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  • J Dent Res 73(5): 1050-1055, May, 1994

    Eugenol Triggers Different PathobiologicalEffects on Human Oral Mucosal FibroblastsJ.H.Jeng2, LJ. Hahn2, FJ. Lu3, Y.J. Wang3, and M.Y.P. Kuo2,42School of Dentistry and 3Graduate Institute of Biochemistry, College of Medicine, National Taiwan University, 1 Chang-Te Street, Taipei,Taiwan 10016, ROC;

    Abstract. Pathobiological effects of eugenol (4-allyl-2-methoxyphenol), a major constituent of betel quid (BQ),were studied on oral mucosal fibroblasts. At aconcentration higher than 3 mmol/L, eugenol wascytotoxic to oral mucosal fibroblasts in a concentration-and time-dependent manner. Cell death was associatedwith intracellular depletion of glutathione (GSH). Most ofthe GSH was depleted prior to the onset of cell death. Atconcentrations of 3 mmol/L and 4 mmol/L, eugenoldepleted about 45% and 77% of GSH after one-hourincubation. In addition, eugenol decreased cellular ATPlevel in a concentration- and time-dependent manner.Eugenol also inhibited lipid peroxidation. Inhibition oflipid peroxidation was partially explained by its dose-dependent inhibition of xanthine oxidase activity. The IC50of eugenol on xanthine oxidase activity was about 0.3mmol/L. No DNA strand break activity for eugenol wasfound at concentrations between 0.5 and 3 mmol/L. Takentogether, frequent exposure of oral mucosa to a highconcentration of eugenol during the chewing of BQ mightbe involved in the pathogenesis of oral submucous fibrosisand oral cancer via its cytotoxicity. In contrast, eugenol at aconcentration less than 1 mmol/L might protect cells fromthe genetic attack of reactive oxygen species via inhibitionof xanthine oxidase activity and lipid peroxidation.

    Key words. Eugenol, Betel Quid Chewing, Cytotoxicity,Glutathione Depletion, Fibroblast.

    'J.HJ. received the IADR Edward H. Hatton Award (FirstPlace, Post-doctoral Category) for this research at the 71stIADR General Session in Chicago on March 11, 1993. 4Towhom correspondence and reprint requests should beaddressed.Received August 27,1993; Accepted November 23,1993

    IntroductionBetel quid (BQ) chewing, an oral habit which has beenlinked to a high incidence of oral submucous fibrosis (OSF)and oral cancer, is popular in India and many SoutheastAsian countries (Pindborg et al., 1965; Kwan, 1976; Shiau andKwan, 1979; Sanghvi et al., 1981). However, there isconsiderable variation in constituents of BQ in differentareas. In Taiwan, most people consume BQ by cutting thefresh betel nut (Areca catechu, BN) into halves andsandwiching them with a piece of inflorescence from Piperbetle (PB) and lime mixture. The BQ is chewed as such, orwith PB leaf. This chewing method differs from that inother parts of the world. Several studies have shown thatBN constituents are genotoxic and carcinogenic (Ashby etal., 1979; Stich et al., 1983; Sundqvist et al., 1989). Lime caninduce the generation of reactive oxygen free-radicals fromBN extract and cause DNA damage in vitro (Nair et al.,1987). However, little is known about the pathobiologicaleffects of the inflorescence of PB (IPB). Therefore, we havebegun a series of studies on the pathobiological effects ofthe constituents of IPB and their possible mechanisms.

    The IPB contains several phenolic compounds (Hwanget al., 1992), among which eugenol (4-allyl-2-methoxyphenol) has been widely used in dentistry asperiodontal dressing, impression materials, and endodonticmedicaments. Despite its extensive clinical use in dentistry,eugenol can inhibit cellular respiration and is cytotoxic toseveral types of cells (Cotmore et al., 1979; Lindqvist andOtteskog, 1981; Hume, 1984; Thompson et al., 1991). Topicalapplication of eugenol on rat labial mucosa can causeprotein denaturation, cell necrosis, and striated muscledissolution (Kozam and Mantell, 1978). Eugenol has beenshown to be mutagenic in Salmonella typhimurium and inmouse cells (Myhr et al., 1985; Woolverton et al., 1986).

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  • Effects ofEugenol on Human Buccal Cells

    Increased frequencies of chromosomal aberrations andsister-chromatid exchanges were also observed inmammalian cells (Stich et al., 1981; NTP, 1983). However,evidence for the carcinogenic activity of eugenol isequivocal. Eugenol lacks DNA-binding activity and did notshow carcinogenic activity in a number of animal tests(Maura et al., 1989; Phillips, 1990). In addition, eugenol cansuppress af latoxin Bi and N-methyl-N'-nitro-N-nitrosoguanidine-induced mutagenicity in Salmonellatyphimurium TA100 (Francis et al., 1989), anddimethylbenzanthracene-induced mutagenesis in TA98(Amonkar et al., 1986).

    In Taiwan, there are two million people who have the BQchewing habit, and approximately 80% of all oral cancerdeaths are associated with this habit (Kwan, 1976; Ko et al.,1992). Although many experiments have been carried out,little is known about the effects of eugenol on the oralmucosal cells. Therefore, the present study has beenundertaken to address the effects of eugenol and its possiblemechanism(s) on human oral mucosal fibroblasts.

