Areca nut extracts suppress the differentiation and functionality of human monocyte-derived dendritic cells

Wang C‐C, Chen T‐Y, Wu H‐Y, Liu T‐Y, Jan T‐R. Areca nut extracts suppress the differentiation and functionality of human monocyte‐derived dendritic cells. J Periodont Res 2012; 47: 198–203. © 2011 John Wiley & Sons A/S Background and Objective: Areca quid chewing, a major risk factor contributing to the occurrence of oral cancer and precancer, has been reported to be associated with the severity and high prevalence of periodontal diseases in areca quid chewers. As dendritic cells are critically involved in the regulation of innate and adaptive immunity in oral mucosa, the objective of the present study was to investigate the effect of areca nut extracts (ANE) on the differentiation and reactivity of dendritic cells derived from monocytes. Material and Methods: Human peripheral blood monocytes were cultured in the presence of granulocyte–monocyte colony‐stimulating factor and interleukin‐4 for 7 d to generate dendritic cells. To examine the effect of ANE on the generation of dendritic cells, the monocytes were exposed to ANE throughout the 7 d culture period. In addition, the effect of ANE on the maturation of monocyte‐derived dendritic cells induced by lipopolysaccharide (LPS) was examined. Results: Monocytes cultured in granulocyte–monocyte colony‐stimulating factor and interleukin‐4 exhibited a typical phenotype of dendritic cells, as evidenced by the heightened expression of human leukocyte antigen (HLA)‐DR, CD11c and the co‐stimulatory molecules CD40, CD80 and CD86. Exposure of the monocytes to ANE did not influence the expression of HLA‐DR and CD11c, but markedly attenuated the proportion of CD40‐positive cells and the mean fluorescence intensity of CD86. The expression of co‐stimulatory molecules in LPS‐activated dendritic cells was not affected, whereas the mRNA expression of interleukin‐12 induced by LPS was markedly suppressed by ANE treatment in a concentration‐dependent manner. Conclusion: These results suggest that ANE exposure interfered with the differentiation of dendritic cells from monocytes. Moreover, the functionality of mature monocyte‐derived dendritic cells was attenuated in the presence of ANE.     

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 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.     

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.     

Areca nut extracts enhance the development of CD11b+Gr‐1+ cells with the characteristics of myeloid‐derived suppressor cells in antigen‐stimulated mice

J Oral Pathol Med (2011) 40 : 769–777 Background: Areca quid chewing is an etiological factor contributing to the development of oral cancer and pre‐cancers, whose pathophysiology has been linked to inflammation and immune deterioration. Myeloid‐derived suppressor cells (MDSC) play a key role in the regulation of immunity under certain pathological conditions, such as inflammation and cancer. As areca nut extracts (ANE) have been reported to induce a proinflammatory effect in antigen‐stimulated mice, we hypothesized that ANE might enhance the development of MDSC. Methods: Ovalbumin (OVA)‐sensitized BALB/c mice were daily administered with ANE (5–50 mg/kg), polyphenol‐enriched ANE (PANE; 25 mg/kg) or arecoline (5 mg/kg) by intraperitoneal injection for 10 doses. The mouse footpads were then subcutaneously challenged with OVA to induce local inflammatory responses. Results: ANE and PANE treatment significantly increased the spleen index and the population of CD11b⁺Gr‐1⁺ cells in the spleen and peripheral blood, whereas arecoline was inactive. In addition, ANE and PANE treatment enhanced the expression of cytokines and enzymes associated with the immunosuppressive function of MDSC, including IL‐10, arginase‐I and iNOS in splenic CD11b⁺ cells. Concordantly, ANE and PANE treatment augmented the infiltration of Gr‐1⁺IL‐10⁺ cells in the footpads challenged with OVA. Conclusions: Our results suggested that areca nut constituents, in particular, polyphenols enhanced the development of myeloid‐derived suppressor cells in vivo, which may be a critical mechanism linking inflammation and the compromised immunity reported to be associated with the pathophysiology of areca‐related oral diseases.     

