Effects of transforming growth factor-ß1 on plasminogen activation in stem cells from the apical papilla: role of activating receptor-like kinase 5/Smad2 and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signalling.

AIM: To study the effects of TGF-ß1 on the plasminogen activation (PA) system of stem cells from the apical papilla (SCAP) and its signalling. METHODOLOGY: SCAP cells were isolated from the apical papilla of immature permanent teeth extracted for orthodontic reasons. They were exposed to various concentration of TGF-ß1 with/without pretreatment and coincubation by SB431542 (ALK/Smad2/3 inhibitor), or U0126 (MEK/ERK inhibitor). MTT assay, Western blotting and enzyme-linked immunosorbent assay (ELISA) were used to detect their effects on cell viability, and the protein expression of plasminogen activator inhibitor-1 (PAI-1), urokinase-type plasminogen activator (uPA), uPA receptor (uPAR) and their secretion. The paired Student's t-test was used for statistical analysis. RESULTS: TGF-ß1 significantly stimulated PAI-1 and soluble uPAR (suPAR) secretion of SCAP cells (P < 0.05), whereas uPA secretion was inhibited. Accordingly, TGF-ß1 induced both PAI-1 and uPAR protein expression of SCAP cells. SB431542 (an ALK5/Smad2/3 inhibitor) pretreatment and coincubation prevented the TGF-ß1-induced PAI-1 and uPAR of SCAP. U0126 attenuated the TGF-ß1-induced expression/secretion of uPAR, but not PAI-1 in SCAP. SB431542 reversed the TGF-ß1-induced decline of uPA. CONCLUSIONS: TGF-ß1 may affect the repair/regeneration activities of SCAP via differential increase or decrease of PAI-1, uPA and uPAR. These effects induced by TGF-ß1 are associated with ALK5/Smad2/3 and MEK/ERK activation. Elucidation the signalling pathways and effects of TGF-ß1 is useful for treatment of immature teeth with open apex by revascularization/revitalization procedures and tissue repair/regeneration.

Horizontal root fractures in posterior teeth without dental trauma: tooth/root distribution and clinical characteristics.

AIM: To describe the clinical characteristics and radiographic findings of horizontal root fractures (HRF) in posterior teeth without a history of dental trauma. METHODOLOGY: A total 24 patients and 31 HRF cases in 28 posterior teeth were collected from 2006 to 2015. Clinical examinations and radiographic imaging were evaluated. Value of confidence intervals of the proportions was calculated for data presentation. RESULTS: The number of males (54%) was similar to females (46%). The patients were predominantly between 50 and 70 years of age (75%). Most HRF cases were found in nonendodontically treated teeth (79%), without crown and bridge restorations (82%), and maxillary molars (54%). Many roots of maxillary molars had developed HRF, and the probability was nearly equal. Fractured teeth usually presented with periodontal and apical bone loss, and most patients (92%) were diagnosed with full mouth chronic periodontitis. Tooth wear was another common clinical feature amongst these patients. CONCLUSIONS: HRF in posterior teeth without dental trauma occurred mainly in patients aged between 50 and 70, in nonendodontically treated teeth, teeth with attrition but without crown and bridge restorations, maxillary molars and with periodontal and periapical bony destruction. Periodontal condition, occlusal wear and patients' age at diagnosis were the possible related factors. HRF in posterior teeth without dental trauma is a diagnostic challenge and even misdiagnosed. A thorough clinical examination, radiographic analysis and recognition of the clinical characteristics are helpful in the early diagnosis and treatment of HRF.

Differential regulation of collagen, lysyl oxidase and MMP-2 in human periodontal ligament cells by low- and high-level mechanical stretching.