    Materials and methods

    Culture of oral mucosal fibroblastsNormal oral mucosa was obtained from a dental studentduring surgical removal of impacted lower third molarswith the consent of the patient. Explants were cultured inDulbecco's modified Eagle's medium (DMEM, GibcoLaboratories, Grand Island, NY, USA) containing 10% fetalcalf serum (FCS, Gibco), 100 U/mL penicillin, and 100gg/mL streptomycin (Gibco). Confluent cells were detachedwith 0.025% trypsin and 0.05% EDTA (Gibco), diluted withculture medium, and then subcultured in a ratio of 1:2. Cellcultures between the fourth and tenth passages were used inthis study. For measurement of cytotoxicity, cellular GSH,cellular ATP, and thiobarbituric acid (TBA) reactivesubstance, fibroblasts were incubated at a concentration of 1x 106 cells/mL with different concentrations of eugenol(Sigma Chemical Co., St. Louis, MO, USA) for up to 4 h inrotating Eppendorf tubes at 37C. Eugenol was dissolved indimethyl-sulfoxide (DMSO, Sigma) before addition to theincubations in a final volume not exceeding 1% (v/v).Cytotoxicity assayCellular toxicity was measured either by the trypan bluedye exclusion method or by the release of cytoplasmicenzyme lactate dehydrogenase (LDH). For the dye exclusionassay, 12 gL of cell suspension was mixed with 12 pL 0.4%trypan blue in phosphate-buffered saline (PBS) for 5 min.Viable cells (which exclude trypan blue) and non-viablecells (which uptake trypan blue dye) were counted byphase-contrast microscopy. For measurement of LDHrelease, 50 ,uL of the cell suspension was pelleted at eachtime point. The LDH released into the supernatants was

    measured by the reduced absorbance at 340 nm due toNADH consumption during the reaction of pyruvate tolactate catalyzed by LDH (Wroblewski and LaDue, 1955).Measurement of reduced GSHThe amount of reduced GSH in the cell was determined by theformation of 2-nitro-5-thiobenzoic acid during the reaction ofGSH and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), aspreviously described by Moron et al. (1979). At each time point,1 mL (106 cells) of the incubated cells was pelleted and thenlysed with 275 gL of 0.2% ice-cold Triton X-100. An aliquot (25,tL) of the cellular homogenate was taken to measure proteinconcentration with the BioRad protein determination kit(BioRad Laboratories, Richmond, CA, USA). Cellular GSH wasextracted by the addition of 11 jtL of 50% sulfosalicylic acid(Sigma) into the remaining 250 ,L of cellular homogenate,followed by centrifugation at 12,000 rpm for 10 min at 4C.Reaction of GSH and DTNB was performed in 3 mL of reactionmixture containing 200 .L of acid-soluble supernatant, 0.8 mLof 0.2 M phosphate buffer (pH 8.0), and 2 mL of 0.6 mmol/LDTNB (Ellman's reagent, Sigma). The absorbance of 2-nitro-5-thiobenzoic acid was measured at 412 nm. A known amount ofGSH was used for calibration of the GSH level, and the resultswere expressed as nmol GSH/mg protein.

    Cellular ATP levelThe amount of ATP in the cell was determined by themethod of Adams (1963). At each time point, 1 mL (106 cells)of the cell incubation was first pelleted, and the cellularATP was extracted by incubation with 6% ice-coldtrichloroacetic acid (TCA, Sigma) for 5 min. ATP was thenmeasured by reduced absorbance at 340 nm because ofNADH consumption, according to the instructions for theATP determination kit from Sigma. The cellular ATP levelof eugenol-treated cells was compared with that of DMSO-treated cells and expressed as a percentage of control.

    Measurement of lipid peroxidation by TBA methodLipid peroxidation was measured according to the method ofWills (1987). The method determines aldehydes formed byspontaneous degradation of lipid hydroperoxides, which reactwith TBA to form pink-colored products. Briefly, after one-hour incubation with various concentrations of eugenol, 1 mL(106 cells) of cells was pelleted, re-suspended in 425 tL of ice-cold PBS, and sonicated on ice. An aliquot (25 pL) of cellhomogenate was used to measure the concentration of proteinas described above. The TBA reaction was performed byadding 400 tL of cell homogenate, 200 pL of 20% TCA, and400 pL of 0.67% TBA (Sigma) into a 1.5-mL Eppendorf tubeand then heated in a boiling water bath for 10 min. The pink-colored products were measured at 535 nm and expressed asOD535/mg protein.Xanthine oxidase activitiesEffect of eugenol on xanthine oxidase activities was

    j Dent Res 73(5) 1994 1051

  • J Dent Res 73(5) 1994

    100_____l

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    -0: 2 mmol/L \40 *-*:3mmol/L\- 2 C :4 mmol/L

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    tv 0 12 3 4Incubation Time (Hours)

    Figure 1. Effects of various concentrations of eugenol on humanoral mucosal fibroblasts as measured by trypan blue dye exclusion.Three separate experiments were performed. The percentages ofviable cells which exclude trypan blue are shown as mean SEM(bars).

    measured by the meth