Effects of arecoline, safrole, and nicotine on collagen phagocytosis by human buccal mucosal fibroblasts as a possible mechanism for oral submucous fibrosis in Taiwan

BACKGROUND: Oral submucous fibrosis (OSF) is associated with the betel quid chewing habit, and 86% of betel quid chewers in Taiwan are also smokers. Arecoline and safrole are major principles in the composition of betel quid, and nicotine is the main toxic ingredient of cigarettes. METHODS: To explore the pathogenesis of OSF, flow cytometry was used to compare collagen phagocytosis by fibroblasts from the normal and the OSF region of the same 15 OSF patients. RESULTS: The results indicated that heterogeneity of fibroblasts existed because collagen phagocytosis by fibroblasts from the normal region was higher than from the OSF region in the same patient. The percentage of phagocytic cells was significantly inhibited by 10, 25 and 50 microg/ml arecoline, safrole and nicotine in normal fibroblast cultures, respectively, and the percentage of phagocytic cells was significantly reduced by 25, 25 and 50 microg/ml arecoline, safrole and nicotine in OSF fibroblast cultures, respectively. Collagen phagocytosis by fibroblasts exhibited prominent dose-dependent inhibition as the concentration of arecoline, safrole, and nicotine increased. Besides, nicotine had a synergistic effect on arecoline- or safrole-inhibited collagen phagocytosis. CONCLUSIONS: The present study concludes that even in the same person, the collagen phagocytosis by fibroblasts is different between normal and OSF region. The deficiency in collagen phagocytosis by fibroblasts of the lesion might participate in the pathogenesis of OSF. Arecoline, safrole and nicotine, which are released in saliva during BQ chewing plus cigarette smoking, inhibit collagen phagocytosis by fibroblasts in a dose-dependent manner and may induce OSF formation in Taiwan's patients.     

Stimulation of human buccal mucosa fibroblasts in vitro by betel-nut alkaloids

Oral submucous fibrosis (OSF) is characterized by excessive collagen production by mucosal fibroblasts and is associated with the habitual chewing of betel-nuts (Areca catechu); nut extracts stimulate fibroblast activity in vitro. The metabolism of arecoline, the major alkaloid in the nut, by human buccal mucosa fibroblasts in vitro was investigated; alkaloid metabolites extracted from culture media were analysed by gas chromatography and thin-layer chromatography. [3H]-arecoline was metabolized predominantly to [3H]-arecaidine and this was accompanied by a concentration-dependent stimulation of collagen synthesis and cell proliferation. Arecaidine was a more potent stimulator than arecoline. The rate of hydrolysis of a series of synthetic arecaidine esters (methyl, ethyl, butyl, propyl and pentyl) by fibroblasts was closely correlated with the extent of stimulation of collagen synthesis. Thus fibroblasts are responsive to the major metabolite of arecoline and hydrolysis of the ester group may be necessary for this action. Exposure of buccal mucosa fibroblasts to these alkaloids in vivo may contribute to the accumulation of collagen in OSF.     

Therapeutic Interventions in Oral Submucous Fibrosis: An Experimental and Clinical Study

Introduction

Oral submucous fibrosis (OSF) is a chronic debilitating disease and premalignant condition of the oral cavity and is a serious public health issue in India and many parts of the world. The treatment is still elusive and empirical because of poorly understood etiopathogenesis, which is believed to be multifactorial including areca nut chewing, ingestion of chillies, genetic and immunologic processes, nutritional deficiencies, and many others. The present investigations was focused to understand the possible therapeutic interventions of anti-OSF agents in arecoline induced experimental in vitro model of OSF and clinical application of these anti-OSF agents in the restoration of various grade of the disease.

