Abstract
The liquid of 2-ethoxybenzoic acid cements is composed of 2-ethoxybenzoic acid and eugenol (4-allyl-2-methoxyphenol). Recently, eugenol was reported to produce radicals at a higher pH, which consequently directly damages cells. We examined here whether eugenol radicals are generated from the mixture of eugenol/calcium hydroxide, and also whether 2-ethoxybenzoic acid or acetylsalicylic acid scavenges radicals, using electron spin resonance spectroscopy. Radicals were generated from the mixture of eugenol/calcium hydroxide in 50% dimethylsulfoxide solution. The radical intensity of eugenol in 50% dimethylsulfoxide with 0.1 M sodium bicarbonate buffer (pH 9.5) was dose-dependently reduced by 2-ethoxybenzoic acid, whereas it was enhanced by acetylsalicylic acid. Next, we investigated the cytotoxic effect of eugenol on 2-ethoxybenzoic acid, acetylsalicylic acid, or calcium hydroxide on human pulp fibroblasts or a human submandibular gland cancer cell line. The cytotoxicity of EBA was decreased, whereas that of acetylsalicylic acid was increased by eugenol. In contrast, that of calcium hydroxide was not affected by eugenol. Human pulp fibroblast but not human submandibular gland cells showed a high resistance against calcium hydroxide. The generation of eugenol radicals in the liquid of 2-ethoxybenzoic acid cements caused by oxidation may be suppressed by 2-ethoxybenzoic acid.
INTRODUCTION
2-Ethoxybenzoic acid cements consist of both powder (zinc oxide and aluminum oxide) and liquid components (2-ethoxybenzoic acid and eugenol) (von Fraunhofer, 1975). 2-Ethoxybenzoic acid cements were developed as temporary restorative materials, but currently, they have been successfully adopted as root-filling or retrograde-filling materials in addition to use as temporary restorative materials (Pantschev et al., 1994; Fulkerson et al., 1996; Tassery et al., 1997). Recently, the cytotoxicity of eugenol was reported to be induced by eugenol radicals, oxygen-centered radicals (Jeng et al., 1994; Fujisawa et al., 2002). Eugenol generates radicals under alkaline conditions, as reported by investigators using Electron Spin Resonance (ESR) Spectroscopy (Satoh et al., 1998a). In addition, the cytotoxicity of eugenol is reduced by the addition of an anti-oxidant such as vitamin C (Satoh et al., 1998b; Fujisawa et al., 1999) but is significantly enhanced by the irradiation of light in air and the elevation of pH due to the generation of radicals (Atsumi et al., 2001).
We recently found that eugenol became a darkened yellowish color during storage and exhibited a higher radical intensity than when fresh, suggesting the occurrence of eugenol oxidation. Yellowish products also were previously reported to be generated during eugenol oxidation (Thompson et al., 1989), suggesting that they are cytotoxic quinone methide intermediates (Thompson, 1998). In contrast, 2-ethoxybenzoic acid may scavenge eugenol radicals, because benzoic acid is a well-known hydroxy-radical scavenging agent (Sagone et al., 1980).
In the present investigation, using ESR spectroscopy, we studied whether eugenol generates radicals in calcium hydroxide, and also whether 2-ethoxybenzoic acid or acetylsalicylic acid scavenges eugenol radicals. In addition, the cytotoxic effects of eugenol on 2-ethoxybenzoic acid, acetylsalicylic acid, and calcium-hydroxide-treated human pulp fibroblasts and human submandibular gland cells were investigated.
MATERIALS & METHODS
Materials
Chemicals and reagents were obtained from the companies indicated: calcium hydroxide (Junsei Kagaku Co., Ltd., Tokyo); eugenol (Lot GF01) and 2-ethoxybenzoic acid (Lot FIA01) (Tokyo Kasei Co. Ltd., Tokyo); dimethylsulfoxide, ethylenediaminetetraacetic acid sodium salt (EDTA) and acetylsalicylic acid (Wako Pure Chem. Ind. Ltd., Osaka, Japan); Eagle’s minimum essential medium (MEM), Eagle’s minimum essential medium alpha modification (α-MEM) (Sigma Chemical Co., St. Louis, MO, USA); fetal bovine serum (FBS) (Biosciences, Lenexa, KS, USA); and Cell Titer 96 Aqueous One of MTT [3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] (Promega Co., Madison, WI, USA).
