Modulation of phagocytosis, chemotaxis, and adhesion of neutrophils by areca nut extracts

BACKGROUND: A higher prevalence of periodontal diseases among areca chewers than non-areca chewers has been demonstrated. Neutrophils, representing the first line of the host defense mechanism against microbial infection, play important roles in maintaining periodontal health. This study determined the possible effects of areca nut on phagocytosis, chemotaxis, and adhesion of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE) and fresh and tender areca nut with husk (tANE) were examined for their effects on neutrophil phagocytosis using flow cytometry and confocal laser scanning microscopy. The effects of rANE and tANE on chemotaxis and adhesion of neutrophils to human aortic endothelial cells were examined using fluorescence-labeled neutrophils. RESULTS: Both rANE and tANE inhibited the phagocytic activity of neutrophils in a dose-dependent manner. The levels of internalized fluorescent bacteria in neutrophils decreased after ANE treatment. However, exposure of neutrophils to rANE and tANE stimulated the chemotaxis activity of neutrophils to N-formyl-Met-Leu-Phe (fMLP) and enhanced adhesion of neutrophils to human aortic endothelial cells in a dose-dependent manner. Moreover, treatment of neutrophils with rANE was more effective than incubation with tANE. CONCLUSIONS: Components of areca nut inhibited phagocytosis activity of neutrophils but enhanced chemotaxis and adhesion of neutrophils. Alterations in functions of neutrophils may lead to signs of clinical diseases associated with areca chewing. The components in ANEs that are responsible for these observations remain to be elucidated.     

Synergistic effects of nicotine on arecoline-induced cytotoxicity in human buccal mucosal fibroblasts

Areca quid chewing has been linked to oral submucous fibrosis and oral cancer. Arecoline, a major areca nut alkaloid, is considered to be the most important etiologic factor in the areca nut. In order to elucidate the pathobiological effects of arecoline, cytotoxicity assays, cellular glutathione S-transferase (GST) activity and lipid peroxidation assay were employed to investigate cultured human buccal mucosal fibroblasts. To date, there is a large proportion of areca quid chewers who are also smokers. Furthermore, nicotine, the major product of cigarette smoking, was added to test how it modulated the cytotoxicity of arecoline. At a concentration higher than 50 microg/ml, arecoline was shown to be cytotoxic to human buccal fibroblasts in a dose-dependent manner by the alamar blue dye colorimetric assay (P<0.05). In addition, arecoline significantly decreased GST activity in a dose-dependent manner (P<0.05). At concentrations of 100 microg/ml and 400 microg/ml, arecoline reduced GST activity about 21% and 46%, respectively, during a 24 h incubation period. However, arecoline at any test dose did not increase lipid peroxidation in the present human buccal fibroblast test system. The addition of extracellular nicotine acted synergistically on the arecoline-induced cytotoxicity. Arecoline at a concentration of 50 microg/ml caused about 30% of cell death over the 24 h incubation period. However, 2.5 mM nicotine enhanced the cytotoxic response and caused about 50% of cell death on 50 microg/ml arecoline-induced cytotoxicity. Taken together, arecoline may render human buccal mucosal fibroblasts more vulnerable to other reactive agents in cigarettes via GST reduction. The compounds of tobacco products may act synergistically in the pathogenesis of oral mucosal lesions in areca quid chewers. The data presented here may partly explain why patients who combined the habits of areca quid chewing and cigarette smoking are at greater risk of contracting oral cancer.     