Materials and Methods

The 127 subjects were selected from patients who visited the OPD of Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, K.G. Medical University, Lucknow. Further the subjects were divided in two groups on the basis of clinical examination. Group-1 subjects showed presence of fibrosis bands in the labial and/or buccal mucosa, loss of elasticity, difficulty to open the mouth and had a habit chewing areca-nut in some form. Group-2 subjects had no habit of chewing areca-nut, were apparently healthy with no mucosal disorder. The samples were collected and were immediately transported to Indian Institute of Toxicology Research, Lucknow, for isolation and cultivation of primary cultures of mucosal fibroblast cells. Then isolation and cultivation of oral mucosal fibroblast, identification of non-cytotoxic doses of arecoline, real time PCR, immunocytochemistry, cytokine determination in culture cells, western blot analyses, functional activity of collagenase, lysyl oxidase enzyme activity, collagen beads assay, cyclooxygenase (COX-2) expression analysis was done.

Results and Conclusions

This study, shows that the reduction of phagocytic cells was strongly related to the arecoline levels in fibroblast culture when we exposed arecoline to normal oral mucosal cells (NOMC) and cells from OSF patient. An enhancement of phagocytic cells was observed following the pre exposure of cells to 1 μM dexamethasone, a glucocorticoids, In this study, histologic evidence is presented which supports the finding that COX-2 expression is upregulated in OSF specimens compared to normal oral submucosal cells. Strong immunostaining for COX-2 was detected in arecoline exposed NOMC and cells from OSF patient. Areca nut extract up-regulates prostaglandin production, cyclooxygenase-2 mRNA and protein expression of human oral keratinocytes. The number of phagocytic cells and phagocytic activity in cultured human oral fibroblasts from OSF site was lower than the fibroblasts from the normal regions of the same person.     

An in-vitro comparison of human fibroblasts from normal and oral submucous fibrosis tissue

Fibroblasts cultured in vitro from normal buccal tissue and from tissue from oral submucous fibrosis (OSF) associated with betel-nut chewing showed no significant difference in their rates of proliferation in culture, nor in the rate at which they hydrolysed the betel nut alkaloid arecoline to arecaidine. Basal rates of collagen synthesis were slightly higher in the OSF cells but, on addition of arecoline, the rate of collagen synthesis in normal and OSF cells was stimulated to the same level.     

Growth of oral and skin fibroblasts from patients with oral submucous fibrosis

The purpose of the investigation was to evaluate and compare the proliferation (growth) of mouth fibroblasts and skin fibroblasts from patients with oral submucous fibrosis (OSF). Material comprised fibroblasts from fibrous bands situated in the buccal mucosa and from the inner aspect of the forearm of 8 patients with classic features of OSF as well as fibroblasts from 6 buccal mucosa and 8 skin biopsy specimens from healthy non-areca nut chewing individuals. Cells were cultured for 8 days according to standard techniques. Their growth was monitored daily, under optimal conditions as well as exposure to concentrations of arecoline. The data were analyzed using regression analysis, analysis of variance and the Kruskal-Wallis test. We found no statistically significant differences between the proliferation patterns of oral and skin fibroblasts from patients or between those from patients and controls. The reaction of the cells exposed to concentrations of arecoline was similar; at low concentrations (0.1–10 μg/ml) normal growth was maintained, while 100 μg/ml inhibited growth. It is concluded that fibroblasts from mouths affected by OSF have proliferation patterns which fall within normal parameters, that the excessive collagen formation in established OSF is not due to increased fibroblast proliferation and that arecoline does not stimulate fibroblast proliferation.     

Arecoline and oral keratinocytes may affect the collagen metabolism of fibroblasts

