Kuo-Wei Tai

Increased tissue inhibitor of metalloproteinase-1 expression and inhibition of gelatinase A activity in buccal mucosal fibroblasts by arecoline as possible mechanisms for oral submucous fibrosis

Oral submucous fibrosis (OSF) is a pre-malignant fibrotic lesion of the mouth in areca quid chewers. It is probably a consequence of disturbances in the hemeostatic equilibrium between synthesis and degradation of extracellular matrix molecules (ECM). To date, there has been little research about the role of tissue inhibitors of metalloproteinase (TIMPs) and matrix metalloproteinases (MMPs) in the pathogenesis of OSF. In the present study, we examined the activity of TIMPs from cells cultured from OSF and normal buccal mucosa. OSF specimens were found to have higher TIMP-1 expression than normal buccal mucosal fibroblasts (BMFs) by Western blots. To verify whether arecoline, a major areca nut alkaloid, could affect TIMP or MMP production by human BMFs, Western blots and gelatine zymography were used. Arecoline was found to elevate TIMP-1 expression at the concentration level under 20 microg/ml in a dose-dependent manner. The amount of TIMP-1 was about 2.7 fold at a concentration level of 10 microg/ml compared with control. From gelatin zymograms, the main gelatinolytic proteinase secreted by the human BMFs was MMP-2, and only minimal amounts of MMP-9 could be detectable from zymogram. In addition, arecoline was found to inhibit MMP-2 secretion and production at the concentration level of 40 microg/ml. The gelatinolytic activity of MMP-2 was about 54% at a concentration level of 80 microg/ml compared with control. Taken together, it was found that arecoline acted not only as an inhibitor on gelatinolytic activity of MMP-2, but also a stimulator for TIMP-1 activity. These synergistic effects may contribute to the ECM components accumulation in the areca quid associated OSF.

Cytotoxic and nongenotoxic effects of phenolic compounds in human pulp cell cultures

Phenolic compounds are widely used in clinical dentistry as sedatives for the dental pulp, as disinfectants for caries, and as root canal medications. The pathobiological effects of various phenolic compounds on human dental pulp fibroblasts were investigated with Hoechst 33258 fluorescence assay and DNA precipitation assay. All phenolic compounds showed cytotoxicity in Hoechst 33258 fluorescence assay by inhibiting cellular DNA in a concentration-dependent manner. The 50% inhibition concentrations required to decrease the cellular DNA contents by guaiacol, phenol, eugenol, and thymol were 9.8, 4.5, 0.9, and 0.5 mM, respectively. However these phenolic compounds did not cause DNA single-strand breaks in cultured human pulp fibroblasts. These results indicate that phenolic compounds are cytotoxic agents but are without genotoxic effects on human pulp fibroblasts in vitro. However care should be taken to reduce the possibility of pulpal as well as periapical irritations from inadvertent extrusion of these substances in clinical usage.

Mutation of p53 gene codon 63 in saliva as a molecular marker for oral squamous cell carcinomas

The inactivation of tumor suppressor gene (TSG) is important during multistage carcinogenesis. The p53 TSG is frequently mutated in oral squamous cell carcinomas. These mutations can serve as very specific markers for the presence of tumor cells in a background of normal cells. In this study, 10 oral squamous cell carcinoma patients and 27 normal dental students were collected from Chung Shan Medical and Dental College Hospital, Taichung, Taiwan. Extractions of DNA from saliva were obtained. Exon 4 and intron 6 within the p53 gene were amplified with polymerase chain reactions (PCRs) followed by DNA sequence analysis. DNA sequence analysis of PCR products revealed that five of eight (62.5%) tumor saliva samples and five of 27 (18. 52%) healthy saliva samples contained p53 exon 4 codon 63 mutations. These results were significantly different by using Chi-square test (P<0.05). The majority of the base substitutions were C deletions. Probable hot spots for the mutation were identified at exon 4 codon 63, which has not been observed before in head and neck cancers. Our study indicated that mutation of p53 codon 63 in saliva might be a molecular marker for oral squamous cell carcinomas. In addition, the amount of DNA recovered from saliva in most cases is sufficiently large and its quality suitable to enable PCR amplification which could be used in the search for mutations. The protocol described is rapid, cheap, and easy to perform, and may be useful for epidemiological studies for oral carcinogenesis.

Proinflammatory cytokines induce cyclooxygenase-2 mRNA and protein expression in human pulp cell cultures.

