Mei Chi Chang

Role of areca nut in betel quid-associated chemical carcinogenesis : current awareness and future perspectives

Betel quid (BQ)-chewing is a popular oral habit with potential links to the occurrence of oral cancer. Many of the literature-based studies reveal that areca nut (AN) extract may demonstrate mutagenic and genotoxic effects, in addition to inducing preneoplastic as well as neoplastic lesions in experimental animals. Areca nut should, thus, be highly suspected as a human carcinogen. Toxicity studies relating to AN-contained polyphenols and tannins are not conclusive, with both carcinogenic and anti-carcinogenic effects being reported. The mutagenicity and genotoxicity of areca alkaloids has been detected by many short-term assays. However, their genotoxicity to oral fibroblasts and keratinocytes, the target cells of BQ, has not been identified. It would thus appear that AN toxicity is not completely due to its polyphenol, tannin and alkaloid content. The single agent which is responsible for AN carcinogenicity awaits further clarification. Reactive oxygen species produced during auto-oxidation of AN polyphenols in the BQ-chewers saliva, are crucial in the initiation and promotion of oral cancer. Nitrosation of areca alkaloids also produces AN-specific nitrosamines, that have been demonstrated to be mutagenic, genotoxic and are capable of inducing tumors in experimental animals. Arecaidine and AN extract are further suggested to be tumor promoters. Antioxidants such as glutathione and N-acetyl-L-cysteine can potentially prevent such AN-elicited cytotoxicity. Further studies are needed to delineate the metabolism of AN ingredient and their roles in the multi-step chemical carcinogenesis, in order to enhance the success of the future chemoprevention of oral cancer and oral submucous fibrosis.

Inducing the cell cycle arrest and apoptosis of oral KB carcinoma cells by hydroxychavicol: roles of glutathione and reactive oxygen species

Hydroxychavicol (HC; 10 – 50 μM), a betel leaf component, was found to suppress the 2% H2O2‐induced lucigenin chemiluminescence for 53 – 75%. HC (0.02 – 2 μM) was also able to trap superoxide radicals generated by a xanthine/xanthine oxidase system with 38 – 94% of inhibition. Hydroxyl radicals‐induced PUC18 plasmid DNA breaks was prevented by HC (1.6 – 16 μM). A 24‐h exposure of KB cells to HC (0.5, 1 mM) resulted in 54 – 74% cell death as analysed by a 3‐(4,5‐dimethyl‐thiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide (MTT) assay. HC (10, 50 μM) further suppressed the growth of KB cells (15 and 76%, respectively). Long‐term colony formation of KB cells was inhibited by 51% with 10 μM HC. Pretreatment of KB cells with 100 μM HC inhibited the attachment of KB cells to type I collagen and fibronectin by 59 and 29%, respectively. Exposure of KB cells to 0.1 mM HC for 24 h resulted in cell cycle arrest at late S and G2/M phase. Increasing the HC concentration to 0.25 and 0.5 mM led to apoptosis as revealed by detection of sub‐G0/G1 peaks with a concomitant decrease in the number of cells residing in late S and G2/M phase. Inducing the apoptosis of KB cells by HC was accompanied by marked depletion in reduced form of GSH (>0.2 mM) and the increasing of reactive oxygen species production (>0.1 mM) as analysed by CMF‐ and DCF‐single cell fluorescence flow cytometry. These results indicate that HC exerts antioxidant property at low concentration. HC also inhibits the growth, adhesion and cell cycle progression of KB cells, whereas its induction of KB cell apoptosis (HC>0.1 mM) was accompanied by cellular redox changes.

Cytotoxicity of sodium fluoride on human oral mucosal fibroblasts and its mechanisms

Because sodium fluoride (NaF) is widely used for prevention of dental caries, pathobiological effects of NaF were investigated on human oral mucosal fibroblasts. The results showed that NaF was cytotoxic to oral mucosal fibroblasts at concentrations of 4 mmol/L or higher. Exposure of cells to NaF for 2 h also inhibited protein synthesis, cellular ATP level and functional mitochondrial activities in a dose-dependent manner. However, incubation of cells with NaF up to 12 mmol/L for 2 h depleted only 13% of cellular glutathione level. The IC50 of NaF on cellular ATP level was about 5.75 mmol/L. Preincubation of the cells with pyruvate and succinate did not protect cells from NaF-induced ATP depletion. At concentrations of 4 mmol/L, 8 mmol/L and 12 mmol/L, NaF inhibited 31%, 56% and 57% of mitochondrial functions, respectively, after 2 h incubation. No significant inhibition for NaF was found at concentrations lower than 2 mmol/L (40 ppm). These results indicate that NaF can be toxic to oral mucosal fibroblasts in vitro by its inhibition of protein synthesis, mitochondrial function and depletion of cellular ATP. Because of repeated and long-term usage of NaF, more detailed studies should be undertaken to understand its toxic effects in vitro and in vivo.