Assay for Radical Intensity of the Mixture of Eugenol and Calcium Hydroxide
Saturated calcium hydroxide solution prepared by an excess amount (0.14 g/dL of distilled water) of this compound was mixed with an equal volume of 200 mM eugenol in 50% dimethylsulfoxide solution. Calcium hydroxide solution after 4 hrs’ dissolution was filtered prior to use. The pH of 10, 1, 0.1, and 0.01 mM calcium hydroxide was 11.4, 9.8, 7.8, and 6.8, respectively. The radical intensity was measured at 25°C, 45 sec, or 3 min later, by ESR spectroscopy (JEOL JES RE1X, X-band, 100 kHz modulation frequency). Radicals derived from eugenol (100 mM) were also measured in 50% dimethylsulfoxide in 0.1 M potassium hydroxide (KOH), pH 12.6. Instrument settings: center field, 336.0 ± 5.0 mT; microwave power, 8 mW; modulation amplitude, 0.1 mT; gain, 500; time constant, 0.1 sec; scanning time, 4 min. Radical intensity was defined as the ratio of the peak height of eugenol radicals to that of maganese oxide.The standard deviation of radical intensities (n = 3) was < 10%.
Assay for Radical Intensity of the Mixture of Eugenol and 2-ethoxybenzoic acid (or Acetylsalicylic Acid)
The mixture of eugenol (100 mM) and the indicated concentrations of 2-ethoxybenzoic acid (or acetylsalicylic acid) was dissolved in dimethylsulfoxide. The radical intensity for 2-ethoxybenzoic acid and acetylsalicylic acid was assayed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 12 min and 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 17, and 20 min, respectively, after the samples were mixed in 50% dimethylsulfoxide with 0.1 M NaHCO3/Na2CO3 buffer, pH 9.5.
Cell Culture
Human pulp fibroblast cells were obtained from a four-year-old female undergoing extraction of a supernumerary tooth with the informed consent of the patient and her parents. Ethical clearance for the study was obtained from the ethics committee of Meikai University, School of Dentistry. The tissue was cut into 1- to 2-mm3 pieces, washed twice in phosphate-buffered saline (PBS) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin, and placed into tissue culture dishes. The explants were incubated in culture medium consisting of α-MEM, 30% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, at 37°C in a humidified atmosphere of 5% carbon dioxide in air. When outgrowth was observed in the cultures, the medium was replaced twice until the cells reached confluence. The cells were then detached from the monolayer by brief treatment with 0.05% trypsin/0.004 M EDTA and recultured in α-MEM containing 30% FBS until confluent monolayers were again obtained. Human pulp fibroblast cells were maintained with α-MEM containing 10% FBS. Cells between the fifth and seventh passages were used in the experiments described below.
A human submandibular gland cell line (Shirasuna et al., 1981) was obtained from Prof. Sato at Tokushima University and has been maintained in our laboratory for 5 yrs. Human submandibular gland cells were maintained as monolayer cultures at 37°C in MEM supplemented with 10% FBS in a humidified 5% carbon dioxide atmosphere.
Assay for Cytotoxic Activity
Human submandibular gland and human pulp fibroblast cells were seeded in 96-microwell plates at a density of 5 × 103 cells/well in 0.1 mL of MEM or α-MEM with 10% FBS and were cultured at 37°C for two days. Before the addition of test materials, the cells were washed twice with serum-free medium. A stock solution of 100 mM test compound was prepared in and diluted with dimethylsulfoxide. These test compounds were added to the wells at a 1/100 vol in the order, first, 2-ethoxybenzoic acid or acetylsalicylic acid, and then calcium hydroxide, and finally eugenol, and then incubated at 37°C for 24 hrs. Dimethylsulfoxide (1%)-treated cells served as the control. After each well had been washed with fresh medium, a 20-μL quantity of Cell Titer 96 Aqueous One Solution was added to each well, and the cells were incubated for 3 to 6 hrs, after which the absorbance was measured at 492 nm with a microplate reader (Biochromatic, Helsinki, Finland). The relative viable cell number was expressed as the percentage of the number of experimental wells relative to that of the control (without compound) wells. Values were expressed as means ± SD (n = 8). Statistical analyses were performed by Student’s t test. The 50% cytotoxic concentration (CC50) was determined from the dose-response curve in the concentration range of 0 to 1 mM (i.e., 0, 0.1, 0.3, 0.6, and 1 mM).