Background:  The characteristic of oral submucous fibrosis (OSF) is related with the disturbance of synthesis and degradation of extracellular matrix. Arecoline, the areca nut (betel nut) component of betel quid, plays a major role in pathogenesis of OSF. But the exact mechanism how arecoline influences the collagen metabolism is unclear.
Methods:  Oral keratinocytes and fibroblasts were cocultured and keratinocytes were pre-treated by arecoline. Fibroblasts alone, fibroblasts stimulated by arecoline, fibroblasts cocultured with keratinocytes and fibroblasts cocultured with keratinocyte pre-treated by arecoline were included as the four groups in the present study. The concentration of collagen, the content and activity of matrix metalloproteinase (MMP) and the concentration of tissue inhibitor of metalloproteinase (TIMP) were assessed.
Results:  The collagen production of fibroblasts decreased when cocultured with keratinocytes; when cocultured with keratinocytes pre-treated by arecoline, fibroblasts produced more soluble collagen than non-pretreated coculture group. MMP-9 was produced only in coculture groups. There was no significant difference in the two coculture groups. The activation ratio of pro-MMP-2 in arecoline pre-treated keratinocytes-fibroblasts coculture group was significantly higher than that of non-coculture groups, but no significant difference existed in the two coculture groups. TIMP-1 produced by arecoline pre-treated keratinocytes-fibroblasts coculture group was significantly higher than those by the other three groups.
Conclusion:  TIMP-1 and the interaction of oral keratinocytes and fibroblasts play important role in pathogenesis of OSF.     

The fibroblast population in oral submucous fibrosis

The purpose of the investigation was to compare the morphology of fibroblasts cultured from healthy oral mucosa and mucosa of patients with oral submucous fibrosis (OSF) and to collate the occurrence of cell types of similar morphology. Cells cultured from biopsy specimens from the buccal mucosa of six subjects who did not chew the areca nut and six patients with OSF who chewed areca nut were grown according to standard techniques. Ninety cells per cell line were recorded daily for 8 days, classified into types F1, F2 and F3 according to their morphology, and the results statistically analyzed. We found that there was a relative increase of F3 cells in relation to Fl cells in OSF resulting in the ratio of F3 to F1 cells being significantly larger in OSF than the ratio in the controls. As it has been reported that F3 cells m rat connective tissues produce significantly more collagen types I and III than F1 cells, we concluded that a change of fibroblast population has occurred in OSF and that this relative increase of F3 cells in humans, which could be committed to the production of large quantities of collagen, can be an explanation for the excessive collagen formation in OSF.     

Copper stimulates human oral fibroblasts in vitro: a role in the pathogenesis of oral submucous fibrosis

Copper is implicated in the pathogenesis of several fibrotic disorders. Areca nut has been shown to have a high copper content and areca chewing is associated with oral submucous fibrosis (OSF). The effects of copper on human oral fibroblasts were investigated in vitro. Human oral fibroblasts were incubated with copper chloride (CuCl2) at concentrations ranging from 0.01 microM to 500 microM for 24 h, and in vitro cell proliferation was assayed by incorporation of tritiated-thymidine; soluble and non-soluble collagen synthesis was assayed using tritiated-proline. Addition of copper chloride at concentrations ranging from 0.1 microM to 50 microM increased the collagen synthesis by the oral fibroblasts compared with growth without copper (P<0.05). The addition of copper chloride neither increased the synthesis of non-collagenous proteins by the fibroblasts nor influenced their proliferation rate. We conclude that copper upregulates collagen production in oral fibroblasts. This appears to be concentration dependent, with peak collagen synthesis at 50 microM CuCl2. These in vitro results taken together with the recent findings of copper in oral biopsies from OSF subjects support the hypothesis that copper in areca nut acts as a mediator of OSF.     

Phagocytosis of fibronectin and collagens type I, III, and V by human gingival and periodontal ligament fibroblasts in vitro.