Scavenging property of three cresol isomers against H2O2, hypochlorite, superoxide and hydroxyl radicals.

Formocresol has long been used for pulpotomy of primary teeth and as an intracanal medicament. Little is known, however, about the pharmacological effect of tricresols. This study showed that three cresol isomers, o-cresol, m-cresol and p-cresol, are H2O2 scavengers with a 50% inhibitory concentration (IC50) of 502, 6.7 and 10.16 microM, respectively. o-, m- and p-cresol were also shown to be effective scavengers of superoxide radicals generated by xanthine/xanthine oxidase with an IC50 of 282, 153 and > 4000 microM, respectively, as analyzed by luminometer. o-, m- and p-cresol showed protective effects on the DNA breaks generated by H2O2/FeCl2 and FeCl3/ascorbate/H2O2 systems at concentrations ranging from 70 microM to 1.43 mM, o-, m- and p-cresol also showed differential protective effects against DNA breaks induced by 0.17% NaOCl with 100% inhibitory concentration (IC100) of about 10, 1 and 10 mM, respectively. In addition, reaction with 3% H2O2 and 0.17% NaOCl completely prevented NaOCl-induced DNA breaks. The results indicate that the three cresol isomers are effective ROS scavengers and may prevent ROS induced damage when used as pulpotomy agents or as intracanal medicaments. Owing to the difference in the position of the functional hydroxyl group in the three cresol isomers, m-cresol is the most effective ROS scavenger. Concomitant use of H2O2 for root canal irrigation may diminish both the tissue dissolving capacity of NaOCl and NaOCl-induced DNA damage.

Carboxylesterase expression in human dental pulp cells: Role in regulation of BisGMA-induced prostanoid production and cytotoxicity

Biocompatibility of dentin bonding agents (DBA) and composite resin may affect the treatment outcome (e.g., healthy pulp, pulpal inflammation, pulp necrosis) after operative restoration. Bisphenol-glycidyl methacrylate (BisGMA) is one of the major monomers present in DBA and resin. Prior studies focused on salivary esterase for metabolism and degradation of resin monomers clinically. This study found that human dental pulp cells expressed mainly carboxylesterase-2 (CES2) and smaller amounts of CES1A1 and CES3 isoforms. Exposure to BisGMA stimulated CES isoforms expression of pulp cells, and this event was inhibited by catalase. Exogenous addition of porcine esterase prevented BisGMA- and DBA-induced cytotoxicity. Interestingly, inhibition of CES by bis(p-nitrophenyl) phosphate (BNPP) and CES2 by loperamide enhanced the cytotoxicity of BisGMA and DBA. Addition of porcine esterase or N-acetyl-l-cysteine prevented BisGMA-induced prostaglandin E(2) (PGE(2)) and PGF(2α) production. In contrast, addition of BNPP and loperamide, but not mevastatin, enhanced BisGMA-induced PGE(2) and PGF(2α) production in dental pulp cells. These results suggest that BisGMA may induce the cytotoxicity and prostanoid production of pulp cells, leading to pulpal inflammation or necrosis via reactive oxygen species production. Expression of CES, especially CES2, in dental pulp cells can be an adaptive response to protect dental pulp against BisGMA-induced cytotoxicity and prostanoid release. Resin monomers are the main toxic components in DBA, and the ester group is crucial for monomer toxicity.

Signaling mechanism of thrombin‐induced gingival fibroblast‐populated collagen gel contraction