BACKGROUND AND OBJECTIVE: Mechanical stretching modulates extracellular matrix (ECM) protein synthesis by periodontal ligament (PDL) cells. However, the mechanoregulation of lysyl oxidase (LOX), a key enzyme for collagen cross-linking, is not fully understood. In the present study, we hypothesized that low-level and high-level mechanical stretching differentially regulates collagen deposition and the expression of LOX and the enzymes responsible for ECM degradation, such as MMP-2 in PDL cells. MATERIAL AND METHODS: Human PDL cells were cultured on flexible-bottom culture plates and subjected to cyclic mechanical stretching (3% and 10% elongation at 0.1 Hz) for 24 and 48 h in a Flexercell FX-4000 strain unit. The levels of expression of type I collagen alpha 1 (COL1A1), type III collagen alpha 1 (COL3A1), lysyl oxidase (LOX), MMP2 and TIMP2 mRNAs were analyzed using an RT-PCR technique. The cell layer and the culture medium were separately collected and processed for detection of the following ECM-related molecules: (i) total collagen content using a Sircol dye-binding method; (ii) LOX protein expression by western blotting; (iii) LOX activity using a fluorometric assay; and (iv) MMP-2 enzyme activity by gelatin zymography. RESULTS: Low-level (3%) mechanical stretching of PDL cells upregulated the expression of COL1A1, COL3A1 and LOX mRNAs, enhanced the production of collagen and increased the LOX activity but did not change the level of expression of MMP2 or TIMP2 mRNA. The collagen content and LOX activity showed obvious elevation in the medium, but not in the cell layer. High-level (10%) mechanical stretching downregulated COL1A1 mRNA but upregulated COL3A1 mRNA; however, the effect on COL3A1 was smaller, and occurred earlier, compared with the effect on the COL1A1 gene. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs but did not change collagen production or LOX activity. Moreover, high-level mechanical stretching increased the level of pro-MMP-2, especially in the cell layer. CONCLUSIONS: This study substantiates the mechanoregulation of the expression of ECM-related molecules in PDL cells. High-level mechanical stretching upregulated the expression of MMP2 and TIMP2 mRNAs, but did not affect collagen production or LOX activity. In addition to increasing the transcription of COL1A1, COL3A1 and LOX genes, low-level mechanical stretching enhanced total collagen production and LOX activity, which should favor ECM stabilization. As an effective regulator of ECM remodeling, mechanical stretching can be exploited in periodontal regeneration and ligament tissue engineering via application of appropriate mechanical stimulation.

Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. MATERIAL AND METHODS: Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively. RESULTS: Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. CONCLUSION: These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.

A fractal dimensional approach to successful evaluation of apical healing.

AIM: To evaluate whether the initial healing of apical radiolucencies 1 year after root canal treatment could be quantitatively identified by the change in fractal dimension (FD) values for the eventually completely healed cases. METHODOLOGY: Twenty-six patients with successful root canal treatment were recruited. All teeth were associated with complete healing either before or at 1 year following treatment (six of 26) or still undergoing healing at 1 year after treatment but completely healed thereafter (20 of 26). Two radiographs were selected for the same patient, one taken before treatment and the other taken 1 year after treatment. Eight regions of interests (ROIs) were selected from each radiograph, two as the experimental group located close to the infected root apex, two as the control group in the healthy bone and the other four in the healthy bone ensuring the image quality. RESULTS: Based on the FD values of the four ROIs in the healthy bone, the two radiographs were confirmed to have been taken with similar projection angles and exposure. The FD values were shown to significantly increase (P = 0.006) and decrease (P = 0.000) around the root apex and the neighbouring region of the apical lesion, respectively. CONCLUSION: Changes in fractal dimension values may serve as a necessary condition to quantitatively indicate the initial healing status 1 year after root canal treatment.

Effect of triethylene glycol dimethacrylate on the cytotoxicity, cyclooxygenase-2 expression and prostanoids production in human dental pulp cells.

AIM: To evaluate the effect of TEGDMA on cell cycle progression as well as alterations of cell cycle-related gene and protein expression. METHODOLOGY: Human dental pulp cells were exposed to 0-5 mmol L(-1) TEGDMA for 24 h. Cytotoxicity was evaluated by 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Cell cycle progression was analysed by propidium iodide (PI) flow cytometry. Cell death pathway was surveyed by annexin V/PI dual-staining flow cytometry. The mRNA expression of cell cycle-related genes (cdc2, cyclinB1 and p21) and COX-2 was evaluated by reverse transcriptase-polymerase chain reaction, and their protein expression was evaluated by Western blotting. The production of PGE(2) and PGF(2α) in the culture medium was determined by enzyme-linked immunosorbent assay. RESULTS: Triethylene glycol dimethacrylate inhibited cellular growth and induced cell cycle deregulation in dental pulp cells. High-dose exposure provoked both necrotic and apoptotic cell death. The gene and protein expression of cdc2, cyclin B1 and cdc25C declined obviously whilst cells treated with 2.5 mmol L(-1) TEGDMA concurrent with the elevated expression of p21. The mRNA and protein expression of COX-2, along with production of PGE(2) and PGF(2α), are drastically raised by 2.5-5 mmol L(-1) TEGDMA. CONCLUSIONS: Triethylene glycol dimethacrylate induced cytotoxicity, cell cycle arrest and apoptosis in dental pulp cells, which was associated with the decline of cdc2, cyclin B1, cdc25C expression and elevation of p21 expression. Concomitantly, COX-2 expression, PGE(2) and PGF(2α) production increased. These effects may contribute to explain the pulpal damage and inflammation induced by TEGDMA after operative procedures.