BACKGROUND: Electron microscopic studies have suggested that the volume density of collagen-containing vacuoles in fibroblasts is higher in the periodontal ligament (PDL) than in the gingiva. Whether this difference reflects intrinsic differences in phagocytic capacity among the cells in these tissues is not known. METHODS: PDL and gingival fibroblasts were isolated from subjects and cultured under identical conditions in the presence of fluorescent beads coated with collagen type I, III, or V or fibronectin. Control beads were coated with bovine serum albumin or an enamel matrix protein mixture that does not constitute part of the extracellular matrix of PDL and gingiva. After various time intervals (1 to 24 hours), the percentage of cells that had internalized beads was assessed by flow cytometry. Since alkaline phosphatase activity has been suggested to play a role in collagen phagocytosis, the activity of this enzyme was determined for all cell populations. RESULTS: The results demonstrated the following order in the percentage of cells internalizing protein-coated beads: fibronectin > collagen type I > III > V. Internalization of collagen type I-coated beads exceeded that of beads coated with bovine serum albumin or enamel matrix proteins by 6 and 3 times, respectively. No differences were observed in collagen phagocytic activity between PDL and gingival fibroblasts, and no relationship could be demonstrated between collagen phagocytosis and alkaline phosphatase activity. CONCLUSIONS: We conclude that differences in collagen phagocytosis between PDL and gingiva, as observed in vivo, are not likely to be explained in terms of intrinsic phagocytic capacities of these cells.     

槟榔提取物抑制人类口腔黏膜成纤维细胞生长的实验研究

目的通过比较正常口腔黏膜和口腔黏膜下纤维性变(OSF)组织中成纤维细胞(FB)增殖差异、检测槟榔提取物(ANE)对成纤维细胞增殖的影响,来探讨OSF的发病机理.方法对人类口腔黏膜成纤维细胞进行分离培养,然后用四唑盐(MTT)比色试验法检测OSF患者和正常人口腔黏膜FB增殖状况,并且观察ANE对FB增殖的影响.结果表示增殖水平的OD值在OSF-FB为0.254±0.045,高于NM-FB的OD值0.236±0.012(P<0.05),ANE以浓度-效应依赖关系抑制FB增殖.结论 OSF-FB细胞增殖率较NM-FB高;ANE对口腔黏膜FB有细胞毒作用,提示槟榔及其有效成分不完全是通过直接刺激FB增殖而诱发OSF.     

Actinobacillus actinomycetemcomitans lipopolysaccharide stimulates collagen phagocytosis by human gingival fibroblasts

Introduction:  Collagen phagocytosis by fibroblasts is involved in the intracellular pathway related to collagen breakdown in soft connective tissues. The possible role of lipopolysaccharide (LPS) in regulating this fibroblast function has not been elucidated so we investigated the effect of LPS from Actinobacillus actinomycetemcomitans , a periodontopathic bacterium, on collagen phagocytic activity in human gingival fibroblasts and associated regulatory mechanisms.
Methods:  LPS pretreatment stimulated binding of collagen-coated beads to cells and, subsequently, their internalization.
Results:  The LPS-activated collagen phagocytic process was enhanced in the presence of the soluble form of CD14 (sCD14) or LPS-binding protein (LBP), while the LPS/LBP treatment activated Akt and induced actin reorganization. Furthermore, these LPS/LBP-induced effects were partially suppressed by adding phosphatidyl-inositol-3 kinase (PI3K) inhibitors.
Conclusion:  These results suggest that A. actinomycetemcomitans LPS disturbs the homeostasis of collagen metabolism within gingival tissue by facilitating collagen phagocytosis by gingival fibroblasts, and serum sCD14 and LBP positively regulate the action of LPS. In addition, the PI3K/Akt signaling is thought to partially mediate the LPS/LBP-stimulated collagen phagocytic pathway, which may be dependent on actin cytoskeletal rearrangement.     

