Cytotoxic effect of Salvadora persica extracts on human gingival fibroblast cells.

OBJECTIVES: To assess the cytotoxic potential of Salvadora persica (S. persica) extracts on human gingival fibroblast (HGF) cells. METHODS: This study was conducted between January and May 2012 in collaboration with Dental Caries Research Chair, College of Dentistry, King Saud University, Riyadh, Saudi Arabia. Extracts of S. persica using hexane, ethylacetate, and ethanol as solvents at concentrations of 0.5 mg/ml and 1 mg/ml were evaluated for their cytotoxic activity against HGFs using the 3 cytotoxic assays: (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) (MTS), lactic dehydrogenase (LDH), and crystal violet (CV). International standards for the evaluation of medical materials recommended cut-off value of cell survival >70% was used for interpretation of the results. RESULTS: Ethanol extract of S. persica at 0.5 mg/ml and 1 mg/ml and hexane extract of S. persica at 0.5 mg/ml were completely devoid of cytotoxic activity, hexane extract at 1 mg/ml in comparison with controls  demonstrated some cytotoxicity with cell survival of 88% (p=0.045) in MTS, 86% (p=0.01) in LDH, and 88% (p=0.002) in CV assays. Similarly, ethyl acetate extract of S. persica at 0.5 mg/ml maintained cell viability of 91% in MTS, 81% in LDH, and 80% in CV assays. Maximum cytotoxicity against HGFs was observed with ethyl acetate extract of S. persica at 1 mg/ml with cell survival of 60% in MTS, 40% in LDH, and 66% CV assays (p=0.0001).   CONCLUSION: The acceptable level of cytotoxicity associated with S. persica ethanol and hexane extracts requires further evaluation to be used as irrigation solutions in endodontic treatment. 

Inhibitory effect of Zingiber cassumunar extracts on lipopolysaccharide-induced cyclooxygenase-2 and matrix metalloproteinase expression in human gingival fibroblasts.

BACKGROUND AND OBJECTIVE: Lipopolysaccharides (LPS) induce the production of proinflammatory mediators such as prostaglandins and matrix metalloproteinases (MMPs) in human gingival fibroblasts (HGFs). Zingiber cassumunar is a medicinal plant that possesses anti-inflammatory properties. The aim of this study was to determine the effects of the Z. cassumunar extract on the expression of cyclooxygenase (COX)-1, COX-2 and MMP-2 in HGFs challenged with LPS. MATERIAL AND METHODS: HGFs were treated with LPS in the presence or absence of Z. cassumunar extracts. The levels of expression of COX-1, COX-2 and MMP-2 mRNAs and of COX-1, COX-2 and MMP-2 proteins were detected by reverse transcription-polymerase chain reaction and western blotting, respectively. MMP-2 activities in cell-culture supernatants were determined using gelatin zymography. MAPK activation was evaluated by western blotting. RESULTS: LPS treatment of HGFs resulted in the activation of ERK1/2, p38 and JNK. Z. cassumunar extracts significantly inhibited the phosphorylation of ERK1/2 and JNK in HGFs stimulated with LPS. A lesser inhibitory effect was observed for the phosphorylation of p38. RT-PCR and western blot analyses showed that Z. cassumunar extracts inhibited the LPS-induced expression of COX-2 mRNA and COX-2 protein, respectively, but not of COX-1 mRNA or COX-1 protein. Pretreatment of HGFs with Z. cassumunar also attenuated the induction of MMP-2 with LPS. CONCLUSION: Our results indicate that Z. cassumunar extracts inhibit COX-2 and MMP-2 production by LPS-activated human gingival fibroblasts through blocking the proinflammatory signaling pathway involving ERK1/2, JNK and p38.

[The role of retinal-hypotalamic-pineal system in pro- and antioxidant processes in the gingival tissues of adult male albino rats].

