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

Eugenol Triggers Different Pathobiological Effects on Human Oral Mucosal Fibroblasts

J.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-2methoxyphenol), 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 concentrationand 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 dosedependent 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.
Key words. Eugenol, Betel Quid Chewing, Cytotoxicity, Glutathione Depletion, Fibroblast.

Introduction
Betel quid (BQ) chewing, an oral habit which has been linked to a high incidence of oral submucous fibrosis (OSF) and oral cancer, is popular in India and many Southeast Asian countries (Pindborg et al., 1965; Kwan, 1976; Shiau and Kwan, 1979; Sanghvi et al., 1981). However, there is considerable variation in constituents of BQ in different areas. In Taiwan, most people consume BQ by cutting the fresh betel nut (Areca catechu, BN) into halves and sandwiching them with a piece of inflorescence from Piper betle (PB) and lime mixture. The BQ is chewed as such, or with PB leaf. This chewing method differs from that in other parts of the world. Several studies have shown that BN constituents are genotoxic and carcinogenic (Ashby et al., 1979; Stich et al., 1983; Sundqvist et al., 1989). Lime can induce the generation of reactive oxygen free-radicals from BN extract and cause DNA damage in vitro (Nair et al., 1987). However, little is known about the pathobiological effects of the inflorescence of PB (IPB). Therefore, we have begun a series of studies on the pathobiological effects of the constituents of IPB and their possible mechanisms. The IPB contains several phenolic compounds (Hwang et al., 1992), among which eugenol (4-allyl-2methoxyphenol) has been widely used in dentistry as periodontal dressing, impression materials, and endodontic medicaments. Despite its extensive clinical use in dentistry, eugenol can inhibit cellular respiration and is cytotoxic to several types of cells (Cotmore et al., 1979; Lindqvist and Otteskog, 1981; Hume, 1984; Thompson et al., 1991). Topical application of eugenol on rat labial mucosa can cause protein denaturation, cell necrosis, and striated muscle dissolution (Kozam and Mantell, 1978). Eugenol has been shown to be mutagenic in Salmonella typhimurium and in mouse cells (Myhr et al., 1985; Woolverton et al., 1986).

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

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Increased frequencies of chromosomal aberrations and sister-chromatid exchanges were also observed in mammalian cells (Stich et al., 1981; NTP, 1983). However, evidence for the carcinogenic activity of eugenol is equivocal. Eugenol lacks DNA-binding activity and did not show carcinogenic activity in a number of animal tests (Maura et al., 1989; Phillips, 1990). In addition, eugenol can suppress af latoxin Bi and N-methyl-N'-nitro-Nnitrosoguanidine-induced mutagenicity in Salmonella typhimurium TA100 (Francis et al., 1989), and dimethylbenzanthracene-induced mutagenesis in TA98 (Amonkar et al., 1986). In Taiwan, there are two million people who have the BQ chewing habit, and approximately 80% of all oral cancer deaths 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 oral mucosal cells. Therefore, the present study has been undertaken to address the effects of eugenol and its possible mechanism(s) on human oral mucosal fibroblasts.

measured by the reduced absorbance at 340 nm due to NADH consumption during the reaction of pyruvate to lactate catalyzed by LDH (Wroblewski and LaDue, 1955).
Measurement of reduced GSH The amount of reduced GSH in the cell was determined by the formation of 2-nitro-5-thiobenzoic acid during the reaction of GSH and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), as previously described by Moron et al. (1979). At each time point, 1 mL (106 cells) of the incubated cells was pelleted and then lysed with 275 gL of 0.2% ice-cold Triton X-100. An aliquot (25 ,tL) of the cellular homogenate was taken to measure protein concentration with the BioRad protein determination kit (BioRad Laboratories, Richmond, CA, USA). Cellular GSH was extracted 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 reaction mixture containing 200 .L of acid-soluble supernatant, 0.8 mL of 0.2 M phosphate buffer (pH 8.0), and 2 mL of 0.6 mmol/L DTNB (Ellman's reagent, Sigma). The absorbance of 2-nitro-5thiobenzoic acid was measured at 412 nm. A known amount of GSH was used for calibration of the GSH level, and the results were expressed as nmol GSH/mg protein.

