induces apoptotic cell death in aorta smooth muscle cells and in other tissues. Apoptosis plays an important role in the onset and progress of periodontitis. The objective of this study was to determine whether hydrogen sulfide causes apoptosis in human gingival fibroblasts. MATERIAL AND METHODS: Necrotic cells were detected using a lactate dehydrogenase assay. Apoptosis was ascertained using a histone-complexed DNA fragment assay and flow cytometry. The level of caspase 3, a key enzyme in apoptotic signaling, was also measured, and the effects of hydrogen sulfide on reactive oxygen species and superoxide dismutase were assessed. DNA damage caused by hydrogen sulfide was examined by means of single-cell gel electrophoresis. RESULTS: After 72 h of incubation with 100 ng/mL of hydrogen sulfide, necrosis was found in less than 10% of human gingival fibroblasts, whereas apoptosis was significantly increased (p < 0.05). Superoxide dismutase activity was strongly inhibited, and reactive oxygen species production was enhanced, after 48 and 72 h of incubation. Caspase 3 activity was also increased after 72 h of incubation (p < 0.01). Tail length, percentage of DNA in tail, and tail moment, measured by single-cell gel electrophoresis, were also intensified after 72 h of incubation (p < 0.001). CONCLUSION: Hydrogen sulfide caused apoptosis and DNA damage in human gingival fibroblasts. An increased level of reactive oxygen species stimulated by hydrogen sulfide may induce apoptosis and DNA strand breaks.

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.

Classification and examination of halitosis.

In this paper, the classification of halitosis and the examination procedures used in diagnosing halitosis are outlined. Halitosis is classified into categories of genuine halitosis, pseudo-halitosis and halitophobia. Genuine halitosis is subclassified into physiologic halitosis and pathologic halitosis. Pathologic halitosis itself is subdivided into oral and extraoral halitosis. Patients diagnosed with pseudo-halitosis and halitophobia usually complain about having oral malodour that does not really exist. Pseudohalitosis can be treated by dental practitioners, but halitophobic patients must be referred to psychological specialists. Oral malodour can be measured using an organoleptic measurement or a gas chromatography analysis. The organoleptic measurement is the most practical procedure with which one can evaluate oral malodour. Gas chromatography (GC) analysis using a flame photometric detector has been shown to be the gold standard for measuring oral malodour, owing its reputation to its objectivity and reproducibility. Moreover, GC is specific for volatile sulphur compounds (VSC), which are the main causes of oral malodour. It has been demonstrated that there is a high correlation between the intensity of oral malodour and the VSC concentration as measured by GC.

Treatment needs (TN) and practical remedies for halitosis.

Dental practitioners have traditionally neglected halitosis despite its high priority for the public, but practitioners' interest in halitosis has recently increased. Although oral pathologic or physiologic halitosis is easily reduced by a suitable treatment based on the treatment needs, systemic and psychological conditions sometimes confuse practitioners. Since a halitophobic patient never agrees with the result that his/her oral malodour has been reduced or eliminated after treatment, this may cause a dilemma for practitioners. Generally, halitosis patients, even genuine ones, have different psychological characteristics concerning their own breath than other individuals. Adverse psychological aspects of these patients are often promoted by the practitioner's mismanagement. Treatment Needs (TN) were, therefore, established to prevent practitioners' mismanagement of halitosis patients. By following these TN, patients can receive proper treatments for halitosis. However, to choose proper treatment measures, practitioners must refer to articles published in peer-reviewed journals, then use critical thinking to judge whether a product is effective in reducing oral malodour. Although it is challenging for dental practitioners to deal with patients with psychological conditions such as pseudo-halitosis or halitophobia, if appropriate treatments are administered accurately the practitioner does not risk mismanagement.

Tongue brushing and mouth rinsing as basic treatment measures for halitosis.

