ebsco eugenol

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. Kuo2,4

2School 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

Introduction

Betel 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 37°C. 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 4°C.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 method

Lipid 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 activities

Effect of eugenol on xanthine oxidase activities was

j Dent Res 73(5) 1994 1051

J Dent Res 73(5) 1994

100 _____l

- 80a,

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

O

tv 0 12 3 4

Incubation 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 method of Noro et al. (1983). Each assaymixture contained 0.5 mL of different concentrations ofeugenol, 1.45 mL of 66 mmol/L phosphate buffer (pH 7.5),and 0.0S mL of enzyme solution (0.08 U/mL, Sigma). Thereaction was initiated by the addition of I mL of 0.15mmol/L xanthine (Sigma) into the reaction mixture. Theprogress of the xanthine/xanthine oxidase reaction couldbe followed by monitoring the formation of urate, whichcould be measured at wavelength 290 nm.

Assay of DNA damageDNA damage was assayed according to the method of Olive(1988), with modification by Martins et al. (1991). Cells wereplated at S x 105 cells per well into 6-well culture plates. Afterovernight attachment, 0.1 ,uCi/mL of [methyl-3H]-thymidine(Amersham International Plc, Amersham, UK) was added andincubated for 24 h. Cells were treated with variousconcentrations of eugenol for 2 h and then lysed with 400 ,uLof DNA lysing buffer (2% SDS, 10 mmol/L EDTA, 10 mmol/LTris, 0.05 mol/L NaOH, pH 12.4). The cell homogenate wasmixed with 400 ,L of 120 mmol/LKC2 and incubated at 64°Cfor 10 min. After cooling, the reaction mixture wascentrifuged at 3500 rpm for 10 min to pellet the bulk DNA-protein complex. The radioactivities of the supernatant(which contains small DNA strand breaks) and pellet werecounted separately in a liquid sciffent connter (BeckmanInstruments, USA). An aliquot (12pL) of culture medium wascollected for LDH assay to rule out cellular cytotoxicity.Stacstonical analysisAt least three separate experiments were performed for alltests. The results were analyzed by unpaired Student's t test.A P value < 0.05 was considered to be statisticallysignificant.

.*-. 4 mmol/L50

a) 40TE 30-0

20 -ET-

00 1 2 3 4


Figure 2. Depletion of intracellular GSH in oral mucosalfibroblasts after exposure to various concentrations of eugenol. Theintracellular GSH was expressed as nmol GSH/mg protein. Allvalues are means of three experiments + SEM (bars).

Results

Cytotoxicity and GSH depletionIncubation of oral mucosal fibroblast with eugenol higherthan 3 mmol/L elicited a cytotoxic response which wasconcentration- and time-dependent (Fig. 1). Eugenol at aconcentration of 3 mmol/L caused 64% of cell death over thefour-hour incubation period, whereas in the control grouponly 3-8% cell death was detected. Almost no viable cellswere found after incubation with eugenol at a concentrationof 4 mmol/L for 4 h. At cytotoxic concentrations tested (3 and4 mmol/L), the onset of cell death occurred mostly after 2 h.Incubation of cells in eugenol at concentrations below 2mmol/L showed no significant cytotoxic effects comparedwith the control group, as analyzed by both the dye exclusiontechnique (Fig. 1) and LDH assay (data not shown). Eugenolalso depleted intracellular GSH (Fig. 2). At concentrations of 3mmol/L and 4 mmol/L, eugenol depleted about 45% and 77%of GSH after one-hour incubation. The GSH depletioncontinued to the four-hour end-point. Cells in the controlgroup maintained original GSH levels during this four-hourincubation period (data not shown).

Cellular ATP levelEffects of eugenol on cellular ATP level are shown in Fig. 3.One-hour incubation in 1.0 and 2.0 mmol/L of eugenoldecreased 18% and 46% of cellular ATP level, respectively.Incubation of cells for 3 h in 2 mmol/L eugenol decreased75% of cellular ATP level. These effects were dose- andtime-dependent.