Materials and methods

Culture of oral mucosal fibroblasts Normal oral mucosa was obtained from a dental student during surgical removal of impacted lower third molars with the consent of the patient. Explants were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco Laboratories, Grand Island, NY, USA) containing 10% fetal calf serum (FCS, Gibco), 100 U/mL penicillin, and 100 gg/mL streptomycin (Gibco). Confluent cells were detached with 0.025% trypsin and 0.05% EDTA (Gibco), diluted with culture medium, and then subcultured in a ratio of 1:2. Cell cultures between the fourth and tenth passages were used in this study. For measurement of cytotoxicity, cellular GSH, cellular ATP, and thiobarbituric acid (TBA) reactive substance, fibroblasts were incubated at a concentration of 1 x 106 cells/mL with different concentrations of eugenol (Sigma Chemical Co., St. Louis, MO, USA) for up to 4 h in rotating Eppendorf tubes at 37C. Eugenol was dissolved in dimethyl-sulf oxide (DMSO, Sigma) before addition to the incubations in a final volume not exceeding 1% (v/v).
Cytotoxicity assay Cellular toxicity was measured either by the trypan blue dye exclusion method or by the release of cytoplasmic enzyme lactate dehydrogenase (LDH). For the dye exclusion assay, 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-viable cells (which uptake trypan blue dye) were counted by phase-contrast microscopy. For measurement of LDH release, 50 ,uL of the cell suspension was pelleted at each time point. The LDH released into the supernatants was

Cellular ATP level The amount of ATP in the cell was determined by the method of Adams (1963). At each time point, 1 mL (106 cells) of the cell incubation was first pelleted, and the cellular ATP was extracted by incubation with 6% ice-cold trichloroacetic acid (TCA, Sigma) for 5 min. ATP was then measured by reduced absorbance at 340 nm because of NADH consumption, according to the instructions for the ATP determination kit from Sigma. The cellular ATP level of eugenol-treated cells was compared with that of DMSOtreated cells and expressed as a percentage of control.
Measurement of lipid peroxidation by TBA method Lipid peroxidation was measured according to the method of

Wills (1987). The method determines aldehydes formed by spontaneous degradation of lipid hydroperoxides, which react with TBA to form pink-colored products. Briefly, after onehour incubation with various concentrations of eugenol, 1 mL (106 cells) of cells was pelleted, re-suspended in 425 tL of icecold PBS, and sonicated on ice. An aliquot (25 pL) of cell homogenate was used to measure the concentration of protein as described above. The TBA reaction was performed by adding 400 tL of cell homogenate, 200 pL of 20% TCA, and 400 pL of 0.67% TBA (Sigma) into a 1.5-mL Eppendorf tube and then heated in a boiling water bath for 10 min. The pinkcolored products were measured at 535 nm and expressed as OD535/mg protein.
Xanthine oxidase activities Effect of eugenol on xanthine oxidase activities was

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J Dent Res 73(5) 1994

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Figure 2. Depletion of intracellular GSH in oral mucosal fibroblasts after exposure to various concentrations of eugenol. The intracellular GSH was expressed as nmol GSH/mg protein. All values are means of three experiments + SEM (bars).

Figure 1. Effects of various concentrations of eugenol on human oral mucosal fibroblasts as measured by trypan blue dye exclusion. Three separate experiments were performed. The percentages of viable cells which exclude trypan blue are shown as mean SEM (bars).
measured by the method of Noro et al. (1983). Each assay mixture contained 0.5 mL of different concentrations of eugenol, 1.45 mL of 66 mmol/L phosphate buffer (pH 7.5), and 0.0S mL of enzyme solution (0.08 U/mL, Sigma). The reaction was initiated by the addition of I mL of 0.15 mmol/L xanthine (Sigma) into the reaction mixture. The progress of the xanthine/xanthine oxidase reaction could be followed by monitoring the formation of urate, which could be measured at wavelength 290 nm.

Results

Assay of DNA damage DNA damage was assayed according to the method of Olive (1988), with modification by Martins et al. (1991). Cells were plated at S x 105 cells per well into 6-well culture plates. After overnight attachment, 0.1 ,uCi/mL of [methyl-3H]-thymidine (Amersham International Plc, Amersham, UK) was added and
incubated for 24 h. Cells were treated with various concentrations of eugenol for 2 h and then lysed with 400 ,uL of DNA lysing buffer (2% SDS, 10 mmol/L EDTA, 10 mmol/L Tris, 0.05 mol/L NaOH, pH 12.4). The cell homogenate was mixed with 400 ,L of 120 mmol/LKC2 and incubated at 64C for 10 min. After cooling, the reaction mixture was centrifuged at 3500 rpm for 10 min to pellet the bulk DNAprotein complex. The radioactivities of the supernatant (which contains small DNA strand breaks) and pellet were counted separately in a liquid sciffent connter (Beckman Instruments, USA). An aliquot (12pL) of culture medium was collected for LDH assay to rule out cellular cytotoxicity.
Stacstonical analysis At least three separate experiments were performed for all tests. The results were analyzed by unpaired Student's t test. A P value < 0.05 was considered to be statistically significant.