The aim of this study is to evaluate the effects of pineal gland functional state on the prooxydant processes and antioxidant system in the gingival tissues. Male rats were assigned into one of the following groups in accordance with the duration of photoperiod: 1) control--natural daylight; 2) permanent darkness for 14 days; 3) permanent light for 14 days. The following parameters were measured in gingival tissues and the blood serum: 1) prooxidant factors (dieneconjugates--DC and malonic dialdehyde--MD); 2) antioxidant enzymes (superoxide dismutase--SOD and catalase). The present findings indicate that the gingival tissue of rats reacts to the changes in the duration of photoperiod by peroxidation and activity of antioxidant enzymes. The antioxidant-prooxidant index under dark conditions (high function of the pineal gland) was lower than under condition of permanent light ("physiological" pinealectomy). Different durations of photoperiod change the intensity of free radical oxidation and the activity of antioxidant enzymes at the systemic level (blood serum) and much more at the organ level (tissues of gingiva). Our data suggest that the gingival tissues possess rather powerful protective antioxidant system, which depends on the functional state of the pineal gland.

Isolation of protein-tyrosine phosphatase-like member-a variant from cementum.

Cementum has been shown to contain unique polypeptides that participate in cell recruitment and differentiation during cementum formation. We report the isolation of a cDNA variant for protein-tyrosine phosphatase-like (proline instead of catalytic arginine) member-a (PTPLA) from cementum. A cementifying fibroma-derived λ-ZAP expression library was screened by panning with a monoclonal antibody to cementum attachment protein (CAP), and 1435 bp cDNA (gb AC093525.3) was isolated. This cDNA encodes a 140-amino-acid polypeptide, and its N-terminal 125 amino acids are identical to those of PTPLA. This isoform, designated as PTPLA-CAP, results from a read-through of the PTPLA exon 2 splice donor site, truncating after the second putative transmembrane domain. It contains 15 amino acids encoded within the intron between PTPLA exons 2 and 3, which replace the active site for PTPLA phosphatase activity. The recombinant protein, rhPTPLA-CAP, has Mr 19 kDa and cross-reacts with anti-CAP antibody. Anti-rhPTPLA-CAP antibody immunostained cementum cells, cementum, heart, and liver. Quantitative RT-PCR showed that PTPLA was expressed in all periodontal cells; however, PTPLA-CAP expression was limited to cementum cells. The rhPTPLA-CAP promoted gingival fibroblast attachment. We conclude that PTPLA-CAP is a splice variant of PTPLA, and that, in the periodontium, cementum and cementum cells express this variant.

[Effects of Bushenguchiwan on expression of matrix metalloproteinase-13 in rats' periodontium].

OBJECTIVE: To investigate the influence of Bushenguchiwan on expression of matrix metalloproteinase-13 (MMP-13) in periodontium of rats with experimental periodontitis. METHODS: The model of experimental periodontitis of rats was established and treated by Bushenguchiwan with different doses. The periodontal tissues from groups of different doses were immunohistochemically stained by antibody of MMP-13. The expression of MMP-13 was examined and semi-quantitative analysis of signals performed by integrated absorbance. RESULTS: MMP-13 was intensely positive in gingival epithelial cells and periodontal fibroblasts in periodontitis models and negative in normal rat periodontal tissues. After 30 days of Bushenguchiwan treatment with high dose, middle dose and low dose, the expression of MMP-13 (2.9103 ± 0.5534, 3.6588 ± 0.4330, 4.4550 ± 0.4255) was down-regulated respectively compared with model rats (5.3233 ± 0.7993), P < 0.05. After 60 days of treatment the expression of MMP-13 (2.1855 ± 0.5381, 2.8558 ± 0.4759, 3.8980 ± 0.5885) was down-regulated more significantly. with model rats (6.2693 ± 0.4538), P < 0.05. CONCLUSIONS: Bushenguchiwan could down-regulate the expression of MMP-13 in rats' periodontium and the high dose group had better effect.

Suppressive effect of ethanolic Kaempferia pandurata Roxb. extract on matrix metalloproteinase-2 expression in Porphyromonas gingivalis-treated human gingival fibroblasts in vitro.