Effects of areca nut extracts on the functions of human neutrophils in vitro

Aqueous extracts of ripe areca nut without husk (ripe ANE) and fresh and tender areca nut with husk (tender ANE) were examined for their effects on the defensive functions of human neutrophils. Exposure of peripheral blood neutrophils to ripe ANE and tender ANE inhibited their bactericidal activity against oral pathogens, including Actinobacillus actinomycetemcomitans and Streptococcus mutans, in a dose-dependent manner. At the concentrations tested, ripe and tender ANEs did not significantly affect the viability of neutrophils as verified by their ability to exclude trypan blue dye. However, both ANEs inhibited the production of bactericidal superoxide anion by neutrophils as measured by cytochrome c reduction. Moreover, the ripe ANE inhibited neutrophils more effectively than did tender ANE. Arecoline, a major alkaloid of areca nut, only exhibited an inhibitory effect on the functions of neutrophils when high concentrations were used. Therefore, arecoline could not be used to explain the inhibitory effects observed for ANEs. In conclusion, our results demonstrated that ripe and tender ANEs reduced the antibacterial activity and the superoxide anion production of neutrophils. This effect may contribute to a less efficient elimination of bacteria from the periodontal environment. Inhibition of the antimicrobial functions of neutrophils may alter the microbial ecology of the oral cavity, and this may be one possible mechanism by which areca nut compromises the oral health of users of areca nut products.     

Adverse effects of arecoline and nicotine on human periodontal ligament fibroblasts in vitro

BACKGROUND, aims: The habit of betel nut chewing impinges on the daily lives of approximately 200 million people. Betel quid chewers have a higher prevalence of periodontal diseases than non-chewers. This study examined the pathobiological effects of arecoline, a major component of the betel nut alkaloids, on human periodontal ligament fibroblasts (PDLF) in vitro. METHOD: Cell viability, proliferation, protein synthesis, and cellular thiol levels were used to investigate the effects of human PDLF exposed to arecoline levels of 0 to 200 microg/ml. In addition, nicotine was added to test how it modulated the effects of arecoline. RESULTS: Arecoline significantly inhibited cell proliferation in a dose-dependent manner. At concentrations of 10 and 30 microg/ml, arecoline suppressed the growth of PDLF by 20% and 50% (p < 0.05), respectively. Arecoline also decreased protein synthesis in a dose-dependent manner during a 24-h culture period. A 100 microg/ ml concentration level of arecoline significantly inhibited protein synthesis to only 50% of that in the untreated control (p < 0.05). Moreover, arecoline significantly depleted intracellular thiols in a dose-dependent manner. At concentrations of 25 microg/ml and 100 microg/ml, arecoline depleted about 18% and 56% of thiols (p < 0.05), respectively. This suggests that arecoline itself might augment the destruction of periodontium associated with betel nut use. Furthermore, the addition of nicotine acted with a synergistic effect on the arecoline-induced cytotoxicity. At a concentration of 60 microg/ml, arecoline suppressed the growth of PDLF by about 33% and 5 mM nicotine enhanced the arecoline-induced cytotoxic response to cause about 66% cell death. CONCLUSION: During thiol depletion, arecoline may render human PDLF more vulnerable to reactive agents within cigarettes. Taken together, people who combine habits of betel nut chewing with cigarette smoking could be more susceptible to periodontium damage than betel nut chewing alone.     

Inhibition of the migration, attachment, spreading, growth and collagen synthesis of human gingival fibroblasts by arecoline, a major areca alkaloid, in vitro

Because betel quid (BQ) chewing has been linked to a higher prevalence of periodontal diseases, the pathobiological effects of arecoline, a main alkaloid found in areca nut, were investigated in cultured human gingival fibroblasts. At concentrations higher than 0.4 mM, arecoline inhibits cell attachment, cell spreading and cell migration in a dose-dependent manner. These inhibitory effects were associated with intracellular depletion of glutathione (GSH). At concentrations of 0.4 mM and 1 mM. arecoline depleted about 26% and 45% of GSH after 2 h incubation. Exposure of cells to areeoline at concentrations lower than 0.4 mM for 2 h showed no significant decrease in either cell viability or intracellular GSH. However, incubation of cells for 24 h in 1 mM arecoline decreased the cell numbers to only 35% of those in the untreated control. Arecoline also decreased cell growth and collagen synthesis in a dose-dependent manner. Because of repeated and long-term exposure to arecoline. BQ chewers could be more susceptible to periodontal damage and less responsive to new attachment procedures.     