Areca nut extract-treated gingival fibroblasts modulate the invasiveness of polymorphonuclear leukocytes via the production of MMP-2

Background:  Areca nut chewing is associated with an increase in the incidence of oral neoplastic or inflammatory diseases. Aberrations in matrix metalloprotease (MMP) expression are associated with the pathogenesis of oral diseases. This study investigated the potential effects of areca nut extract (ANE) on human gingival fibroblasts and the consequential impacts on inflammatory pathogenesis.
Methods:  Analyses of senescence marker, cell viability, changes of the cell cycle, and cell granularity in gingival fibroblasts together with an assessment of the invasiveness of polymorphonuclear (PMN) leukocytes after treatment with the supernatant of ANE-treated gingival fibroblasts were performed to characterize the phenotypic impacts. Western blotting and gelatin zymography were used to assay the expression and activity of MMP-2.
Results:  Chronic subtoxic (<10 μg/ml) ANE treatment resulted in premature growth arrest, appearance of senescence-associated β-galactosidase activity and various other senescence-associated phenotypes in gingival fibroblasts. Gingival fibroblasts established from older individuals had a higher propensity to become ANE-induced senescent gingival fibroblasts. An activation of MMP-2 was identified in senescent cells. PMN leukocytes treated with the supernatant of ANE-induced senescent cells exhibited a significant increase in invasiveness, which was abrogated by both a MMP-2 blocker and a MMP-2 nullifying antibody.
Conclusions:  This study provides evidence whereby MMP-2 secreted from ANE-induced senescent gingival fibroblasts would facilitate the invasiveness of PMN leukocytes, which could be associated with the oral inflammatory process in areca chewers.     

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.     

Areca nut extract upregulates vimentin by activating PI3K/AKT signaling in oral carcinoma

J Oral Pathol Med (2011) 40 : 160–166 Background: Areca nut is a group I carcinogen. Areca nut extract (ANE) is known to activate signaling pathways in oral epithelial cells. Activation of the serine/threonine protein kinase AKT/pKB (AKT) signaling pathway is known to be important during the neoplastic process. Vimentin is a mesenchymal intermediate filament and a regulator of tumor progression. This study investigated the impact of ANE on PI3K/AKT activation during vimentin expression. Materials and methods: Oral carcinoma cells were treated with ANE to explore the signaling changes underlying vimentin expression. Oral carcinoma tissues were subjected to immunohistochemical analysis to study the implications that vimentin expression has on patient survival. Results: After ANE treatment, the OECM‐1 and Fadu cells developed a fibroblastoid morphology and there was an increase in vimentin expression. The treatment also induced the phosphorylation of AKT and glycogen synthase kinase 3β in OECM‐1 cells. Blockage of phosphatidylinositol 3‐kinase (PI3K)/AKT signaling attenuated vimentin expression when it was induced by ANE. However, it did not affect ANE‐mediated extracellular signal‐regulated kinase (ERK) activation or cyclooxygenase 2 (COX‐2) upregulation. Oral carcinoma tissue samples were found to have significantly higher levels of vimentin and pAKT expression than their controls. Tumors exhibiting no vimentin expression and weak AKT phosphorylation were found to be associated with better survival than groups with high levels of expression. Conclusion: Our results imply that PI3K/AKT activation and vimentin expression are important pathogenic cascades in areca‐associated oral carcinogenesis.     

Nicotine and smokeless tobacco effects on gingival and peripheral blood mononuclear cells