In periodontal disease, gingival fibroblasts activated by the Gram-negative anaerobic bacterium Porphyromonas gingivalis induce overexpression of matrix metalloproteinase-2 (MMP-2), which is involved in inflammatory progression. This process is followed by tissue destruction and bone loss. In the present study, we investigated the in vitro effect of the ethanolic Kaempferia pandurata Roxb. extract on expression of MMP-2 in P. gingivalis-treated human gingival fibroblast-1 (HGF-1) cells. In addition, we utilized gelatin zymography, Western blotting, and reverse transcription-PCR analysis to elucidate the molecular mechanisms underlying MMP-2 inhibition via the mitogen-activated protein kinase (MAPK) and cyclic AMP response element-binding protein (CREB) signaling pathways. Treatment with K. pandurata extract (1-10 µg/ml) dose-dependently suppressed the activity, secretion, and protein expression of MMP-2 in HGF-1 cells exposed to P. gingivalis. At the transcriptional level, inhibition of MMP-2 gene expression by K. pandurata was mediated by phosphorylation of c-Jun N-terminal kinase (JNK) and CREB signaling pathways in P. gingivalis-treated HGF-1 cells. These results suggest that K. pandurata extract suppresses MMP-2 expression at the protein and gene levels via downregulation of the principal JNK and CREB signaling pathways. Due to its efficacy in inhibiting MMP-mediated periodontal destruction, K. pandurata might represent a new, potent periodontal therapy.

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 microg/ml) ANE treatment resulted in premature growth arrest, appearance of senescence-associated beta-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.

The effects of selective COX-2 inhibitor/celecoxib and omega-3 fatty acid on matrix metalloproteinases, TIMP-1, and laminin-5gamma2-chain immunolocalization in experimental periodontitis.

BACKGROUND: Matrix metalloproteinases (MMPs) play important roles in tissue-destruction mechanisms-associated periodontitis. MMP-8 and -13 are the predominant collagenases that are important in the extracellular matrix degradation in periodontal tissues. MMP-14 is a membrane-type MMP, whereas laminin-5 indicates basal membrane modification and epithelial induction. The purpose of the present study was to evaluate the effects of celecoxib and omega-3 fatty acid administration on the gingival tissue expression of MMP-8, -13, and -14, tissue inhibitor of MMP (TIMP)-1, and laminin (Ln)-5gamma2-chain in rat experimental periodontitis induced by Escherichia coli endotoxin (lipopolysaccharide [LPS]). METHODS: Experimental periodontitis was induced in rats by repeated LPS injection. Fifty-one adult male Sprague-Dawley rats were divided into six study groups: saline control, LPS, LPS + celecoxib, LPS + therapeutic omega-3 (TO3), prophylactic omega-3 + LPS + omega-3 (P+TO3), and LPS + celecoxib + omega-3 fatty acid. Celecoxib and omega-3 fatty acid were given as a single agent or as combination therapy for 14 days. On day 15, all rats were sacrificed, and gingival tissues were analyzed immunohistochemically for the expression of MMP-8, -13, and -14, TIMP-1, and Ln-5gamma2-chain. Alveolar bone loss was evaluated morphometrically under a stereomicroscope. Data were tested statistically by Kruskal-Wallis and Mann-Whitney tests and Spearman correlation analysis. RESULTS: Alveolar bone loss was significantly higher in all study groups compared to the saline control group (all P <0.01). MMP-8 expression was significantly higher in the LPS group than in the saline group (P = 0.001). Very low expression of MMP-8 was found in the celecoxib, P+TO3, and combination groups. TO3 increased TIMP-1 expression significantly compared to the LPS group (P <0.05). Individual celecoxib and P+TO3 administration increased MMP-14 significantly compared to saline control and LPS groups (P <0.05). No significant differences were found among the study groups with regard to Ln-5gamma2-chain and MMP-13 expressions (P >0.05). CONCLUSIONS: Selective cyclooxygenase-2 inhibitor, prophylactic omega-3 fatty acid, and a combination of these two agents can inhibit gingival tissue MMP-8 expression. Moreover, the individual administration of therapeutic omega-3 may increase gingival TIMP-1 expression in contrast to no effect on MMP-8, -13, and -14 expressions in experimental periodontitis. These experimental findings in a rat model of LPS-induced periodontitis need to be verified by clinical human studies.

Areca nut extract represses migration and differentiation while activating matrix metalloproteinase-9 of normal gingival epithelial cells.