The increased release of prostaglandins (PG) within pulpal tissues is considered to play a pathogenic role during pulpal disease progression. The rate-limiting step in the formation of PG from arachidonic acid is catalyzed by cyclooxygenase (COX). COX-2 is an inducible enzyme believed to be responsible for PG synthesis at site of inflammation. The effect of proinflammatory cytokines on human pulp cells with special reference to COX-2 expression has not been reported earlier. The aim of the present study was to investigate the effects of interleukin (IL)-1alpha and tumor necrosis factor-alpha (TNF-alpha) on the expression of COX-2 mRNA gene and protein in cultured human pulp cells. Investigations of the time dependence of COX-2 mRNA expression in proinflammatory cytokines-treated human pulp cells revealed a rapid accumulation of the transcript, a significant signal first detectable 1 h after exposure. In addition, both IL-1alpha and TNF-alpha up-regulated COX-2 protein expression by human pulp cells. The kinetics of this response showed that COX-2 was detectable in cell lysates as early as 2 h post proinflammatory cytokines challenge and remained elevated throughout the 24-h incubation period. This suggests that one of the pathogenic mechanisms of pulpal inflammation in vivo may be the synthesis of COX-2 by resident cells in response to a proinflammatory cytokines challenge. COX-2 may play an important role in the regulation of prostanoid formation in the pathogenesis of pulpal inflammation. Taken together, we propose that the use of selective COX-2 inhibitors might provide a valuable tool in the control of pulpal inflammation.

Cytotoxic evaluation of root canal filling materials on primary human oral fibroblast cultures and a permanent hamster cell line.

To date there has been very little data on the cytotoxic response of different cell lines to root canal sealers. The objective of this study was to determine the cytocompatibility of three different extracts of root canal sealers and to compare the cytotoxic response of these materials on two different primary human oral fibroblasts (derived from gingiva and buccal mucosa) and one permanent hamster cell line (V79 cells). Cytotoxicity was judged using an assay of tetrazolium bromide reduction. The results showed that root canal sealers (AH plus, Canals, and N2) were cytotoxic to primary human oral fibroblast cultures and V79 cells. It was found that N2 was the most toxic root canal sealer among those tested in all cultures. The toxicity decreased in an order of N2 > AH plus approximately = Canals. The sensitivity of toxicity depended on the materials tested and the cell culture system used.

Cytotoxicity and arecoline mechanisms in human gingival fibroblasts in vitro

Abstract Betel nut chewing, like cigarette smoking, is a popular oral habit which impinges on the daily lives of a population of approximately 200 million. People who chew betel nuts have a higher prevalence of periodontal diseases than those who do not. Many of the undesirable effects of betel nuts have been attributed to arecoline, a major component of the particular alkaloid in betel nuts. In this in vitro study, we have focused on the effects of arecoline and the role it could play in periodontal breakdown via its direct effects on human gingival fibroblasts. Human gingival fibroblasts were derived from three healthy individuals undergoing crown-lengthening procedures. We found that arecoline is cytotoxic to human gingival fibroblasts at a concentration higher than 50 µg/ml by depleting intracellular thiols and inhibiting mitochondrial activity (P<0.05). In addition, the cells displayed a marked arrest at G2/M phase in a dose-dependent manner. Repeated and long-term exposure to arecoline could impair the gingival fibroblast functions. As they are cytotoxic, the use of betel nut products in conjunction with periodontal therapy may interfere with optimal healing and/or lead to further periodontal breakdown.

Induction of Tissue Plasminogen Activator Gene Expression by Proinflammatory Cytokines in Human Pulp and Gingival Fibroblasts

Plasminogen activator converts plasminogen to plasmin, and plasmin activates the latent matrix metalloproteinases. Tissue plasminogen activator (t-PA) is one of the important proteolysis factors present in human inflamed tissues. However, few studies reported on the mechanisms of tissue destruction via a t-PA proteolysis pathway in pulpal and periapical diseases. The subsequent reactions leading to pulpal and periapical injury after the induction of proinflammatory cytokines remains to be elucidated. The aim of this study was to investigate the effects of interleukin-1alpha and tumor necrosis factor-alpha on the expression of t-PA mRNA gene in cultured human pulp and gingival fibroblasts. The mRNAs for t-PA were measured by reverse transcription-polymerase chain reaction at 2, 6, and 24 h. The results show that both cytokines induced significantly high levels of t-PA mRNA gene expression in human pulp fibroblasts. The peak of t-PA mRNA levels induced by both proinflammatory cytokines was at the 6-h incubation period. Interleukin-1alpha was found to be more effective in induction of t-PA gene expression than tumor necrosis factor-alpha. In addition, a similar induction pattern was also found in human gingival fibroblasts. These results indicate that proinflammatory cytokines can induce t-PA gene expression and such an effect may partially contribute to the destruction of pulpal and periapical tissues through dysregulated pericellular proteolysis. An understanding of the mechanism could not only further define the role of immune events in pulpal and periapical diseases but also have important implication for pharmacological intervention.