Prostaglandin F(2alpha) stimulates MEK-ERK signalling but decreases the expression of alkaline phosphatase in dental pulp cells.

AIM: To study prostaglandin F(2alpha) (PGF(2alpha)) receptor expression and downstream signalling in cultured human dental pulp cells and the effect of PGF(2alpha) on the alkaline phosphatase (ALP) activity of dental pulp cells. METHODOLOGY: Human dental pulp cells were cultured and exposed to PGF(2alpha). The expression of PGF(2alpha) (FP) receptors was analysed by reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting. The activation of extracellular regulated kinase (ERK) and cAMP responsive element binding protein/activating transcription factor-1 (CREB/ATF-1) signalling was determined by Western blotting. The expression of ALP in pulp cells after exposure to PGF(2alpha) was evaluated by ALP staining and PCR. RESULTS: Dental pulp cells expressed FP receptor mRNA and protein. Exposure to PGF(2alpha) revealed little cytotoxicity to pulp cells. PGF(2alpha) induced both ERK and CREB/ATF-1 phosphorylation in pulp cells. Exposure to PGF(2alpha) (>1 micromol L(-1)) further decreased the ALP activity and mRNA expression. However, U0126 (an inhibitor of MEK1) showed little preventive effect on the decline of ALP activity in dental pulp cells by PGF(2alpha). CONCLUSION: PGF(2alpha) may potentially activate FP receptors leading to ERK/CREB-ATF-1 activation during its production in inflamed dental pulp. PGF(2alpha) attenuated the ALP activity of pulp cells possibly via pathways not solely by MEK/ERK activation. PGF(2alpha) is a contributing factor of pulpal inflammation by regulating the activities of pulp cells.

Comparative cytotoxicity of five root canal sealers on cultured human periodontal ligament fibroblasts.

AIM: To evaluate the cytotoxicity of current root canal sealers to periodontal ligament (PDL) fibroblasts. METHODOLOGY: Five root canal sealers (Canals, Canals-N, Topseal, Sealapex, Tubliseal) were prepared and placed into transwells. After initial setting for 1 h, the transwells with sealers were placed into cultured PDL fibroblasts. They were cultured for further 3 or 18 h. Morphological changes were observed. Cell viability was estimated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT) assay. RESULTS: Marked retraction and death of PDL fibroblasts were observed after exposure to Canals or Topseal for 3 h. A 3-h exposure of PDL fibroblasts to Tubliseal stimulated MTT reduction. Canals-N showed little cytotoxicity even after an exposure of 18 h. CONCLUSION: Canals was the most toxic sealer, followed by Topseal. Sealapex and Tubliseal had comparable and moderate cytotoxicity to PDL fibroblasts, whereas Canals-N showed little cytotoxicity. Exposure to Tubliseal may modulate MTT reduction in PDL fibroblasts. Canals-N had good biocompatibility.

Post-tooth extraction sepsis without locoregional infection--a population-based study in Taiwan.

OBJECTIVE: To investigate the incidence and risk factors of post-tooth extraction sepsis in patients without locoregional infection. SUBJECTS AND METHODS: We assessed all claim records of the Taiwanese National Health Insurance program in 2005. Admissions for patients aged > or =16 years containing a discharge diagnosis of sepsis, and who received tooth extraction within 14 days before the admission were identified. Patient charts were reviewed to confirm the diagnosis of sepsis and rule out other infection sources. The relationship between postextraction sepsis (PES) and clinical parameters was analyzed. RESULTS: Thirty-three of the 2 223 971 extraction cases met the criteria of PES, an incidence of 1.48 per 100 000, and seven patients (21.2%) died of the disease. Aging significantly increased the risk of PES (P < 0.001). Pre-existing comorbidities were found in 20 of the 33 cases, with diabetes mellitus and hematologic diseases the most common. The method, number, and position of extraction had no influence on PES incidence. Blood cultures were positive in 25 patients (75.8%) and isolates included species of the Streptococcus, Actinomyces, Klebsiella, Bacteroides, Prevotella, and Enterococcus genera. CONCLUSION: Tooth extraction is associated with a low but significant risk of postoperative sepsis, especially in the elderly and patients with underlying diseases.