BACKGROUND AND OBJECTIVE: Areca (betel) chewing is associated with an increase in the incidence of periodontal diseases. Aberrations in matrix metalloproteinase (MMP) expression have been reported to be associated with periodontal disease. This study investigated the effects of areca nut extract on MMP activity and the phenotype of human gingival epithelial cells. MATERIAL AND METHODS: Reverse transcription-polymerase chain reaction, western blotting and gelatin zymography were used to assay MMPs. Cell viability, mobility and detachment assays were performed to characterize the phenotypic impact. Confocal microscopy was employed to evaluate cell aggregation and the distribution of E-cadherin and F-actin. RESULTS: Treatment of gingival epithelial cells with 10 microg/mL of areca nut extract reduced its cell viability. Treatment with 5 and 10 microg/mL of areca nut extract for 24 h activated MMP-9 but not MMP-2 in gingival epithelial cells. This activation could be nuclear factor-kappaB dependent and was abrogated by 10 microM curcumin. Areca nut extract also reduced the migration and detachment of gingival epithelial cells. The differentiated cell-cell contact of gingival epithelial cells was markedly impaired by areca nut extract. This was accompanied by a disruption of distribution of E-cadherin and F-actin. CONCLUSION: The areca nut extract-mediated activation of MMP-9 in gingival epithelial cells could signify a potential periodontal pathogenesis in areca chewers. The areca nut extract-mediated inhibition of cell viability and migration, together with the changed aggregation in gingival epithelial cells, suggests that impairment of the re-epithelization underlies the process and this, in turn, might exacerbate gingival inflammation.

Down-regulation of iNOS and TNF-alpha expression by kaempferol via NF-kappaB inactivation in aged rat gingival tissues.

The primary objective of this study was to evaluate the ability and mechanism of action of kaempferol, which is contained in extracts from Nelumbo nucifera, a well-known Oriental herb used in traditional medicine, with regard to the inhibition of iNOS and TNF-alpha expression in aged rat gingival tissues. We conducted an investigation into the age-related effects of kaempferol on reactive oxygen species (ROS) and GSH oxidative status in samples of aged gingival tissues. Western blotting was conducted in order to determine the expression of iNOS, TNF-alpha, p38 MAPK, NIK/IKK, p65 and IkappaBalpha in the sample tissues. Electrophoretic mobility shift assays (EMSA) were conducted in an effort to characterize the binding activities of NF-kappaB transcription factors in the aged rat gingival nuclear extracts. Our results indicate that kaempferol reduced ROS levels and augmented GSH levels in a dose-dependent manner in the aged gingival tissues. Kaempferol was shown to effect a significant reduction in iNOS and TNF-alpha protein levels, as compared to control gingival tissue samples. The results of Western blot analysis revealed that kaempferol treatment effected the reduction of iNOS and TNF-alpha expression, decreased nuclear p65 and increased cytosolic p65, down-regulation of Erk, p38, JNK and NIK/IKK expression. The EMSA results also indicated that kaempferol, when administered to the rat tissues, attenuated the NF-kappaB nuclear binding activity. Kaempferol may inhibit ROS generation via the inhibition of iNOS and TNF-alpha expression in aged gingival tissues, via the modulation of the NF-kappaB and mitogen-activated protein kinase (MAPK) pathways.

Lupinus albus, a novel vegetable extract with metalloproteinase inhibitory properties: a potential periodontal therapy.

BACKGROUND: In this study we examine the properties of a vegetable extract from seeds of Lupinus albus (LU 105). In previous works we demonstrated that LU 105 reduced the expression, by gingival fibroblasts, of both matrix metalloproteinase (MMP)-2 and MMP-9. We decided to study the impact of LU 105 on cell proliferation and morphology. Using organ culture media we also studied the MMP and tissue inhibitors of metalloproteinases (timp) expression AND THE cytokines secretion. METHODS: Healthy and inflamed gingival biopsies were placed in appendage culture with or without LU 105. The organ culture media were analyzed using Western blottings (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, MMP-13, TIMP-1, and TIMP-2) and gelatine zymography. A reverse transcription polymerase chain reaction (RT-PCR) was also performed on healthy and inflamed gingival biopsies, which were maintained in culture with or without LU 105 0.1%. Then, we decided to determine the amount of cytokines present in the organ culture media such as interleukin (IL)-1 beta, IL-4, IL-6, transforming growth factor (TGF)-beta, and tumor necrosis factor (TNF)-alpha. RESULTS: When gingival biopsies derived from inflamed tissues were cultured with LU 105 0.1% in the culture media, the MMP and TIMP expression and activity decreased significantly when compared to cultures without LU 105. Moreover, we did not note any statistical difference in the cell proliferation compared with human gingival fibroblast cultures without LU 105. Furthermore, IL-1 beta, IL-6, TGF-beta, and TNF-alpha amounts in the culture media decreased significantly, whereas IL-4 increased significantly when LU 105 0.1% was added to the culture media. CONCLUSION: LU 105, a novel metalloproteinase inhibitor with few consequences on cell proliferation and morphology, is a vegetable extract with potential clinical capacity.