Regulation of interleukin-6 expression by arecoline in human buccal mucosal fibroblasts is related to intracellular glutathione levels

OBJECTIVES: Cytokines play an important role in regulating fibroblast function and is likely to play a key role in regulating the initiation and progression of scarring in any fibrotic disease. Interleukin-6 (IL-6) has been implicated in the development of a variety of fibrotic diseases. The aim of this study was to compare IL-6 expression in fibroblasts cultured from normal human buccal mucosa and oral submucous fibrosis (OSF) specimens and further explore the potential mechanism that may lead to induce IL-6 expression. METHODS: mRNA level of IL-6 in fibroblasts from OSF was compared with normal buccal mucosa. The effects of arecoline, the major areca nut alkaloid, on IL-6 expression in normal human buccal mucosa fibroblasts (BMFs) were measured in vitro. mRNA was quantified with AlphaImager 2000. To determine whether glutathione (GSH) levels were important in the induction of IL-6 by arecoline, we pretreated cells with 2-oxothiazolidine-4-carboxylic acid (OTZ) to boost GSH levels or with buthionine sulfoximine (BSO) to deplete GSH. RESULTS: Fibroblasts derived from OSF exhibited higher IL-6 gene expression than BMF in mRNA levels (P < 0.05). The exposure of quiescent BMF to arecoline resulted in the elevation of IL-6 mRNA expression in a dose-dependent manner (P < 0.05). IL-6 gene regulated by arecoline correlated with intracellular GSH levels in BMF. Arecoline at a concentration of 129 muM induced about 2.7-fold IL-6 mRNA levels over the 6-h incubation period. However, BSO enhanced the IL-6 mRNA levels by 3.9-fold (P < 0.05). In addition, OTZ was found to marginally reduce the arecoline-induced IL-6 expression by about 1.7-fold (P < 0.05). CONCLUSIONS: Taken together, these results suggest that IL-6 expression is significantly upregulated in OSF fibroblasts in areca quid chewers and arecoline may be responsible for the enhanced IL-6 expression. In addition, the regulation of IL-6 expression induced by arecoline is critically dependent on the intracellular GSH concentrations.     

Diffusion of reduced arecoline and arecaidine through human vaginal and buccal mucosa

Because alkaloids from areca nut, arecoline and arecaidine, have been implicated in the development of oral submucous fibrosis, we determined their diffusion kinetics through human buccal and vaginal mucosa. Four clinically healthy vaginal mucosa specimens (mean patient age +/- standard deviation: 47 +/- 15 years; age range: 31-60 years) and 4 buccal mucosa specimens from 2 male patients and 2 female patients (mean patient age +/- standard deviation: 31 +/- 9 years; age range: 17-53 years) were obtained during surgery. In vitro flux rates of reduced arecoline and arecaidine (r-arecoline and r-arecaidine) were determined by use of a flow-through diffusion apparatus. Analysis of variance, a Duncan multiple range test, and an unpaired t-test were used to determine steady state kinetics and flux differences over time intervals. Although statistically significant differences were observed between flux values for both alkaloids and tissues at certain time points, these were not considered to be of biological (clinical) significance. However, the flux rates across both mucosa of r-arecoline were significantly higher statistically than those of rarecaidine. The findings demonstrated the differences in the diffusion kinetics between r-arecoline and r-arecaidine across human buccal and vaginal mucosa, an observation that could be explained in terms of their ionisation characteristics. Additionally, the results obtained further support the hypothesis that human vaginal mucosa can be used as a model for buccal mucosa in studies of permeability to various chemical compounds.     

Cytopathologic effects of arecoline on human gingival fibroblasts in vitro

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. Received: 31 August 1998 / Accepted: 3 November 1998     

核受体共激活因子7在槟榔碱诱导的上皮间充质转化中的生物学功能

目的研究核受体共激活因子7(nuclear receptor coactivator 7,NCOA7)在口腔黏膜下纤维性变(OSF)中的表达及意义,并探讨其在人口腔角质细胞株HOKs间充质转化过程中的作用。方法收集OSF组织30例及正常口腔黏膜组织15例,采用免疫印迹法检测NCOA7、上皮间充质转化相关标记物在OSF组织及正常黏膜组织中蛋白水平的表达情况;以Western blot法检测槟榔碱刺激后HOKs中NCOA7、上皮及间充质标记物的蛋白水平变化。结果 NCOA7在30例OSF组织高表达(P<0.05);同时在细胞基底层出现间充质标记物的阳性表达。NCOA7在槟榔碱刺激的HOKs中高表达,且具有槟榔碱浓度依赖性(P=0.004 3);随着槟榔碱浓度的升高,上皮标志物表达逐渐下调,间充质标记物表达逐渐升高。结论口腔黏膜下纤维性变组织中高表达的NCOA7,可能参与调控上皮细胞间充质转化过程,进一步促进纤维化进程。     