Tongue brushing and mouth rinsing are basic treatment measures for halitosis, and as such are categorised as treatment needs (TN)-1. Although TN-1 is used for treatment of physiologic halitosis treatment, pseudo-, extra oral pathologic or halitophobic patients must also be managed with TN-1 as well as other treatments. Since the origin of physiological halitosis is mainly the dorso-posterior region of the tongue, tongue cleaning is more effective than mouth rinsing. However, practitioners should always instruct their patients on how to brush their tongues to prevent harmful effects. Another approach using a chlorhexidine mouthwash is most effective in reducing oral malodour. However, chlorhexidine should not be used routinely; therefore, zinc-containing mouthwashes have been recommended for use. People can also use chewing gum to reduce oral malodour. Surprisingly, however, it has been noted that sugarless chewing gum increased methyl mercaptan, one of the principal components of oral malodour. Mint did not reduce the concentration of methyl mercaptan either, although these products are widely used for their ability to mask oral malodour. There is a need for the development of a novel food or chewing gum that could considerably reduce VSC levels in mouth air to complement TN-1.

The effects of methyl mercaptan on epithelial cell growth and proliferation.

AIM: Previous studies have demonstrated that methyl mercaptan (CH3SH), one of the main causes of oral malodour, might contribute to the initiation and progression of periodontal disease. These studies suggested that CH3SH may affect the epithelial cells of the gingival crevice, which form a barrier to the penetration of microbial substances. In this study, the effects of CH3SH on the epithelial cells and gingival fibroblasts were investigated. METHOD: Human oral epithelial carcinoma cell line (KB), human oral squamous cell carcinoma cell line (HSC-2), and human gingival fibroblasts (HGF) derived from healthy gingiva were used in this study. These cells were cultured in conditions of 5% CO2/95% air with or without CH3SH (10 ng/ml or 50 ng/ml) for 5 days. Cell numbers, proliferation and cytotoxicity were evaluated. RESULTS: CH3SH inhibited epithelial cell growth and proliferation at the concentration of 50 ng/ml, and a cytotoxic effect of CH3SH was also noted. On the other hand, HGF cells were not affected by 50 ng/ml CH3SH. CONCLUSION: High concentrations of CH3SH such as 50 ng/ml have an inhibitory effect on the growth and proliferation of epithelial cells, but not on those of fibroblasts.

Examination, classification, and treatment of halitosis; clinical perspectives.

Patients with halitosis may seek treatment from dental clinicians for their perceived oral malodour. In this article, an examination protocol, classification system and treatment needs for such patients are outlined. Physiologic halitosis, oral pathologic halitosis and pseudo-halitosis would be in the treatment realm of dental practitioners. Management may include periodontal or restorative treatment or both, as well as simple treatment measures such as instruction in oral hygiene, tongue cleaning and mouth rinsing. Psychosomatic halitosis is more difficult to diagnose and manage, and patients with this condition are often mismanaged in that they receive only treatments for genuine halitosis, even though they do not have oral malodour. A classification system can be used to identify patients with halitophobia. Additionally, a questionnaire can be used to assess the psychological condition of patients claiming to have halitosis, which enables the clinician to identify patients with psychosomatic halitosis. In understanding the different types of halitosis and the corresponding treatment needs, the dental clinician can better manage patients with this condition.

Genuine halitosis, pseudo-halitosis, and halitophobia: classification, diagnosis, and treatment.

Although tongue brushing and appropriate mouthrinses are both important and basic treatment measures for halitosis, other dental treatments are sometimes required. The treatment of genuine halitosis caused by oral conditions is not complex. In addition to genuine halitosis patients, psychosomatic halitosis patients also visit dental practitioners. Although psychosomatic halitosis is out of the treatment realm of dental practitioners, patients with this condition will still seek help from a dental practitioner. They often only receive treatment for genuine halitosis without referral to a psychological specialist. If these psychosomatic halitosis patients are incorrectly managed, the psychological condition might become worse than before the visit. To avoid the mismanagement of halitosis patients, classifications of halitosis patients have been established. Genuine halitosis was subclassified as physiologic halitosis and pathologic halitosis. Pathologic halitosis was further categorized to oral pathologic halitosis and extraoral pathologic halitosis. Both pseudo-halitosis and halitophobia patients complain of the existence of halitosis, which is not offensive. Pseudo-halitosis cannot be treated by dental practitioners, and halitophobia patients must be referred to psychological specialists. Clinicians need to examine the psychological condition of halitosis patients at the initial patient visit. A questionnaire prepared for the clinic at the University of British Columbia was found to be advantageous for this purpose.

Clinical application of a questionnaire for diagnosis and treatment of halitosis.