Epidermal growth factor stimulates urokinase-type plasminogen activator expression in human gingival fibroblasts. Possible modulation by genistein and curcumin.

BACKGROUND: Regulation of the extracellular matrix turnover is a crucial process in wound healing and the progress of periodontal disease. It has been proposed that urokinase-type plasminogen activator (uPA), under the control of growth factors or cytokines, provides the proteolytic potential to the accomplishment of these cellular events. Epidermal growth factor (EGF) is one of the growth factors that has been shown to be active in uPA regulation. METHODS: In this study, we have assessed the effect of EGF on uPA expression in primary cultures of human gingival fibroblasts. We also studied the signaling pathways involved in this process and the role of the dietary phytoestrogens curcumin and genistein as potential modulators of this response. RESULTS: Human gingival fibroblasts expressed a basal uPA activity, which was inhibited by genistein, but not by curcumin. After treatment with 10 ng/ml EGF, uPA production was strongly stimulated. Exposure to genistein and curcumin inhibited EGF-stimulated urokinase production, although only genistein showed a statistically significant inhibitory response. Using more specific inhibitors, we found that the mitogen-activated extracellular kinase and c-Jun N-terminal kinase (JNK) inhibitors PD98059 and SP600125 also blocked the EGF-dependent stimulatory effect. On the other hand, SB203580, inhibitor of the p38 member of mitogen-activated protein kinase family, did not alter this response. In accordance to these findings, EGF stimulated a potent activation of JNK and a mild activation of extracellular signal-regulated kinases 1/2. Finally, EGF stimulated the phosphorylation of its receptor and tyrphostin (AG1478), curcumin and genistein were able to inhibit this stimulatory effect. CONCLUSIONS: These results indicate that EGF constitutes a strong stimuli on uPA expression in human gingival fibroblasts. Our data also shows that EGF-stimulated uPA production involves the activation of the extracellular signal-regulated kinases 1/2 and JNK signaling pathways and might be modulated by the natural phytoestrogens curcumin and genistein.

Effects of a vegetable extract from Lupinus albus (LU105) on the production of matrix metalloproteinases (MMP1, MMP2, MMP9) and tissue inhibitor of metalloproteinases (TIMP1, TIMP2) by human gingival fibroblasts in culture.

This study examined the effects of a vegetable extract from Lupinus albus (LU105) on MMPs and TIMPs secreted by human gingival fibroblasts in culture. LU105 was extracted from seeds of L. albus and is freely soluble in water. Gelatin zymography showed that control human gingival fibroblasts maintained in culture for 48 h express pro-MMP2 (progelatinase A) in the culture medium while the active form of MMP2 (gelatinase A), the active form of MMP9 (gelatinase B), and pro-MMP9 (progelatinase B) are not detected. Fibroblasts derived from inflamed gingiva expressed in the culture medium increased amounts of pro-MMP2 (progelatinase A) compared with controls and significant amounts of pro-MMP9 (progelatinase B). LU105 diminished the expression by gingival fibroblasts derived from inflamed tissue of both pro-MMP2 and pro-MMP9. Furthermore LU105 did not modify the amount of TIMP2 expressed in culture by controls or by gingival fibroblasts derived from inflamed tissue. TIMP1 and MMP1 significantly decreased when LU105 was added in the culture media of gingival fibroblasts derived from inflamed tissue compared with control fibroblasts. Thus LU105 seems to offer an opportunity to restore a correct balance between MMP2, MMP9, MMP1, and their natural inhibitors, i.e., TIMP1 and TIMP2 in human inflamed gingiva.

Effect of avocado and soybean unsaponifiables on gelatinase A (MMP-2), stromelysin 1 (MMP-3), and tissue inhibitors of matrix metalloproteinase (TIMP- 1 and TIMP-2) secretion by human fibroblasts in culture.