Elevated vimentin expression in buccal mucosal fibroblasts by arecoline in vitro as a possible pathogenesis for oral submucous fibrosis

Areca quid chewing is strongly correlated with oral submucous fibrosis (OSF) in Taiwan. The cytotoxicity of arecoline, a major areca nut alkaloid, on human oral fibroblasts has been extensively studied. To date, however, there has been little research exploring the possible effects of arecoline on cytoskeleton components. In this study, in addition to conducting a cytotoxicity assay, we examine the effect of arecoline on vimentin, an intermediate filament, and its expression in human buccal mucosal fibroblasts on exposure to various levels of arecoline (0-200 microg/ml) for 48 h. At a concentration above 50 microg/ml, arecoline demonstrated dose-dependent cytotoxicity (P<0.05) for cultured fibroblasts. Using sodium dodecyl sulphate-polyacrylamide gel electrophoresis, we demonstrated dose-dependent elevation of 57 kDa cytoskeletal-protein levels for arecoline. Evidence from immunoblotting assay indicated this 57 kDa cytoskeletal protein was vimentin. The increase in vimentin with arecoline exposure corresponded to that noted for fibroblasts cultured from OSF patients. Immunohistochemical assay also revealed that vimentin expression was much higher for OSF specimens than for normal buccal mucosa. We suggest these results may advance understanding of the possible pathogenesis for submucous fibrosis through the transformation of normal buccal mucosa as a result of areca quid chewing.

Cytopathologic effects of arecoline on human gingival fibroblasts in vitro

Abstract Arecoline, a major betel nut alkaloid, has been detected in saliva obtained during betel nut chewing in concentrations up to 140 μg/ml, corresponding to 0.9 mM. Arecoline in the millimolar concentration range might participate in the initiation and/or progression of periodontal disease during the long-term effects of betel nut chewing. In this study, cell growth, cell proliferation, assessment of cytoplasmic enzyme lactate dehydrogenase (LDH) and collagen synthesis were used to investigate the effects of human gingival fibroblasts exposed to arecoline levels of 0–200 μg/ml. Control culture exhibited a normal monolayer of long spindle-shaped fibroblast morphology. Arecoline-treated human gingival fibroblasts showed a more rounded appearance and detached at the higher concentrations. At concentrations higher than 75 μg/ml, many cells had detached from the surface of the petri dish and numerous floating cells could be seen under the inverted microscope. At a concentrations higher than 25 μg/ml, arecoline inhibited cell growth, proliferation and collagen synthesis and increased LDH leakage in a dose-dependent manner (P<0.05). These results indicate that arecoline is a cytotoxic agent to human gingival fibroblasts. Repeated and long-term exposure to arecoline could impair gingival fibroblast function. Betel quid chewers might be more susceptible to destruction of the periodontium and less responsive to a regeneration procedures during periodontal therapy.

Resinous Perforation-Repair Materials Inhibit the Growth, Attachment, and Proliferation of Human Gingival Fibroblasts

The choice of repair material is one of the important factors in the prognosis of the endodontically treated tooth with a perforation defect. The cytotoxicity of perforation-repair materials must be investigated to ensure a safe biological response. The aim of this in vitro study was to evaluate the effect of resin-modified, glass-ionomer cement, compomer, and resin on human-gingival fibroblasts. Human gingival fibroblasts from crown lengthening surgery were cultured by using an explant technique with the consent of the patient. Cytotoxicity was judged by using an assay of tetrazolium bromide reduction. The results showed that resin-modified, glass-ionomer cement Fuji II LC, compomer Compoglass, and resin SpectrumTPH (TPH) were cytotoxic to primary human gingival fibroblast cultures by inhibiting cell growth and proliferation. TPH alone had an effect on cell attachment. It was found that TPH was the most cytotoxic repair material among those tested in all cultures. The toxicity decreased in the order of TPH>FLC>CG.