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

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-3 alpha/beta 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.

Areca nut extracts increased expression of inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8, in peripheral blood mononuclear cells.

BACKGROUND AND OBJECTIVE: Cytokines represent a central role in inflammatory tissue destruction and regulate the immune responses that may govern the progression of periodontal diseases. This study investigated the effects of areca nut extracts on the expression of inflammatory cytokines, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 in peripheral blood mononuclear cells. The role of oxidative stress of areca nut extracts was also examined using curcumin. MATERIAL AND METHODS: The expression of cytokines in peripheral blood mononuclear cells treated with extracts of ripe areca nut or extracts of tender areca nut was analyzed using enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction. RESULTS: Both extracts of ripe areca nut (< or = 40 microg/mL) and extracts of tender areca nut significantly enhanced the production of tumor necrosis factor-alpha and interleukin-1beta in peripheral blood mononuclear cells in a dose-dependent and time-dependent manner. The kinetics of mRNA expression of both cytokines was also enhanced by areca nut extracts. The stimulatory effects of areca nut extracts on the secretion of tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 and on the mRNA expression of tumor necrosis factor-alpha, interleukin-1beta and interleukin-6 at 4 h of incubation were reduced by curcumin (20-50 microm). However, the level of interleukin-8 transcripts was not affected by curcumin. Moreover, interleukin-1beta induction by extracts of tender areca nut, but not by extracts of ripe areca nut, was weakened by 10 microm curcumin. The inhibitory effects of curcumin may vary with different cytokines and with different areca nut extract treatments. CONCLUSION: The complex cytokine profile induced by areca nut extracts-treated peripheral blood mononuclear cells implied the possibility of enhanced local inflammation and altered immune functions by the areca chewing habit. The inhibitory effects of curcumin on cytokine expression suggested that oxidative stress might be involved in areca nut extracts-associated immune alteration.

Areca nut extracts-activated secretion of leukotriene B4, and phosphorylation of p38 mitogen-activated protein kinase and elevated intracellular calcium concentrations in human polymorphonuclear leukocytes.

BACKGROUND AND OBJECTIVE: Polymorphonuclear leukocytes are the major source of leukotriene B4, which is synthesized via the 5-lipoxygenase pathway. Activation of the 5-lipoxygenase pathway is regulated by intracellular calcium and the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The impact of areca nut extracts on the biosynthesis of leukotriene B4 by human polymorphonuclear leukocytes was evaluated, and some of the possible mechanisms underlying the responses were examined. MATERIAL AND METHODS: Polymorphonuclear leukocytes were treated with various concentrations of areca nut extracts. The concentrations of leukotriene B4 released into the supernatants were evaluated using enzyme immunoassay. The phosphorylation of p38 MAPK was monitored using immunoblotting, and the cytosolic calcium kinetics were assessed fluorometrically using Fura-2. RESULTS: Exposure of polymorphonuclear leukocytes to areca nut extracts led to a dose-dependent increase in the production of leukotriene B4, with levels peaking at 30 min and decreasing thereafter. Areca nut extracts enhanced the phosphorylation of p38 MAPK, an enzyme known to activate 5-lipoxygenase. Incubation with areca nut extracts also resulted in a rapid elevation of intracellular calcium concentrations in polymorphonuclear leukocytes. The induction of leukotriene B4 by areca nut extracts was suppressed with the p38 MAPK inhibitor, SB203580, or with the intracellular calcium chelator, BAPTA-AM. CONCLUSION: The interaction of areca nut extracts with polymorphonuclear leukocytes activated the arachidonic acid metabolic cascade. Incubation of polymorphonuclear leukocytes with areca nut extracts resulted in the activation of intracellular events, such as phosphorylation of p38 MAPK and Ca2+ mobilization, involved in the release of pro-inflammatory lipid mediators. The results of this study emphasize the potential importance of polymorphonuclear leukocytes as a source of leukotriene B4, which may modulate the inflammatory response in areca chewers.

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.

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.