Thrombin is activated during gingival tissue injury and inflammation. Thrombin (platelet)‐rich plasma has been used for periodontal regeneration with success. Thrombin and other bacterial proteases also affect the functions of adjacent periodontal cells via stimulation of protease‐activated receptors (PARs). We noted that thrombin (0.1–2 U ml−1), human, and frog PAR‐1 agonist peptide (20–240 μM) induced the gingival fibroblast (GF)‐populated collagen gel contraction within 2 h of exposure. However, PAR‐2, PAR‐3, and PAR‐4 agonist peptide (20–240 μM) showed little effect on collagen gel contraction. U73122 (phospholipase C inhibitor) and 2‐APB (IP3 antagonist) were effective in inhibition of GF contraction. Thrombin‐induced GF contraction was inhibited by 5 mM EGTA (an extracellular calcium chelator) and verapamil (an L‐type calcium channel blocker). In addition, W7 (10 and 25 μM, a calcium/calmodulin (CaM) inhibitor), ML‐7 (50 μM, myosin light chain kinase (MLCK) inhibitor), and HA1077 (100 μM, Rho kinase inhibitor) completely inhibited the thrombin‐induced collagen gel contraction. Thrombin also induced the phosphorylation of ERK1/ERK2 and elevated the Rho‐GTP levels in GF. However, U0126 only partially inhibited the thrombin‐induced GF contraction. Similarly, wortmannin (100 nM), LY294002 (20 μM) (two PI3K inhibitor) and genistein also showed partial inhibition. Moreover, NAC was not able to suppress the GF contraction, as supported by the slight decrease in reactive oxygen species production in GF by thrombin. Thrombin also stimulated metalloproteinase‐2 (MMP‐2) and MMP‐3 production in GF. But addition of GM6001 or 1,10‐phenanthroline, two MMP inhibitors, could not inhibit the thrombin‐induced GF contraction. These results indicate that thrombin is crucial in the periodontal inflammation and wound healing by promoting GF contraction. This event is mainly mediated via PAR‐1 activation, PLC activation, extracellular calcium influx via L‐type calcium channel, and the calcium/CaM–MLCK and Rho kinase activation pathway.

Areca nut-induced buccal mucosa fibroblast contraction and its signaling: a potential role in oral submucous fibrosis—a precancer condition

Betel quid (BQ) chewing is an oral habit that increases the risk of oral cancer and oral submucous fibrosis (OSF), a precancerous condition showing epithelial atrophy and tissue fibrosis. Persistent fibroblast contraction may induce the fibrotic contracture of tissue. In this study, we found that areca nut extract (ANE) (200-1200 µg/ml) stimulated buccal mucosa fibroblast (OMF)-populated collagen gel contraction. Arecoline but not arecaidine-two areca alkaloids, slightly induced the OMF contraction. Exogenous addition of carboxylesterase (2U/ml) prevented the arecoline- but not ANE-induced OMF contraction. OMF expressed inositol triphosphate (IP3) receptors. ANE-induced OMF (800 µg/ml) contraction was inhibited by U73122 [phospholipase C (PLC) inhibitor] and 2-aminoethoxydiphenyl borate (IP3 receptor antagonist), respectively. Ethylene glycol tetraacetic acid and verapamil, two calcium mobilization modulators, also suppressed the ANE-induced OMF contraction. ANE induced calcium/calmodulin kinase II and myosin light chain (MLC) phosphorylation in OMF. Moreover, W7 (a Ca(2+)/calmodulin inhibitor), HA1077 (Rho kinase inhibitor), ML-7 (MLC kinase inhibitor) and cytochalasin B (actin filament polymerization inhibitor) inhibited the ANE-induced OMF contraction. Although ANE elevated reactive oxygen species (ROS) level in OMF, catalase, superoxide dismutase and N-acetyl-L-cysteine showed no obvious effect on ANE-elicited OMF contraction. These results indicate that BQ chewing may affect the wound healing and fibrotic processes in OSF via inducing OMF contraction by ANE and areca alkaloids. AN components-induced OMF contraction was related to PLC/IP3/Ca(2+)/calmodulin and Rho signaling pathway as well as actin filament polymerization, but not solely due to ROS production.

Transforming growth factor β1 down-regulates Runx-2 and alkaline phosphatase activity of human dental pulp cells via ALK5/Smad2/3 signaling

OBJECTIVE Transforming growth factor β1 (TGF-β1) plays a role in repair and dentinogenesis in dental pulp. The purpose of this study was to study how TGF-β1 affects 2 differentiation markers, Runt-related transcription factor 2 (Runx-2) and ALP, in dental pulp cells. STUDY DESIGN Primary-cultured human dental pulp cells were treated with TGF-β1 with or without pretreatment and coincubation with 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto)butadiene (U0126, a mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor), Noggin (a bone morphogenetic protein inhibitor), or 4-(5-benzol[1,3]dioxol-5-yl-4-pyrldin-2-yl-1H-imidazol-2-yl)-benzamide hydrate (SB431542, an activin receptor-like kinase (ALK) 5/Smad2/3 inhibitor). The differentiation status of pulp cells was evaluated by ALP staining and quantitative ALP activity assay. Changes in ALP and Runx-2 mRNA expression were determined by reverse-transcription polymerase chain reaction. RESULTS Cells under the treatment of TGF-β1 (5 and 10 ng/mL) showed a decrease in ALP activity and gene expression of ALP and Runx-2. Pretreatment by U0126 and Noggin was not effective to prevent the TGF-β1-induced decline of ALP activity. Interestingly, SB431542 prevented the TGF-β1-induced decline of ALP activity and ALP and Runx-2 gene expression. CONCLUSION TGF-β1 down-regulates Runx-2 and ALP in human dental pulp cells via ALK5/Smad2/3 signaling. These events may play important roles at specific stages of pulpal repair and dentinogenesis.