Increased plasminogen activator inhibitor-1/tissue type plasminogen activator ratio in oral submucous fibrosis

OBJECTIVE: Plasminogen activators and their inhibitors are thought to be key participants in the balance of proteolytic and antiproteolytic activities that regulate extracellular matrix (ECM) turnover. However, little is known about the expression of plasminogen/plasmin system at the site of oral submucous fibrosis (OSF). METHODS: We compared the activities of tissue type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) between fibroblasts derived from normal buccal mucosa and OSF by using an enzyme-linked immunosorbent assay. Furthermore, arecoline, a major areca nut alkaloid, was challenged with normal buccal mucosal fibroblasts (BMFs) to elucidate whether the activities of t-PA and PAI-1 could be affected by arecoline. RESULTS: Both t-PA and PAI-1 were found to be increased in OSF than in BMFs (P < 0.01). In addition, there was a statistically significant difference in PAI-1/t-PA ratio between OSF and BMF (P < 0.01). The addition of arecoline upregulated not only PAI-1, but also t-PA in BMFs (P < 0.05). In addition, the ratio between PAI-1 and t-PA was found to be significantly increased by a linear regression assay (P < 0.01). CONCLUSION: These results suggest that OSF caused by areca quid chewing may be the result of an imbalance in the plasminogen/plasmin system, the net result of which is increased deposition of ECM.     

Increased lysyl oxidase activity in fibroblasts cultured from oral submucous fibrosis associated with betel nut chewing in Taiwan

Growth characteristics and lysyl oxidase activity of fibroblasts derived from human normal mucosa (NM) and oral submucous fibrosis (OSF) associated with betel nut chewing were compared in cell cultures. The growth rates of cultured cells were identified by plating 5×105 cells/35 mm culture dish (Day 0) and every 24 hours cell proliferation was determined by quantifying the cell number (using a hemocytometer). The third to seventh passages were used. A medium without serum but supplemented with 5 mg/ml bovine serum albumin was substituted for the original medium at the subconfluent period and cultured for an additional 24 h. The medium was collected and used for assays of protein content and lysyl oxidase activity. Lysyl oxidase activity was assayed with [4,5-³H] -lysine labelled purified chick - embryo aorta elastin substrate. After incubation for 10 h at 37°C, the enzyme activity was measured from 3HHO (tritiated water) separated by ultrafiltration using Amicon C-10 micro-concentrators. The results showed the mean doubling time of OSF fibroblasts was 3.2 days and of NM fibroblasts was 3.6 days. NM fibroblasts became confluent at day 6 as determined by cell number, while OSF fibroblasts were confluent by Day 5. Furthermore, the immunoenzymatic assay for BrdUrd incorporation revealed that OSF fibroblasts proliferate significantly faster than NM fibroblasts under standard culture conditions. Both total protein content (10.84±1.15 mg/ml) and lysyl oxidase activity (3558.6±345.5 cpm/106 cell) in OSF fibroblasts were greater than in NM fibroblasts (6.35±0.96 mg/ml and 2436.0±352.6 cpm/10⁶ cell). The results of this study provide evidence that fibroblasts derived from oral submucous fibrosis (OSF) tissue and normal mucosa (NM), although similar in many respects, exhibit specific differences in proliferation rates and lysyl oxidase activity. Moreover, collagen deposition in OSF tissue may, at least in part, be ascribed to increased lysyl oxidase activity.     

Cytotoxicity and arecoline mechanisms in human gingival fibroblasts in vitro

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. Received: 16 February 2000 / Accepted: 25 July 2000