Assay of lipid peroxidationBecause GSH depletion by exogenous chemicals has been

1052 Jeng et al.


Table. Effects of eugenol on cellular lipid peroxidationa

Eugenol (mmol/L) OD535/mg protein

DMSO control 0.0467 + 0.00320.5 0.0308 + 0.0031b1.0 0.0287 + 0.0037b1.5 0.0266 + 0.0044b2.0 0.0275 + 0.0028b2.5 0.0255 + 0.0041b3.0 0.0252 + 0.0028b

a Lipid peroxidation was done by measurement of the cellularproduction of TBA-reactive substances. The pink-coloredproducts were measured at 535 nm and expressed as OD535/mgprotein. All values are means + SEM of three separateexperiments.Statistically significant, P < 0.05.

ouuIw) °-> -#-

J 0a- C)o

o -

80

60 .

40 .

20 [

00 2 3


Figure 3. Effects of various concentrations of eugenol on cellularATP levels in human oral mucosal fibroblasts. The cellular ATPlevel of eugenol-treated cells was compared with that of DMSO-treated cells and expressed as a percentage of control. All values aremeans of three experiments + SEM (bars).

shown to increase the intracellular concentration ofreactive oxygen free-radicals and cause subsequent lipi hepatocytes, eugenol is actively oxidized to form a quinoneperoxidation on biological membranes (Anundi et al., 1979), methide intermediate. The quinone methide covalentlythe TBA method was used to test whether GSH depletion by nds to thiol groups on proteins, forms glutathioneeugenol increased cellular lipid peroxidation in oral co\rjugates, and depletes intracellular glutathionemucosal fibroblasts. Unexpectedly, eugenol at any tested (ThoXipson et al., 1990, 1991). These events have beendoses inhibited about 50% of lipid peroxidation in our suggested to be responsible f or the cytotoxicity tofibroblast system (Table). hepatocytes. In the present fibroblast system, depletion of

GSH also showed intimate correlation to the cytotoxicity ofAssay of xanthine oxidase activity eugenol. These results imply that metabolic activation ofAs shown in Fig. 4, xanthine oxidase activity was inhibited eugenol in fibroblasts is necessary for its cellular toxicity,by exposure to eugenol, and the inhibition was dose- and that quinone methide may also play a role in fibroblastdependent. At a concentration of 0.5 mmol/L, eugenol cytotoxicity.inhibited 81% of the xanthine oxidase activity. The IC50 of Incubation of human fibroblasts with 2 mmol/L eugenoleugenol was about 0.3 mmol/L. No pre-incubation was for 3 h depleted 77% of cellular ATP level, possibly fromneeded for this effect.

DNA strand breakage assayEugenol at concentrations between 0.5 and 3 mmol/Lshowed no DNA strand break abilities on fibroblaststhroughout a two-hour incubation period. No difference inthe percentage of DNA precipitated was noted between theDMSO-treated control and eugenol-treated cells (data notshown).

Discussion

Eugenol was found to be toxic to oral mucosal fibroblasts atconcentrations higher than 3 mmol/L within 2 h, asanalyzed by the trypan blue dye exclusion technique. Theseresults are comparable with the results obtained by Hume(1984) in mouse fibroblasts. However, the susceptibility oforal mucosal fibroblasts to eugenol is six times less thanthat of rat hepatocyte obtained by Thompson et al. (1991).This difference may occur because hepatocytes usuallyhave more metabolic enzyme systems than fibroblasts. In

100

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r1)0j 60-0 4-.- 0

E CL. 20

x

0.125 0.250 0.375 0.50

Concentration of Eugenol (mmol/L)

Figure 4. Effects of eugenol on xanthine oxidase activities. Variousconcentrations of eugenol were added to reactions containing0.0013 U/mL xanthine oxidase and 0.05 mmol/L xanthine. Theformation of product absorbing at 290 nm was monitored. Threeseparate experiments were performed. The results are expressed asmean + SEM (bars), percentage of controls.