RESULTS
ESR Study
ESR spectroscopy showed that eugenol produced broad multiple peaks of radicals at pH 9.5 (Fig. 1a). The radical intensity of eugenol declined in 0.1 M KOH at pH 12.7 (Fig. 1b) and in the saturated calcium hydroxide solution, 10 mM (pH 11.4) (Fig. 1c), possibly due to the accelerated degradation of radicals. The radical intensity of eugenol declined with decreasing concentrations of calcium hydroxide, due to the reduction of pH by dilution (Figs. 1d–1f). In each sample, dimethylsulfoxide was added at the final concentration of 50% to dissolve eugenol completely. The ESR signal of eugenol in the calcium hydroxide solution was not sufficiently split, compared with the present findings in NaHCO3/Na2CO3 buffer, pH 9.5. Radicals were detected as early as 45 sec after eugenol and calcium hydroxide were mixed.
The radical intensity of eugenol (100 mM) with increasing concentrations of 2-ethoxybenzoic acid in 0.1 M NaHCO3/Na2CO3 (pH 9.5) containing 50% dimethylsulfoxide as a function of the incubation is shown in Fig. 2A. 2-Ethoxybenzoic acid dose-dependently reduced the radical intensity of eugenol. Acetylsalicylic acid enhanced the radical intensity of eugenol at lower concentrations (0–10 mM) but completely eliminated it at higher concentrations (30 mM) (Fig. 2B).
Cytotoxicity
The CC50 of eugenol against human pulp fibroblast cells (0.77 ± 0.05 mM) was significantly larger than that against human submandibular gland cells (0.52 ± 0.07 mM) (p < 0.001) (data not shown). The cytotoxic effects of 0.1 or 0.3 mM eugenol on 0.3, 0.6, or 1.0 mM 2-ethoxybenzoic acid, acetylsalicylic acid, and calcium-hydroxide-treated human submandibular gland (a) and human pulp fibroblast (b) cells are shown in Figs. 3a and 3b, respectively. The cytotoxicity of 2-ethoxybenzoic acid (0.3 or 1 mM) was decreased in the presence of eugenol (0.1 or 0.3 mM), whereas that of acetylsalicylic acid (0.3 or 1 mM) was increased. In contrast, the cytotoxicity of calcium hydroxide was not affected by eugenol. At the high concentration of 1 mM, the cytotoxic activity of eugenol against both human pulp fibroblast and human submandibular gland cells was the highest among the tested compounds, followed by that for 2-ethoxybenzoic acid. Acetylsalicylic acid was the least cytotoxic (Figs. 3a and 3b). One mM of calcium hydroxide was highly toxic to human submandibular gland cells but not to human pulp fibroblast cells. Human pulp fibroblast cells were highly resistant to alkaline solutions of calcium hydroxide.
DISCUSSION
In general, the cytotoxic effects of chemicals depend on the kinds of cells and the
lengths of exposure time to the cells. The depression of cell respiration in human
dental pulp and mouse fibroblasts was previously reported to be dose-dependent, with a
threshold at about 0.1 mM and a maximum at 1 mM (Hume,
1984). The CC50 of eugenol vs. fibroblast growth was
previously reported to be about 0.5 mM (Lindqvist and Otteskog, 1980). The CC50 of eugenol against human
gingival fibroblast cells was previously reported to be about 0.04 mM (Gerosa et al., 1996). In the present
study, the CC50 of eugenol against human submandibular gland and human pulp
fibroblast cells was about 0.52 mM and 0.77 mM, respectively, in agreement with the
cited values (Hume, 1984). The human pulp fibroblast cells
were significantly more resistant to eugenol. Eugenol showed less cytotoxicity than
2-ethoxybenzoic acid at concentrations lower than 0.3 mM, whereas eugenol showed
markedly higher cytotoxicity at concentrations greater than 1 mM. This suggested that
the cytotoxic activity was rapidly enhanced above the concentration of micelle-formation
of eugenol. The stabilization-lysis action of eugenol, 2-ethoxybenzoic acid, and
acetylsalicylic acid to lecithin-cholesterol liposomes as a model for biological
membranes was previously investigated, suggesting that the lysis occurs at
concentrations of 0.1, 0.3, and 10 mM, respectively, resulting from the turbidity test
(Fujisawa et al., 1982, 1983). The indicated concentration probably
corresponds to that of micelle-formation of these compounds. The concentration for
acetylsalicylic acid is the highest, and acetylsalicylic acid has the least cytotoxicity