Cytotoxicity and GSH depletion Incubation of oral mucosal fibroblast with eugenol higher than 3 mmol/L elicited a cytotoxic response which was concentration- and time-dependent (Fig. 1). Eugenol at a concentration of 3 mmol/L caused 64% of cell death over the four-hour incubation period, whereas in the control group only 3-8% cell death was detected. Almost no viable cells were found after incubation with eugenol at a concentration of 4 mmol/L for 4 h. At cytotoxic concentrations tested (3 and 4 mmol/L), the onset of cell death occurred mostly after 2 h. Incubation of cells in eugenol at concentrations below 2 mmol/L showed no significant cytotoxic effects compared with the control group, as analyzed by both the dye exclusion technique (Fig. 1) and LDH assay (data not shown). Eugenol also depleted intracellular GSH (Fig. 2). At concentrations of 3 mmol/L and 4 mmol/L, eugenol depleted about 45% and 77% of GSH after one-hour incubation. The GSH depletion continued to the four-hour end-point. Cells in the control group maintained original GSH levels during this four-hour incubation period (data not shown).
Cellular ATP level Effects of eugenol on cellular ATP level are shown in Fig. 3. One-hour incubation in 1.0 and 2.0 mmol/L of eugenol decreased 18% and 46% of cellular ATP level, respectively. Incubation of cells for 3 h in 2 mmol/L eugenol decreased 75% of cellular ATP level. These effects were dose- and

time-dependent. Assay of lipid peroxidation Because GSH depletion by exogenous chemicals has been

j Dent Res 73(5) 1994

Eugenol (mmol/L)

Effects of Eugenol on Human Buccal Cells

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Table. Effects of eugenol on cellular lipid peroxidationa

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OD535/mg protein
0.0467 + 0.0308 + 0.0287 + 0.0266 + 0.0275 + 0.0032 0.0031b

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Lipid peroxidation was done by measurement of the cellular production of TBA-reactive substances. The pink-colored products were measured at 535 nm and expressed as OD535/mg protein. All values are means + SEM of three separate experiments. Statistically significant, P < 0.05.

Incubation Time (Hours)

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

shown to increase the intracellular concentration of reactive oxygen free-radicals and cause subsequent lipi peroxidation on biological membranes (Anundi et al., 1979), the TBA method was used to test whether GSH depletion by eugenol increased cellular lipid peroxidation in oral mucosal fibroblasts. Unexpectedly, eugenol at any tested doses inhibited about 50% of lipid peroxidation in our fibroblast system (Table).
Assay of xanthine oxidase activity As shown in Fig. 4, xanthine oxidase activity was inhibited by exposure to eugenol, and the inhibition was dosedependent. At a concentration of 0.5 mmol/L, eugenol inhibited 81% of the xanthine oxidase activity. The IC50 of eugenol was about 0.3 mmol/L. No pre-incubation was needed for this effect.
DNA strand breakage assay Eugenol at concentrations between 0.5 and 3 mmol/L showed no DNA strand break abilities on fibroblasts throughout a two-hour incubation period. No difference in the percentage of DNA precipitated was noted between the DMSO-treated control and eugenol-treated cells (data not

hepatocytes, eugenol is actively oxidized to form a quinone methide intermediate. The quinone methide covalently nds to thiol groups on proteins, forms glutathione co\rjugates, and depletes intracellular glutathione (ThoXipson et al., 1990, 1991). These events have been suggested to be responsible f or the cytotoxicity to hepatocytes. In the present fibroblast system, depletion of GSH also showed intimate correlation to the cytotoxicity of eugenol. These results imply that metabolic activation of eugenol in fibroblasts is necessary for its cellular toxicity, and that quinone methide may also play a role in fibroblast cytotoxicity. Incubation of human fibroblasts with 2 mmol/L eugenol for 3 h depleted 77% of cellular ATP level, possibly from
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Discussion
Eugenol was found to be toxic to oral mucosal fibroblasts at concentrations higher than 3 mmol/L within 2 h, as analyzed by the trypan blue dye exclusion technique. These results are comparable with the results obtained by Hume (1984) in mouse fibroblasts. However, the susceptibility of oral mucosal fibroblasts to eugenol is six times less than that of rat hepatocyte obtained by Thompson et al. (1991). This difference may occur because hepatocytes usually have more metabolic enzyme systems than fibroblasts. In