Preliminary study on wilcoxon learning machines.

As is well known in statistics, the resulting linear regressors by using the rank-based Wilcoxon approach to linear regression problems are usually robust against (or insensitive to) outliers. This motivates us to introduce in this paper the Wilcoxon approach to the area of machine learning. Specifically, we investigate four new learning machines, namely Wilcoxon neural network (WNN), Wilcoxon generalized radial basis function network (WGRBFN), Wilcoxon fuzzy neural network (WFNN), and kernel-based Wilcoxon regressor (KWR). These provide alternative learning machines when faced with general nonlinear learning problems. Simple weights updating rules based on gradient descent will be derived. Some numerical examples will be provided to compare the robustness against outliers for various learning machines. Simulation results show that the Wilcoxon learning machines proposed in this paper have good robustness against outliers. We firmly believe that the Wilcoxon approach will provide a promising methodology for many machine learning problems.

Horizontal/oblique root fractures in the palatal root of maxillary molars with associated periodontal destruction: case reports.

AIM: To report two cases of palatal root fracture in maxillary molars that were successfully managed in the short term by root canal treatment and root amputation. SUMMARY: In the first case, a 48-year-old woman with bony destruction and a deep periodontal pocket on the palatal root of tooth 26 (FDI) underwent root canal treatment. Bleeding into the palatal canal and radiolucent lines over the root suggested a fracture. Further evidence was provided by an electronic apex locator. Subsequent surgery confirmed the presence of a horizontal root fracture and the fractured root was removed. In the second case, a 75-year-old woman presented with pain from the left posterior teeth. Clinical examination revealed an oblique root fracture of tooth 27 palatal roots with abscess formation and a deep periodontal pocket. Palatal root amputation and odontoplasty were performed. This was followed by root canal treatment. Both teeth were preserved in the short term and early healing of these two cases was uneventful. KEY LEARNING POINTS: Horizontal/oblique root fracture of the palatal root in molars is rare. A combination of periodontal and root canal treatment and palatal root amputation may allow short-term preservation of functional teeth.

Hydroxychavicol, a novel betel leaf component, inhibits platelet aggregation by suppression of cyclooxygenase, thromboxane production and calcium mobilization.

BACKGROUND AND PURPOSE: Platelet hyperactivity is important in the pathogenesis of cardiovascular diseases. Betel leaf (PBL) is consumed by 200-600 million betel quid chewers in the world. Hydroxychavicol (HC), a betel leaf component, was tested for its antiplatelet effect. EXPERIMENTAL APPROACH: We tested the effect of HC on platelet aggregation, thromboxane B(2) (TXB(2)) and reactive oxygen species (ROS) production, cyclooxygenase (COX) activity, ex vivo platelet aggregation and mouse bleeding time and platelet plug formation in vivo. The pharmacokinetics of HC in rats was also assessed. KEY RESULTS: HC inhibited arachidonic acid (AA) and collagen-induced platelet aggregation and TXB(2) production. HC inhibited the thrombin-induced TXB(2) production, but not platelet aggregation. SQ29548, suppressed collagen- and thrombin-induced TXB(2) production, but not thrombin-induced platelet aggregation. HC also suppressed COX-1/COX-2 enzyme activity and the AA-induced ROS production and Ca(2+) mobilization. HC further inhibited the ex vivo platelet aggregation of platelet-rich plasma (>100 nmole/mouse) and prolonged platelet plug formation (>300 nmole/mouse) in mesenteric microvessels, but showed little effect on bleeding time in mouse tail. Moreover, pharmacokinetics analysis found that more than 99% of HC was metabolized within 3 min of administration in Sprague-Dawley rats in vivo. CONCLUSIONS AND IMPLICATIONS: HC is a potent COX-1/COX-2 inhibitor, ROS scavenger and inhibits platelet calcium signaling, TXB(2) production and aggregation. HC could be a potential therapeutic agent for prevention and treatment of atherosclerosis and other cardiovascular diseases through its anti-inflammatory and antiplatelet effects, without effects on haemostatic functions.

The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by alpha v beta 3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C.