BACKGROUND: In inflamed periodontal tissues, gingival fibroblasts are able to express matrix metalloproteinases (MMPs) and their natural inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs). They can also respond to growth factors and cytokines. In this study, the in vitro effects of avocado and soybean unsaponifiable residues (ASU), their fractions (avocado unsaponifiable [ASF] or soy unsaponifiable [SSF]) on MMP-2 and MMP-3, and the activity and secretion of their inhibitors TIMP-1 and TIMP-2 were investigated using cultured human gingival fibroblasts. METHODS: Gingival fibroblasts were cultured for 72 hours with ASU, ASF, and SSF at concentrations of 0. 1, 0.5, 2.5, 5, and 10 microgram/ml of culture medium, after pretreatment or no pretreatment for 1 hour with interleukin-1beta (IL-1beta). MMP-2 and MMP-3 were detected and quantified in the culture media after zymography and image analysis. TIMP-1, TIMP-2, MMP-2, and MMP-3 were also evidenced by dot blotting and quantified by image analysis. RESULTS: In the absence of IL-1beta, a slight decrease in the secretion of MMP-2 was observed with lower doses of ASU, ASF, and SSF. The decrease of MMP-3 secretion was clearly marked with all fractions especially at low concentrations (0.1 and 2.5 microgram/ml). A slight decrease in TIMP-2 secretion was seen for low doses of ASU, ASF, and SSF, while a small increase was seen at higher concentrations. Concerning TIMP-1, no significant variation was observed in culture medium for low concentrations, and a decrease was noted for 5 and 10 microgram/ml of ASU, ASF, and SSF. As anticipated, IL-1beta induced a marked release of MMP-2, MMP-3, and TIMP-1, but no variation for TIMP-2 was seen. ASU, ASF, and SSF reversed the IL-1beta effect on gingival fibroblasts for MMP-2 and MMP-3, particularly with doses varying from 0.1 to 2.5 microgram/ml and for TIMP-1, particularly with doses varying from 2.5 to 10 microgram/ml. CONCLUSIONS: These findings suggest a potential role for avocado and soy unsaponifiable extracts to prevent the deleterious effects of IL-1beta that occur during periodontal diseases.

Areca nut extract up-regulates prostaglandin production, cyclooxygenase-2 mRNA and protein expression of human oral keratinocytes.

There are about 600 million betel quid (BQ) chewers in the world. BQ chewing is associated with increased incidence of oral cancer and submucous fibrosis. In this study, areca nut (AN) extract (200-800 microg/ml) induced the prostaglandin E(2) (PGE(2)) production by 1. 4-3.4-fold and 6-keto-PGF(1 alpha) production by 1.1-1.7-fold of gingival keratinocytes (GK), respectively, following 24 h of exposure. Exposure of GK to AN extract (>400 microg/ml) led to cell retraction and intracellular vacuoles formation. At concentrations of 800 and 1200 microg/ml, AN extract induced cell death at 21-24 and 32-52% as detected by MTT assay and cellular lactate dehydrogenase release, respectively. Interestingly, AN-induced morphological changes of GK are reversible. GK can still proliferate following exposure to AN extract. Cytotoxicity of AN extract cannot be inhibited by indomethacin (1 microM) and aspirin (50 microM), indicating that prostaglandin (PG) production is not the major factor responsible for AN cytotoxicity. PGE(2) exhibited little effect on the growth of GK at concentrations ranging from 100-1000 pg/ml. Stimulating GK production of PGs by AN extract could be due to induction of cyclooxygenase-2 (COX-2) mRNA expression and protein production. These results suggest that AN ingredients are critical in the pathogenesis of oral submucous fibrosis and oral cancer via their stimulatory effects on the PGs, COX-2 production and associated tissue inflammatory responses. AN cytotoxicity to GK is not directly mediated by COX-2 stimulation and PG production.

Smokeless tobacco extracts modulate exogenous gene expression in early passage cultured human oral epithelial cells: an in vitro system to study chemical and viral enhancer/promoter interaction.

The increased risk for cancers of the oral cavity from smokeless tobacco use may reflect the interaction of tobacco with genetic factors, such as oncogenes, and other exogenous factors, such as viruses. An in vitro system was developed based on expression of the chloramphenicol acetyltransferase (CAT) reporter gene to study interactions of chemical treatments with viral enhancer/promoters in early passage cell cultures of oral cavity-derived epithelial cells. Expression of CAT in transfected cells was significantly greater with CAT under the control of the cytomegalovirus immediate early enhancer/promoter (pCEP4/CAT) compared to the Rous sarcoma virus long terminal repeat enhancer/promoter (pRSV-cat) and the simian virus 40 (SV40) early promoter (pSV2-cat). No CAT expression was detected using corresponding control plasmids without the CAT reporter gene. Using this system, smokeless tobacco extracts prepared from either dry snuff or moist snuff delayed maximum CAT expression from Day 4 to Day 5, with sustained, significantly increased CAT expression at 12 days compared to the declining CAT expression observed in untreated control cells. Smokeless tobacco extracts can modulate intracellular gene expression. This system provides an in vitro model to test specificity of toxic agents on enhancer/promoter activity and the interaction on exogenous gene expression.