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Figure 4. Effects of eugenol on xanthine oxidase activities. Various concentrations of eugenol were added to reactions containing 0.0013 U/mL xanthine oxidase and 0.05 mmol/L xanthine. The formation of product absorbing at 290 nm was monitored. Three separate experiments were performed. The results are expressed as mean + SEM (bars), percentage of controls.

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eugenol may protect cells from the genetic attack of reactive oxygen species produced endogenously and exogenously. Because of low exposure frequency, duration, and dosage, clinical usage of eugenol is generally safe for humans, and might even have some beneficial effects. However, eugenol at high concentrations, because it acts to deplete GSH and ATP, was cytotoxic to oral mucosal fibroblasts. In BQ chewers, this cytotoxic effect may potentiate the cytotoxic and genotoxic effects of BN-related compounds (Sundqvist et al., 1989), and cause atrophy of the oral mucosal epithelium and alterations of connective tissue, as found in oral submucous fibrosis (Pindborg et al., 1965; Shiau and Kwan, 1979). In addition, because of repeated and long-term exposure, eugenol might also potentiate continued cell turnover by killing cells and thus increase the probability of converting endogenous oxidative DNA damage into mutations and cancers (Ames and Gold, 1990a; Cohen and Ellwein, 1990). However, further studies are needed to elucidate the exact role(s) of eugenol in BQ-related oral lesions.

inhibition of cellular respiration by eugenol (Hume, 1984). However, no cytotoxicity was found at this concentration. These results were in agreement with those reported by Kristensen (1989), who found that although several studies have shown that maintenance of cell membrane integrity is energy-dependent and ATP depletion can result in cell damage, only minimal energy is needed to maintain cell membrane integrity. These results were also consistent with results obtained by Hume (1984), who demonstrated that 1 mmol/L eugenol can rapidly depress cellular respiration in mouse fibroblasts, but 3H-thymidine uptake is inhibited only after exposure to this concentration for one day or more. In addition, susceptibility to depress respiration by eugenol has been shown to be associated with metabolic activity in the cell (Hume, 1984). Thus, thiol depletion by quinone methide may also play a role in decreasing the cellular ATP level. GSH is present in food and is one of the major antioxidants and anticarcinogens in the cells. It provides protection from reactive oxygen free-radicals generated by cellular oxygen metabolism as by-products and by various exogenous agents (Ames, 1983). Recently, it has been shown that DNA hits per cell per day from endogenous oxidants are approximately 104 in humans (Shigenaga et al., 1989; Ames and Gold, 1990b). This massive DNA damage can be converted to stable mutations during cell division and plays a major role in the development of cancer (Ames and Gold, 1990a,b). The reactive oxygen radicals can also cause lipid peroxidation of cellular membranes and modulate gene expression related to growth and differentiation (Cerutti, 1985). It has been shown that GSH depletion by exogenous chemicals 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 et al., 1982); however, depletion of GSH by eugenol did not cause DNA single strand breaks and cellular lipid peroxidation in the present fibroblast test system. On the contrary, eugenol could inhibit cellular lipid peroxidation, as revealed by decreasing the cellular production of TBA-reactive substances. There are two possible explanations for this: (a) Phenolic compounds have been shown to have the properties of anti-oxidant and oxygen free-radical scavengers (Kuehl et al., 1977). A free phenolic hydroxy group is essential for scavenging oxygen free-radicals. Thus, the reactive oxygen produced in the cell could be captured by eugenol. (b) The second possibility is that eugenol could inhibit cellular-free radical-producing enzymes. Xanthine/xanthine oxidase, a well-established free-radical-generating system in the cell, was used to examine this possibility, and it was found that eugenol could inhibit the xanthine oxidase activity. In conclusion, the effects of eugenol on oral tissue depended on exposure dose, frequency, and duration. The lack of DNA strand break activity and the inhibition of lipid peroxidation and xanthine oxidase activity by eugenol suggested that a low concentration (less than 1 mmol/L) of

Acknowledgment
This study was supported by grants from the National Science Council (NSC 81-0412-B002-613 and NSC 82-0412B002-456-M14), ROC.

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