Abstract. The pathogenesis of tobacco-related periodontal diseases is not well understood. The purpose of this study was therefore to investigate smokeless tobacco extract (ST) and nicotine effects on prostaglandin E₂ (PGE₂) and interleukin-1β (IL-1β) secretion by peripheral blood mononuclear cells (PBMC, consisting of monocytes and lymphocytes) and gingival mononuclear cells (GMC). Both peripheral blood and gingival tissue adjacent to the alveolar crest were taken from non-smoking adult periodontitis patients. Gingival tissue was treated with collagenase and deoxyribonuclease and GMC and PBMC were isolated by Ficoll-Hypaque centrifugation. GMC and PBMC (100,000 cells/200 μl) were cultured for 24 hours in supplemented RPMI 1640 alone (control), or in supplemented RPMI 1640 containing 1% ST, 100μg/ml nicotine, 1 μg/ml Porphyromonas gingivalis LPS, or 1 μg/ml P. gingivalis LPS and either 100 μg/ml nicotine or 1% ST. Enzyme immunoassays were used to quantity PGE₂ and IL-1β. Treatments were compared by repeated measures ANOVA. 100 μg/ml nicotine (7-fold, p<0.02) and 1% ST (3.5-fold, p<0.004) significantly increased secretion of PGE₂ by PBMC relative to control cultures. 100 μg/ml nicotine and 1% ST, however, had no effect on IL-1β secretion by PBMC. Enhanced PGET secretion also was seen when PBMC were treated with P. gingivalis LPS+100 μg/ml nicotine relative to P. gingivalis LPS alone (p<0.007). In contrast, 100 μg/ml nicotine significantly downregulated IL-1β secretion by GMC relative to medium alone (p<0.008) and had no effect on PGE₂ secretion by GMC, These data indicate that while nicotine and ST can stimulate PBMC to secrete PGE₂, they cannot activate further mononuclear cells extracted from gingiva, possibly due to maximal previous stimulation in the periodontitis lesion.     

Nicotine inhibits human gingival fibroblast migration via modulation of Rac signalling pathways

AIM: Cigarette smoking is a risk factor in the development of periodontal diseases. In addition, a delayed healing process has been shown in smokers compared with non-smokers after periodontal treatment. Cell migration is a key process of wound healing and it is highly regulated by a variety of signalling pathways. The small G protein, Rac, is necessary for cell migration. Our aim was to determine if nicotine disrupted Rac and its downstream signalling proteins, p21-activated kinase 1/2 (PAK1/2), and p44/42 mitogen-activated protein kinase (MAPK) (extracellular regulated kinase 1/2). MATERIAL AND METHODS: Primary human fibroblasts from healthy gingival tissues were cultured and grown to confluence. Cells were serum starved for 24 h, and then treated with nicotine (0 or 0.5 microM) prior to in vitro wounding. Cell migration was analysed in live cell assays following in vitro wounds. Rac activity, phosphorylation levels of PAK1/2, and p44/42 MAPK were assessed in cultures treated with or without nicotine after multiple wounds. RESULTS: Nicotine decreased cell migration rates by 50% compared with controls. In addition, nicotine altered the activation patterns of Rac and PAK 1/2 and up-regulated p44/42 MAPK. CONCLUSION: Decreased cell migration in periodontal wounds exposed to nicotine may be mediated through the Rac and PAK1/2 signalling pathways.     

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.     

Areca nut extract treatment elicits the fibroblastoid morphological changes, actin re-organization and signaling activation in oral keratinocytes

BACKGROUND: Areca (named as betel) is an important etiological factor linked with the high prevalence of oral squamous cell carcinoma (OSCC) in South-Asian countries. This in vitro study investigated the cellular changes and signaling activation in oral keratinocytes in response to areca nut extract (ANE) treatment. METHODS: Normal human oral keratinocyte (NHOK) and oral epidermoid carcinoma cell, Meng-1 (OECM-1) OSCC cell line were treated with variable dosages of ripen ANE. The morphological and cytoskeletal changes, as well as the activation of GTPase proteins and signaling kinases, were analyzed. RESULTS: Most NHOK cells in culture were polygonal, with only <5% cells exhibiting fibroblastoid morphology. However, 10 microg/ml ANE elicited fibroblastoid morphological change, genesis of lamellipodia, loss of subcortical actin, and stress-fiber formation in approximately 25% cultivated NHOK cells. Similar morphological changes were observed in nearly all OECM-1 cells following the ANE treatment. The activation of Rac and Rho GTPase, together with the prominent phosphorylation of a stress-activated kinases, particularly JNK1, was identified in treated OECM-1 cells. CONCLUSION: The novel evidences from the study that ANE impairs the actin organization and activates the signals in oral keratinocytes might bestow further insight into the impacts of ANE in oral pathogenesis.     

Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils

BACKGROUND: Areca quid chewers have a higher prevalence of periodontal disease than non-chewers. Little is known about the influence of areca quid on the immune system. This study was to determine the possible effects of the areca nut on phagocytic activity of human neutrophils. METHODS: Aqueous extracts of ripe areca nut without husk (rANE), fresh and tender areca nut with husk (tANE), a major alkaloid (arecoline), and a phenolic component ([+]-catechin) of areca nut were examined for their effects on cellular viability using trypan blue exclusion assay. The possible effects on the phagocytic activity of neutrophils against a periodontal pathogen, Actinobacillus actinomycetemcomitans ATCC 33384, were determined using flow cytometry and confocal laser scanning microscopy. RESULTS: At the concentrations tested, rANE, tANE, arecoline, and (+)-catechin did not significantly affect viability of neutrophils. However, rANE, tANE, arecoline, and (+)-catechin inhibited the phagocytic activity of neutrophils in a dose-dependent manner. Approximately 50% of the relative phagocytic activity of neutrophils was affected when 50 microg/ml of rANE, 400 microg/ml of tANE, 20,000 microg/ml of arecoline, or 2,500 microg/ml of (+)- catechin was used. Decreased levels of internalized fluorescent bacteria were also demonstrated. However, arecoline or (+)-catechin alone could not be used to explain the inhibitory effects observed for rANE and tANE. CONCLUSIONS: Components of areca nut reduced the uptake of A. actinomycetemcomitans ATCC 33384 by human neutrophils. The inhibition of areca nut on phagocytosis of neutrophils may be one possible mechanism by which the areca nut compromises the periodontal health of areca quid chewers.     

Simvastatin alters fibroblastic cell responses involved in tissue repair

Cáceres M, Romero A, Copaja M, Díaz‐Araya G, Martínez J, Smith PC. Simvastatin alters fibroblastic cell responses involved in tissue repair. J Periodont Res 2011; 46: 456–463. © 2011 John Wiley & Sons A/S Background and Objective: Statins have been used to control hypercholesterolemia. However, these drugs also exert pleiotropic effects that include the modulation of inflammation and cell signaling. The present study has analyzed the effects of simvastatin on several cell responses involved in tissue repair, including cell adhesion, cell migration and invasion, actin cytoskeleton remodeling and cell viability. Material and Methods: Primary cultures of gingival fibroblasts were stimulated with simvastatin. Cell adhesion was evaluated using a colorimetric assay. Cell spreading was evaluated microscopically. Cell migration and invasion were assessed using a scratch wound‐healing assay and a bicameral cell culture system, respectively. Changes in actin cytoskeleton and focal adhesion assembly were evaluated through immunofluorescence for actin, vinculin and active β1 integrin. Rac activation was evaluated by means of a pull‐down assay. Cell viability was assessed using a colorimetric assay that determines mitochondrial functionality. Data analysis was performed using the Mann–Whitney U‐test. Results: Simvastatin diminished cell adhesion and spreading over a fibronectin matrix. It also altered the closure of scratch wounds induced on cell monolayers and cell invasion through a Transwell system. Simvastatin‐treated cells displayed an altered lamellipodia with poorly developed focal adhesion contacts and reduced levels of β1 integrin activation. During cell spreading, simvastatin diminished Rac activation. Conclusion: The present study shows that simvastatin may alter cell migration by disrupting the cell signaling networks that regulate the actin cytoskeleton dynamics. This mechanism may affect the response of gingival mesenchymal cells during wound healing.     

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.     

Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis

Background and Objective: Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. This study was designed to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. Material and Methods: Ligature‐induced periodontitis was applied in periodontitis‐susceptible Fischer 344 rats. The animals were either given daily intraperitoneal injections of the nicotinic acetylcholine receptor antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous injections in the neck skin of nicotine (0.8 mg/kg), or treated with the same amount of saline intraperitoneally and nicotine subcutaneously, or treated with mecamylamine and saline. Control animals received intraperitoneal and subcutaneous injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 wk. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 μg/kg, intraperitoneally) to induce a robust immune and stress response. Results: Compared with saline/saline‐treated control animals, saline/nicotine‐treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor‐α, transforming growth factor‐1β and interleukin‐10. Mecamylamine pretreatment of nicotine‐treated rats abrogated the increased periodontal bone loss and the LPS‐induced decrease in tumour necrosis factor‐α, but had no significant effects on the levels of transforming growth factor‐1β and interleukin‐10, or the stress hormone corticosterone. Conclusion: The results indicate that nicotine enhances the susceptibility to periodontitis via nicotinic acetylcholine receptors, which may act by suppressing protective immune responses through the cholinergic anti‐inflammatory pathway.     

Cytogenetic alterations in buccal mucosa cells of chewers of areca nut and tobacco

Objective

The rationale of the study was to evaluate the cytological alterations especially micronucleus (MN) and other nuclear anomalies in buccal mucosa cells of chewers to understand the genotoxic and clastogenic potential of chewing mixture (containing areca nut and tobacco as main ingredients).

Methods

The buccal cytome assay involves the examination of epithelial smear to determine micronucleated cell and other nuclear anomalies after the Feulgen plus light green staining. The assay was applied to exfoliated buccal mucosa cells of 262 subjects [non-chewers – 161 and chewers – 101 (includes 20 subjects with OSMF)] and 1000 cells per individual were examined microscopically. Nuclear anomalies were compared among chewers, non-chewers and OSMF subjects and correlated with consumption of quids per day and duration of chewing in years.

Results

MN cells were found significantly (p < 0.0001) higher among chewers and OSMF subjects as compared to non-chewers. Further analysis indicated that MN was significantly higher in OSMF subjects with respect to even chewers. Nuclear buds were significantly higher (p < 0.0001) in OSMF subjects as compared to chewers as well as non-chewers. Nuclear anomalies viz. binucleated, karyorrhexis and karyolysis were also considerably higher in OSMF subjects as compared to non-chewers.

Conclusion

The MN and other nuclear anomalies reflected genetic damage and cytotoxicity, associated with tobacco and areca nut consumption. Further, these data reveal a risk for development of OSMF among chewers of mixture containing areca nut and/or tobacco, as all the OSMF subjects were chewers.     

Nicotinic acetylcholine receptor activation mediates nicotine-induced enhancement of experimental periodontitis

Background and Objective: Smokers have an increased risk of developing periodontitis as well as showing more rapid progression and resistance to treatment of the disease, but the biological mechanisms are poorly understood. Our objective was to investigate putative biological mechanisms by which nicotine may enhance the susceptibility and thus the course of periodontitis in an animal model. Material and Methods: Ligature‐induced periodontitis was applied in periodontitis‐susceptible Fischer 344 rats. The animals were given daily intraperiotonal (i.p.) injections of the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine (1 mg/kg) 45 min before subcutaneous (s.c.) injections in the neck skin with nicotine (0.8 mg/kg), or treated with the same amount of saline i.p. and nicotine s.c., or with mecamylamine and saline. Control rats received i.p. and s.c. injections of saline only. Periodontal bone loss was assessed when the ligatures had been in place for 3 weeks. Two hours before decapitation, all rats received lipopolysaccharide (LPS; 100 μg/kg, i.p.) to induce a robust immune and stress response. Results: Compared with saline/saline‐treated control rats, saline/nicotine‐treated rats developed significantly more periodontal bone loss, and LPS provoked a significantly smaller increase in circulating levels of the cytokines tumour necrosis factor α (TNF‐α), transforming growth factor 1β (TGF‐1β) and interleukin‐10 (IL‐10). Mecamylamine pretreatment of nicotine‐treated rats abrogated the increased periodontal bone loss and the LPS‐induced TNF‐α decrease, but had no significant effects on the levels of TGF‐1β and IL‐10, or the stress hormone corticosterone. Conclusion: The results indicate that nicotine enhances susceptibility to periodontitis via nAChRs, which may act via suppressing protective immune responses through the cholinergic anti‐inflammatory pathway.