Human gingival fibroblast collagenase: purification and properties of precursor and active forms.

Human gingival fibroblast procollagenase has been purified to apparent homogeneity from serum-free and serum-supplemented fibroblast culture medium by a combination of ammonium sulfate precipitation, CM-cellulose chromatography, and gel-filtration on Bio-Gel P-150. Sodium dodecylsulfate polyacrylamide gel electrophoretic studies suggests that the purified fibroblast proenzyme is comprised of two closely related zymogens with the estimated Mr of 57,000 and 52,000. Upon densitometric scanning of the gels, the ratio of the two proenzyme forms was about 1 : 4 (57 : 52 kdal). Limited proteolysis of the fibroblast procollagenase with trypsin resulted in the conversion of both proenzyme forms into active enzyme forms of Mr 48,000 and 44,000, respectively. Amino acid analysis of the active enzymes and proenzyme forms revealed that the active enzymes contained fewer basic amino acids than do the proenzyme forms. The purified trypsin-activated fibroblast collagenase hydrolyzed type I collagen fibrils, cleaved tropocollagen in solution at 24 degrees C into TCA and TCB fragments, and cleaved the synthetic peptide substrate, DNP-peptide III, at the Gly-Ile bond. The gingival fibroblast collagenase exhibited a pH optimum of 7.5, was completely inhibited by EDTA or dithioerythritol but was not inhibited by N-ethylmaleimide or phenylmethylsulfonyl fluoride, and appeared to cleave human type III collagen approximately 10-fold faster than homologous type I collagen. In addition, comparison of the biochemical properties of the precursor and active forms of human gingival fibroblast collagenase with the precursor and active forms of human skin fibroblast collagenase, previously characterized by Stricklin and co-workers (Biochemistry 17: 2331-2337, 1978), revealed that they were similar in Mr, amino acid composition, and substrate specificity. Furthermore, the human gingival and skin fibroblast procollagenases were immunologically identical.

Effects of local anesthesia on gingival cAMP levels.

The basal levels of cAMP in the attached gingiva of Rhesus monkeys and the changes in tissue cAMP levels produced by infiltration anesthesia with lidocaine and lidocaine containing 1:100,000 epinephrine were studied. The basal level of cAMP in uninjected monkey gingiva ranged from 12 to 20 picomoles of cAMP per mg of gingival protein. This level was 75 times greater than the cAMP content of monkey blood plasma. Infiltration of the attached gingiva with saline or plain lidocaine for 5 minutes did not produce any significant changes in tissue cAMP levels. Infiltration of the gingiva with lidocaine containing 1:100,000 epinephrine, on the other hand, caused a very marked increase in tissue cAMP levels. Thirty seconds after infiltration with lidocaine with 1:100,000 epinephrine there was a 250% increase in cAMP content of the anesthetized tissue versus the uninjected control tissues. The maximal increase in tissue cAMP levels was observed 5 minutes after infiltration when the cAMP content of the gingiva was 1000 to 1100% above the control level. It is proposed that regulation of tissue cAMP levels by epinephrine or other agents may prove of therapeutic usefulness in regulating inflammation and healing of tissues after surgery or other trauma.

Bioenergetics in clinical medicine-X. Survey of the adjunctive use of coenzyme Q with oral therapy in treating periodontal disease.

Approximately 60% of young adults and 80% of middle-aged Americans have periodontal disease. Effective treatment of this widespread affliction is needed. It is generally believed that bacterial plaque on teeth and gingiva is the only direct cause of periodontal disease, and that control of plaque makes possible a normal periodontium in all age brackets. The fallacy in this common belief is the knowledge that effective oral physiotherapy can be ineffective in a small percentage of patients in periodontal practice. There is increasing knowledge and awareness that good nutrition is related to good periodontal health, and that many known vitamins influence the biochemistry of teeth, periodontium, and related bone.