Areca nut extracts reduce the intracellular reactive oxygen species and release of myeloperoxidase by human polymorphonuclear leukocytes

BACKGROUND AND OBJECTIVE: Polymorphonuclear leukocytes (PMN) represent the first line of host defense. Areca nut extract inhibits the bactericidal activity of, and the release of superoxide anion (O2- ) by, PMN. This study investigated the effects of areca nut extract on the intracellular production of reactive oxygen species (ROS) and on the extracellular release of lysosomal enzyme, myeloperoxidase (MPO), by PMN. The effects of arecoline, a principal component of areca nut, were also examined. MATERIAL AND METHODS: Human PMN were treated with various concentrations of areca nut extract or arecoline followed by treatment with Hanks' balanced salt solution, with or without cytochalasin B and fMet-Leu-Phe (CB/fMLP). The viability of PMN was determined using propidium iodide staining and flow cytometry. The presence of intracellular ROS was determined using 2',7'-dichlorofluorescin diacetate and fluorometry. MPO release was determined using a substrate assay. RESULTS: Areca nut extract (25 and 50 microg/ml) significantly decreased the viability of PMN. The intracellular levels of ROS and the extracellular release of MPO were induced in PMN by CB/fMLP. Exposure of PMN to areca nut extract (up to 25 microg/ml) or to arecoline (up to 2 mg/ml) did not directly affect the levels of ROS and MPO activity. However, under conditions that did not affect the viability of PMN, the ability of CB/fMLP to trigger production of intracellular ROS and release of MPO in human PMN was significantly suppressed by areca nut extract and arecoline. CONCLUSION: Areca nut impaired the activation of PMN by CB/fMLP that might decrease the effectiveness of PMN in the host defense. Alternatively, exposure of PMN to areca nut extract could decrease the capacity of PMN to damage tissues.     

Inhibitory Effects of Areca Nut Extract on Expression of Complement Receptors and Fc Receptors in Human Neutrophils

Background: Chewing of areca quid increases the prevalence of periodontal diseases. Areca nut extract (ANE) inhibits the phagocytic activity of human neutrophils. This in vitro study investigates the effects of ANE on complement‐ and antibody‐opsonized phagocytosis by neutrophils. Expression of complement receptors, Fc receptors, and F‐actin in ANE‐treated neutrophils is also analyzed. Methods: The viability of ANE‐treated neutrophils was determined using the propidium iodide staining method. The possible effects of ANE on the expression of complement receptors and Fc receptors were examined using an immunofluorescence staining method followed by flow cytometry and confocal laser scanning microscopy. The phagocytic activity of neutrophils against complement or immunoglobulin (Ig)G‐opsonized fluorescent beads was analyzed using flow cytometry. Expression of F‐actin was determined using confocal laser scanning microscopy. Results: ANE significantly inhibited the production of complement receptors (CR1, CR3, and CR4) and Fc receptors (FcγRII and FcγRIII) in a concentration‐dependent manner. Treatment of neutrophils with ANE significantly impaired their ability to phagocytose fluorescent beads. ANE also inhibited phagocytosis of fluorescent beads that were opsonized by complement or IgG. Moreover, expression of F‐actin was inhibited after ANE treatment. Conclusions: ANE inhibits the complement‐ and IgG‐mediated neutrophil phagocytosis that may result from reduction of the expression of complement receptors, Fc receptors, and F‐actin formation after ANE treatment. The findings suggest that areca nut chewing may jeopardize the defensive functions of neutrophils and affect periodontal health.     

Effects of areca nut extract on the apoptosis pathways in human neutrophils

Ho W‐H, Lee Y‐Y, Chang L‐Y, Chen Y‐T, Liu T‐Y, Hung S‐L. Effects of areca nut extract on the apoptosis pathways in human neutrophils. J Periodont Res 2010; 45: 412–420. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: Areca nut, a major component in area quid, possesses genotoxic and carcinogenic activities. Areca nut extract (ANE) may affect the defensive functions of neutrophils. Recent studies suggest that areca nut chewing is associated with a higher prevalence of periodontal disease as a result of the detrimental effects of ANE on the host defense system. This study examined the effects of ANE on the apoptosis pathways in human neutrophils. Material and Methods: Apoptosis/necrosis of neutrophils was determined using flow cytometry. Proteins involved in the apoptosis pathway were determined using western blotting analysis. Results: The results indicated that ANE reduced early apoptosis, but increased the primary necrosis of neutrophils. ANE may arrest neutrophils in the G0/G1 phase and reduce the apoptotic hypodiploid DNA contents. The levels of cleaved forms of poly(ADP‐ribose) polymerase, and of caspase‐3 and caspase‐8 were decreased by treatment with ANE. Moreover, glycogen synthase kinase‐3α/β may be involved in the ANE‐modulated effects of neutrophils. Conclusion: Areca nut may regulate death pathways in neutrophils. This may be one mechanism by which areca nut compromises the periodontal health of areca nut chewers.     