A\ A.1 II1

ElI

*-* :0.5 mmol/L EA-A: 1.0 mmol/LEl-El 2.0 mmol/L

j Dent Res 73(5) 1994 1053

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JDent Res 73(5)1994

inhibition of cellular respiration by eugenol (Hume, 1984).However, no cytotoxicity was found at this concentration.These results were in agreement with those reported byKristensen (1989), who found that although several studieshave shown that maintenance of cell membrane integrity isenergy-dependent and ATP depletion can result in celldamage, only minimal energy is needed to maintain cellmembrane integrity. These results were also consistent withresults obtained by Hume (1984), who demonstrated that 1mmol/L eugenol can rapidly depress cellular respiration inmouse fibroblasts, but 3H-thymidine uptake is inhibitedonly after exposure to this concentration for one day ormore. In addition, susceptibility to depress respiration byeugenol has been shown to be associated with metabolicactivity in the cell (Hume, 1984). Thus, thiol depletion byquinone methide may also play a role in decreasing thecellular ATP level.

GSH is present in food and is one of the major anti-oxidants and anticarcinogens in the cells. It providesprotection from reactive oxygen free-radicals generated bycellular oxygen metabolism as by-products and by variousexogenous agents (Ames, 1983). Recently, it has been shownthat DNA hits per cell per day from endogenous oxidants areapproximately 104 in humans (Shigenaga et al., 1989; Amesand Gold, 1990b). This massive DNA damage can beconverted to stable mutations during cell division and plays amajor role in the development of cancer (Ames and Gold,1990a,b). The reactive oxygen radicals can also cause lipidperoxidation of cellular membranes and modulate geneexpression related to growth and differentiation (Cerutti,1985). It has been shown that GSH depletion by exogenouschemicals can increase lipid peroxidation (Anundi et al., 1979)and the sensitivity of cells to the effects of irradiation(Dethmers and Meister, 1981) and oxidative stress (Arrick etal., 1982); however, depletion of GSH by eugenol did not causeDNA single strand breaks and cellular lipid peroxidation inthe present fibroblast test system. On the contrary, eugenolcould inhibit cellular lipid peroxidation, as revealed bydecreasing the cellular production of TBA-reactivesubstances. There are two possible explanations for this: (a)Phenolic compounds have been shown to have the propertiesof anti-oxidant and oxygen free-radical scavengers (Kuehl etal., 1977). A free phenolic hydroxy group is essential forscavenging oxygen free-radicals. Thus, the reactive oxygenproduced in the cell could be captured by eugenol. (b) Thesecond possibility is that eugenol could inhibit cellular-freeradical-producing enzymes. Xanthine/xanthine oxidase, awell-established free-radical-generating system in the cell,was used to examine this possibility, and it was found thateugenol could inhibit the xanthine oxidase activity.

In conclusion, the effects of eugenol on oral tissuedepended on exposure dose, frequency, and duration. The lackof DNA strand break activity and the inhibition of lipidperoxidation and xanthine oxidase activity by eugenolsuggested that a low concentration (less than 1 mmol/L) of

eugenol may protect cells from the genetic attack of reactiveoxygen species produced endogenously and exogenously.Because of low exposure frequency, duration, and dosage,clinical usage of eugenol is generally safe for humans, andmight even have some beneficial effects. However, eugenolat high concentrations, because it acts to deplete GSH andATP, was cytotoxic to oral mucosal fibroblasts. In BQchewers, this cytotoxic effect may potentiate the cytotoxicand genotoxic effects of BN-related compounds (Sundqvistet al., 1989), and cause atrophy of the oral mucosalepithelium and alterations of connective tissue, as found inoral submucous fibrosis (Pindborg et al., 1965; Shiau andKwan, 1979). In addition, because of repeated and long-termexposure, eugenol might also potentiate continued cellturnover by killing cells and thus increase the probabilityof converting endogenous oxidative DNA damage intomutations and cancers (Ames and Gold, 1990a; Cohen andEllwein, 1990). However, further studies are needed toelucidate the exact role(s) of eugenol in BQ-related orallesions.

AcknowledgmentThis study was supported by grants from the NationalScience Council (NSC 81-0412-B002-613 and NSC 82-0412-B002-456-M14), ROC.

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