To treat halitosis, clinicians must examine the psychologic condition of a patient as well as the disease history and plausible causes of oral malodor, such as periodontal disease. However, it is not easy for a practitioner to carry out a psychologic examination other than the normal inquiry concerning oral malodor itself. Hence, a questionnaire that appears to be a normal inquiry, rather than one containing psychologic questions, was composed to survey the causes of halitosis and psychosomatic tendencies.

Clinical dilemmas posed by patients with psychosomatic halitosis.

Patients affected by psychosomatic halitosis never wish to visit a psychologic specialist, because they cannot recognize their own psychosomatic condition. They also never doubt that they have offensive oral malodor. Other people's behavior, such as covering the nose or averting the face, is interpreted by these patients as an indication that their breath is offensive, and these behaviors or attitudes reinforce their belief that they have a strong oral malodor. To clarify whether the patient's perception of another individual's attitude is affected by his or her delusion, this article is focused on the relationship between the behavior toward oral malodor and the psychologic profiles of patients with psychosomatic halitosis. If a patient expects simple avoidance behavior from other individuals, the development of psychosomatic halitosis may be accelerated, as it becomes a self-fulfilling prophecy. Individuals who are concerned with their own oral malodor but exhibit no oral malodor may have latent psychosomatic tendencies and may be mentally immature. A protocol for referring a patient to a psychologic specialist is presented.

Effect of methyl mercaptan on synthesis and degradation of collagen.

Measurements of the conversion of [14C]-proline to [14C]-hydroxyproline were employed to assess the effect of methyl mercaptan on intra- and extracellular metabolism of collagenous proteins in human gingival fibroblast cultures. Following a 30-min pulse, 10 ng of methyl mercaptan per ml of 95% air/5% CO2 head-space suppressed collagen synthesis by 39% and increased the intracellular degradation of newly synthesized collagen from 26% to 42%. Parallel cultures assayed for proline transport demonstrated a 29% inhibition of [14C]-proline uptake. A similar analysis of cultures exposed to methyl mercaptan for 12 h revealed an increase in intracellular degradation (20% control vs. 30% test) and a marked increase in extracellular collagenolysis (4% control vs. 55% test). While pulsing, collagen synthesis was decreased by 39%. Slab gel electrophoresis also demonstrated that treatment with methyl mercaptan caused reductions both in mature alpha 1 and alpha 2 chains of type I collagen and in type III procollagen. Identities of the procollagen species were confirmed by pepsin digestion. Reverse transcribed polymerase chain reaction was utilized to compare expression of alpha 1 chains of type I procollagen with type III procollagen and indicated suppression of mRNA synthesis for type III procollagen in cultures exposed to methyl mercaptan.

Effect of volatile thiol compounds on protein metabolism by human gingival fibroblasts.

The levels of volatile sulphur compounds (VSC) in periodontal pockets and mouth air have been found to correlate with severity of the disease process. The purpose of this study was to examine the influence of hydrogen sulphide and methyl mercaptan on protein metabolism of human gingival fibroblasts. The incorporation of labelled amino acids into protein was used to evaluate effects on total protein content. Changes in collagenous protein concentration were monitored by release of radioactivity following collagenase digestion as well as direct analysis of hydroxyproline. Both thiols were found to reduce total protein synthesis, with mercaptan exerting a greater adverse effect. In cultures exposed to mercaptan, total protein was reduced by 35%. The changes in total protein were accompanied by a corresponding decrease in collagenase-digestible protein. Hydroxyproline analysis of CH3SH-exposed cultures confirmed the changes associated with collagenous proteins. It indicated that in comparison to the controls the CH3SH-exposed cultures had a 70% reduction in collagen which resulted from a combined effect of suppressed synthesis and increased rate of collagen degradation. The possibility of thiol reaction with collagen was determined using in vitro systems in which type I collagen was reacted with varying concentrations of [35S]-H2S. The carboxymethyl (CM) cellulose assays of resulting reaction mixtures indicate that [35S]-radioactivity was incorporated directly into alpha 1, alpha 2, beta 11, beta 12 peptide chains. Furthermore, upon exposure of collagen to elevated H2S concentrations, the H2S converted some of the acid-soluble collagen to a more soluble product which could be extracted in neutral salt and analyzed by CM-cellulose chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)