Modulation of KGF-1 gene expression in oral fibroblasts by ripe areca nut extract

BACKGROUND: Areca chewing is a common habit of Asians, leading to a high propensity for a variety of oral diseases in this population. This research aimed to study the expression level of genes in oral fibroblast cell lines in response to exposure to ripe areca nut extract (rANE). METHODS: Fifteen oral fibroblast cell lines obtained from individuals aged 20-77 years were established. Treatment of a cell line with 40 micro g/ml rANE for 24 h was performed to achieve RNA for cDNA microarray analysis. RESULTS: Among some 320 genes exhibiting detectable expression levels, 14 were up-regulated and 26 were down-regulated more than 2.5-fold. Semi-quantitative RT-PCR analysis suggested that up-regulation of IL-6 expression and down-regulation of PDGFR, APP-1 and KGF-1 expressions in multiple cell lines assayed, were compatible with the results of the microarray analysis. Using quantitative real-time RT-PCR analysis, a remarkable down-regulation of KGF-1 expression in response to 40 microg/ml rANE, ranging 1.5-ninefold as compared to controls, was found in 60% (9/15) of the cell lines. CONCLUSION: This study established a novel toxicogenomic database for rANE. The down-regulation of KGF-1 expression in oral fibroblast cell lines potentially impairs the proliferation of overlying keratinocytes, which could partially explain the frequent epithelial atrophy observed in chronic areca chewers in vivo.     

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     

Association between betel quid chewing, periodontal status and periodontal pathogens

The present investigation examined whether an association exists between betel quid chewing and signs of periodontal disease and determined the prevalence of Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis by polymerase chain reaction. The periodontal status of 34 betel quid chewers and 32 non-betel quid chewers were compared. A significantly higher prevalence of bleeding on probing was found in betel quid chewers than non-chewers among the subjects with higher plaque level, greater gingival inflammation, deeper probing depth or greater attachment loss. Also, the results suggested that betel quid chewers may harbor higher levels of infection with A. actinomycetemcomitans and P. gingivalis than non-betel quid chewers. The association persists after adjusting for severity of the clinical parameters. In conclusion, betel quid chewing was associated with a higher prevalence of bleeding on probing where higher clinical levels of disease existed, and with a likelihood of subgingival infection with A. actinomycetemcomitans and P. gingivalis.     

Heat shock protein 27 expression in areca quid chewing-associated oral squamous cell carcinomas

Oral Diseases (2012) 18 , 713–719 Objectives: Heat shock protein (HSP) 27 is a low‐molecular‐weight protein that functions as a molecular chaperone and plays a cytoprotective role through its antioxidant activity during cell stress. Areca quid chewing is associated with the high incidence of oral squamous cell carcinomas (OSCCs) in Taiwan. The aim of this study was to compare heat shock protein 27 (HSP27) expression in OSCCs and the normal oral tissues. Methods: Forty‐eight OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by immunohistochemistry for HSP27. The normal human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot for HSP27. Furthermore, epigallocatechin‐3 gallate (EGCG), glutathione precursor N‐acetyl‐l ‐cysteine (NAC), cyclooxygenase‐2 inhibitor NS‐398, HSP inhibitor quercetin, extracellular signal‐regulated protein kinase (ERK) inhibitor PD98059, and p38 inhibitor SB203580 were added to find the possible regulatory mechanisms. Results: Heat shock protein 27 exhibited higher expression in OSCCs than normal specimens (P < 0.05). Arecoline was found to elevate HSP27 expression in a dose‐ and time‐dependent manner (P < 0.05). The additions of pharmacological agents were found to inhibit arecoline‐induced HSP27 expression (P < 0.05). Conclusions: Heat shock protein 27 expression is significantly elevated in areca quid chewing‐associated OSCCs. Arecoline‐induced HSP27 expression was downregulated by EGCG, NS398, NAC, quercetin, PD98059, and SB203580.     