Prevention of the areca nut extract-induced unscheduled DNA synthesis of gingival keratinocytes by vitamin C and thiol compounds

There are about 600 million betel quid (BQ) chewers in the world. BQ chewing is the major risk factor of oral cancer in India, Taiwan, South Africa and numerous other countries. Areca nut (AN) extract, the main component of BQ, exerts cytotoxicity and genotoxicity to several types of cells. In the present study, AN extract induced the unscheduled DNA synthesis (UDS) of gingival keratinocytes (GK). Vitamin C, at concentration of 50 and 200 microg/ml prevented the AN-induced UDS by 41 and 56%, respectively. Glutathione (GSH, 1-3 mM) and N-acetyl-L-cysteine (NAC, 1-3 mM) also protected the AN-induced UDS by 89-100 and 76-90%. These preventive effects were not due to cytotoxicity as analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Deferoxamine (20 and 30 mM), an iron chelator and a free radical scavenger, also prevented AN extract induced UDS of GK by 30-55%. On the contrary, banthocuproine (50-200 microM, a copper chelator) and 1,10-phenanthroline (50, 100 microM, a lipid permeable iron chelator), lacked preventive effects. Specific reactive oxygen species scavengers such as dimethyl-sulfoxide (2%), mannitol (10-20 mM), dimethylthiourea (10-20 mM), pyruvate (10 mM), catalase (200 and 400 U/ml), and superoxide dismutase (50 and 200 U/ml) also lacked these preventive effects. Moreover, higher concentrations of H(2)O(2) (0.5-1 mM) inhibited the basal levels of UDS by 19-37%. Interestingly, NAC, GSH, Vitamin C and deferoxamine cannot prevent the AN-induced morphological changes of GK at similar concentrations. These results reveal that AN extract-induced UDS of GK is associated with free radical reactions. Possibly different ingredients of AN is responsible for genotoxicity and cytotoxicity. Vitamin C, GSH and NAC may be potentially used in the future for chemoprevention of BQ chewing related oral mucosal lesions.

Regulation of vascular cell adhesion molecule-1 in dental pulp cells by interleukin-1β: The role of prostanoids

INTRODUCTION Vascular cell adhesion molecule (VCAM-1) plays a critical role in the inflammatory processes by stimulating the recruitment, extravasation, and migration of leukocytes. Its expression and regulation in the dental pulp is not well elucidated. METHODS Primary dental pulp cells were exposed to prostaglandin E(2) (PGE(2)), prostaglandin F(2α) (PGF(2α)), or interleukin 1β (IL-1β) with/without aspirin. VCAM-1 messenger RNA expression was analyzed by reverse transcriptase-polymerase chain reaction. Soluble VCAM-1 (sVCAM-1) in the culture medium was determined by enzyme-linked immunosorbent assay, and the number of viable cells was estimated by (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS IL-1β induced VCAM-1 gene expression of pulp cells. IL-1β also stimulated sVCAM-1 production. The IL-1β-induced sVCAM-1 production was not inhibited but rather enhanced by aspirin, a cyclooxygenase (COX) inhibitor. PGE(2) and PGF(2α) decreased the VCAM-1 expression and sVCAM-1 production of pulp cells. U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), a mitogen-activated protein kinase kinase (MEK) inhibitor, attenuated IL-1β-induced sVCAM-1 production. However, no marked cytotoxicity was noted in these experimental conditions as analyzed by MTT assay. CONCLUSIONS IL-1β may be involved in the pulpal inflammatory processes via stimulation of VCAM-1 expression and sVCAM-1 production. This event is not mediated by COX activation and prostanoid production but is associated with MEK signaling. PGE(2) and PGF(2α) may potentially regulate inflammatory processes by the inhibition of VCAM-1.