The mechanism of morphologic change of human cultured umbilical vein endothelial cells (HUVECs) caused by fibrin was investigated. Ancrod, a thrombin-like enzyme, did not cause morphologic alteration of HUVEC by itself at concentrations ranging from 0.01 to 10 U/ml. However, when 0.02 U/ml of ancrod was added to cultured HUVEC monolayers in the presence of citrated plasma, it caused pronounced morphologic change of HUVEC after 6-10 h incubation period. Gly-Pro-Arg-Pro (4 mg/ml), an inhibitor of fibrin polymerization, prevented the morphologic alteration, indicating that the morphologic alteration was caused by the polymerized fibrin. The morphologic change of HUVEC caused by ancrod-generated fibrin was not observed in the presence of an intracellular calcium mobilization inhibitor TMB-8 (50 microM), and the morphologic alteration was also less pronounced with BAPTA(15 microM)-loaded HUVECs and HUVECs pretreated with EGTA (1.2 mM). Ancrod (in Medium 199) itself did not stimulate phosphoinositide breakdown of HUVEC. However, when ancrod was present in plasma, it caused an increase of [3H]IP1 of HUVECs preloaded with [3H]myoinositol. This IP1 increment was inhibited by Gly-Pro-Arg-Pro. The increase of IP1 was significantly inhibited by the pretreatment of monoclonal antibodies 23C6 and 7E3 directed against alpha v beta 3 integrin. Neomycin (1 mM) and pertussis toxin (100 ng/ml), but not aspirin or mepacrine, blocked this enhanced phosphoinositide breakdown. The morphologic change was also prevented by the monoclonal antibodies, 23C6 and 7E3. These results suggest that both intra- and extra-cellular calcium participate in the event of morphologic change of HUVEC caused by ancrod-generated fibrin, and the morphologic change is mediated, at least in part, by fibrin binding to integrin alpha v beta 3 on HUVECs, causing the subsequent activation of the endogenous G-protein coupled phospholipase C.

Cell-mediated immunity and head and neck cancer: with special emphasis on betel quid chewing habit.

Betel quid (BQ) chewing is popular in Taiwan, India, and many southeast-Asian countries. BQ chewing has strong association with the risk of oral leukoplakia (OL), oral submucous fibrosis (OSF), and oral cancer (OC). BQ components exhibit genotoxicity and may alter the structure of DNA, proteins and lipids, resulting in production of antigenicity. BQ ingredients are also shown to induce keratinocyte inflammation by stimulating the production of prostaglandins, TNF-alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in keratinocytes. These events may provoke tissue inflammation, early cell-mediated immunity (CMI), and immune surveillance in BQ chewers. However, BQ components also directly affect the functional activities of immunocompotent cells, and moreover tumor cells may hypo-respond to the CMI via diverse mechanisms such as induction of apoptosis of lymphocytes, induction of production of suppressor T cells, downregulation of MHC molecules in tumor cells, etc. Clinically, an alteration in lymphocyte subsets, a decrease in total number of lymphocytes, and a reduction in functional activities of CMI have been observed in isolated peripheral blood mononuclear cells (PBMC) and tumor infiltrated lymphocytes (TIL) in patients with OSF, OL or OC. Adaptation of tumor cells to immune system may promote clonal selection of resistant tumor cells, leading to immune tolerance. Future studies on effects of BQ components on CMI and humoral immunity in vitro and in vivo can be helpful for chemoprevention of BQ-related oral mucosal diseases. To elucidate how virus infection, tobacco, alcohol and BQ consumption, and other environmental exposure affect the immune status of patients with oral premalignant lesions or OC will help us to understand the immunopathogenesis of OC and to develop immunotherapeutic strategies for OC.

Effects of TGF-beta s on the growth, collagen synthesis and collagen lattice contraction of human dental pulp fibroblasts in vitro.

Transforming growth factor-beta (TGF-beta) is important in regulating the repair and regeneration of damaged dental pulp. For further elucidating the roles of different isoforms of TGF-beta in the healing and inflammatory processes of human dental pulp, we found that TGF-beta1, TGF-beta2 and TGF-beta3 inhibited the growth of two human dental pulp cell strains in vitro by 19-29, 18-25 and 23-26%, respectively, at a concentration of 0.5 ng/ml. TGF-beta also differentially stimulated the collagen synthesis of pulp cells. Collagen synthesis increased by 1 ng/ml of TGF-beta1 and TGF-beta2 by 42 and 51%, respectively. TGF-beta3 (0.1-1 ng/ml) lacked of stimulatory effect on collagen synthesis of pulp cells. Pulp cells have the intrinsic capacity to contract collagen lattice, leading to decreasing of lattice diameter. An 8 h exposure to TGF-beta1 and TGF-beta2 enhanced the pulp cell-populated collagen lattice contraction at concentrations ranging from 0.2 to 3 ng/ml. At similar concentrations, TGF-beta3 lacked of this stimulatory effect. When collagen lattice were detached after 24 h of exposure, TGF-beta1 and TGF-beta2 (0.6-3 ng/ml) induced the pulp cells-populated collagen lattice contraction within 4-8h of gel detachment. These results indicate that TGF-beta-induced collagen lattice contraction is a late cellular event. These in vitro results indicate that effects of TGF-beta isoforms on the growth, collagen synthesis and collagen lattice contraction of pulp cells may play crucial roles in the pathobiological processes of dental pulp.