Effect of chewing a mixture of areca nut and tobacco on periodontal tissues and oral hygiene status

The present study was conducted to clarify the effects of chewing a quid containing areca nut and tobacco on periodontal tissue and oral hygiene status. A total of 365 subjects (168 chewers and 197 non-chewers with a mean age of 32.5 +/- 0.7 and 30.4 +/- 0.8 years, respectively) were enrolled. Clinical data on periodontal tissues, oral hygiene status, as well as information on bleeding from gums, ulcers in the oral cavity, or a burning sensation in the soft tissues, were collected as indicators of the possible presence and extent of periodontal lesions. The results indicated that a significantly higher number of quid-chewers suffered bleeding from the gums, halitosis, difficulty in opening the mouth and swallowing solid food, a burning sensation in the soft tissues, and ulcers in the oral cavity than non-chewers. There was no significant difference between quid-chewers and non-chewers with respect to oral hygiene measures adopted. However, clinical examination using the oral hygiene index score indicated that the oral hygiene status of quid-chewers was significantly deteriorated. The effect of quid-chewing on the periodontium, i.e. the occurrence of periodontal pockets, gingival lesions and gum recession, were significantly higher in quid-chewers than in non-chewers. Age, sex and smoking adjusted odds ratios for quid-chewers against non-chewers using logistic regression analysis indicated that, in general, chewers were at significantly higher risk for various oral complaints and periodontium status. The present data indicate that chewing quid comprising areca nut and tobacco has adverse effects on periodontal tissues, oral hygiene and incidence of oral lesions.     

Oral lichenoid contact lesions induced by areca nut and betel quid chewing: a mini review

Betel quid (BQ) and areca nut chewing is widely prevalent in many parts of Asia and Asian‐migrant communities throughout the world. Global reports estimate 600 million users. Sufficient evidence of carcinogenicity has been found for BQ and its main ingredient, areca nut. BQ areca nut users have an increased risk of potentially malignant disorders. Among chewers, BQ remains in contact with the oral mucosa for prolonged periods. This review examines the clinical and pathological aspects of lichenoid lesions caused by areca nut and BQ, a condition that has received little attention in the published literature.     

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.     

Salivary arecoline levels during areca nut chewing in human volunteers

J Oral Pathol Med (2010) 39 : 465–469 Background: Arecoline stimulates cultured cells above 0.1 μg/ml and is cytotoxic above 10 μg/ml. Although this alkaloid seems important for areca nut induced oral carcinogenesis, little is known of the levels achieved during chewing. Materials and methods: Saliva was collected in 3‐ to 5‐min intervals over 50 min in 32 habitual chewers: before, for 25 min during, and for 20 min after chewing areca nut (0.5 g) without any other additives. Salivary arecoline was quantitated by HPLC‐MS. Controls comprised six subjects who denied areca nut use, and who were given rubber‐base material to chew during experiments instead. Results: Arecoline was detected before chewing in 22 subjects, exceeding the 0.1 μg/ml threshold in 20 cases. Salivary arecoline exceeded either the 0.1 or 10 μg/ml thresholds in all participants during chewing (P < 0.001). Maximum concentrations ranged from 5.66 to 97.39 μg/ml. All subjects reached 0.1 μg/ml salivary arecoline in at least 85% of time points studied (P < 0.0001), whereas 10 μg/ml was reached in 11 participants in at least 30% of the time points (P < 0.003). Arecoline concentrations varied greatly over time between individuals, and levels were much lower when peak concentrations were reached before 3 min, than in cases where arecoline peaked later (P < 0.02). No salivary arecoline was found in control saliva. Conclusions: Areca nut users have persistent background salivary arecoline levels long after chewing, whereas concentrations achieved are highly variable and consistent with a role in oral pre‐malignancy and malignancy.