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 microM), a betel leaf component, was found to suppress the 2% H(2)O(2)-induced lucigenin chemiluminescence for 53 - 75%. HC (0.02 - 2 microM) 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 microM). 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 microM) further suppressed the growth of KB cells (15 and 76%, respectively). Long-term colony formation of KB cells was inhibited by 51% with 10 microM HC. Pretreatment of KB cells with 100 microM 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-G(0)/G(1) peaks with a concomitant decrease in the number of cells residing in late S and G(2)/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.

Genotoxic and non-genotoxic effects of betel quid ingredients on oral mucosal fibroblasts in vitro.

To understand the role of betel quid (BQ) in the pathogenesis of oral submucous fibrosis (OSF) and oral cancer, we used DNA damage, cytotoxicity, and cell proliferation assays to study the pathobiological effects of aqueous extracts of three BQ constituents [betel nut (Areca catechu, BN), inflorescence of Piper betle (IPB), and lime], one BN alkaloid (arecoline), and one BN polyphenol [(+)-catechin] on cultured oral mucosal fibroblasts. Extracts of BN and IPB induced DNA strand break formation in a dose-dependent manner. Extracts of BN and IPB, (+)-catechin, and arecoline decreased cell survival and proliferation in a dose-dependent manner. However, aqueous extract of lime (50-800 micrograms/mL) increased cell proliferation by 20-40%. These results indicate that BQ contains not only genotoxic and cytotoxic agents, but also compounds which stimulate cell proliferation. These compounds may act synergistically in the pathogenesis of OSF and oral cancer in BQ chewers. In addition, five anti-oxidants [glutathione (GSH), cysteine, mannitol, catalase, and superoxide dismutase (SOD)] were tested for their protective effects against the cytotoxicity of BQ constituents. GSH (1.95 and 2.6 mmol/L) and cysteine (4 and 8 mmol/L) prevented the arecoline-induced cytotoxicity. In contrast, mannitol, catalase, and SOD did not decrease the arecoline-induced cytotoxicity. These results indicate that thiol depletion, but not the attack of oxygen free radicals, could be the mechanism for arecoline cytotoxicity. GSH could also protect cells from the cytotoxicity of IPB extract. Increasing dietary intake of GSH-rich foods or dietary supplements of GSH may have chemopreventive potential to reduce BQ-associated oral lesions.

Eugenol triggers different pathobiological effects on human oral mucosal fibroblasts.

Pathobiological effects of eugenol (4-allyl-2-methoxyphenol), a major constituent of betel quid (BQ), were studied on oral mucosal fibroblasts. At a concentration higher than 3 mmol/L, eugenol was cytotoxic to oral mucosal fibroblasts in a concentration- and time-dependent manner. Cell death was associated with intracellular depletion of glutathione (GSH). Most of the GSH was depleted prior to the onset of cell death. At concentrations of 3 mmol/L and 4 mmol/L, eugenol depleted about 45% and 77% of GSH after one-hour incubation. In addition, eugenol decreased cellular ATP level in a concentration- and time-dependent manner. Eugenol also inhibited lipid peroxidation. Inhibition of lipid peroxidation was partially explained by its dose-dependent inhibition of xanthine oxidase activity. The IC50 of eugenol on xanthine oxidase activity was about 0.3 mmol/L. No DNA strand break activity for eugenol was found at concentrations between 0.5 and 3 mmol/L. Taken together, frequent exposure of oral mucosa to a high concentration of eugenol during the chewing of BQ might be involved in the pathogenesis of oral submucous fibrosis and oral cancer via its cytotoxicity. In contrast, eugenol at a concentration less than 1 mmol/L might protect cells from the genetic attack of reactive oxygen species via inhibition of xanthine oxidase activity and lipid peroxidation.

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-chewer's 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.

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.