(Fig. 3). In addition, 2-ethoxybenzoic acid and acetylsalicylic acid
preferably impregnate these molecules in biological membranes when they exist at lower
pH than their Eigenvalues—e.g., benzoic acid, pKa 4.2, and
acetylsalicylic acid, pKa 3.5 (O’Neil et
al., 2001)
If 2-ethoxybenzoic acid scavenges eugenol radicals and eugenol-induced generation of reactive-oxygen species (Atsumi et al., 2001), the cytotoxicity of the mixture of 2-ethoxybenzoic acid/eugenol may be reduced. The cytotoxicity of 2-ethoxybenzoic acid was reduced by the addition of eugenol (Fig. 3), suggesting that interactions of 2-ethoxybenzoic acid with eugenol may produce less-toxic species. Since the toxic mechanism of 2-ethoxybenzoic acid is not well-understood, the toxicity of 2-ethoxybenzoic acid is possibly associated with reactive-oxygen species generation that can be scavenged by eugenol (0.1 mM). Eugenol is a well-known scavenger of reactive-oxygen species (Thompson et al., 1989), but after scavenging reactive-oxygen species, eugenol is promptly converted to eugenol radical and subsequently becomes a quinonemethide and/or dimer. Eugenol acts as a pro-oxidant at high concentrations and as an anti-oxidant at low concentrations, and the pro-oxidative activity of eugenol is influenced by light, oxygen, and elevations of pH (Fujisawa et al., 2002). In the present cell-free study, eugenol produced radicals in calcium hydroxide solutions at pH 11.4. The possibility of cytotoxicity induced by eugenol radicals cannot be excluded in the case of the direct interaction between eugenol/calcium hydroxide and pulp tissues. However, the pH ~ 7 values of the culture medium did not change following the addition of 1 mM calcium hydroxide. The cytotoxicity of calcium hydroxide was not increased by the addition of eugenol. This may be due to the formation of calcium salts such as calcium carbonate that are an inert substance in the culture media. The effect of alkaline earth salts on the NMR spectra of eugenol was previously investigated (Yokoyama, 1975), suggesting the complex formation between eugenol and alkaline earth salts such as calcium chloride, calcium carbonate, and magnesium chloride. As demonstrated previously, calcium hydroxide does not affect phospholipid-cholesterol liposomes (Fujisawa and Kadoma, 1988).
In contrast, the cytotoxicity of acetylsalicylic acid was increased by the addition of eugenol. Acetylsalicylic acid is hydrolyzed to salicylic acid in enzymatic and non-enzymatic manners. This compound is preferably de-acetylated to form a salicylic acid sodium salt due to hydrolysis (Fig. 2B), and this sodium salt produces radicals under alkaline conditions. The radical intensity of eugenol was enhanced by the addition of 3–10 mM acetylsalicylic acid, whereas it was completely reduced by its concentration of 30 mM. The reduction of radicals may be due to lowering of the pH by the higher concentration of acetylsalicylic acid. Similarly, we previously found dose-dependent enhancing and inhibiting effects of gallic acid on the intensity of eugenol radicals under alkaline conditions (Satoh et al., 1998b). Recently, salicylic-acid-induced generation of reactive-oxygen species has been reported (Kawano and Muto, 2000). In the present study, the interactive cytotoxicity of the mixture of eugenol/acetylsalicylic acid may be connected with the production of salicylic acid free radicals. In addition, salicylates were recently reported to react with hydroxy free radicals to form stable products that may be cytotoxic (Diez et al., 2001). Since acetylsalicylic acid and 2-ethoxybenzoic acid showed enhancing or inhibiting effects on eugenol radical intensity, the difference in the functional groups such as −OC2H5 and −OCOCH3 may be responsible for this disparity. The radical oxidation of acetylsalicylic acid in the presence of eugenol radicals preferably undergoes esterolysis of acetylsalicylic acid, and, subsequently, acetylsalicylic acid is decomposed into salicylic acid; consequently, salicylic acid probably acts as a pro-oxidant. In contrast, since the ethoxy group of 2-ethoxybenzoic acid is not capable of decomposing by radical oxidation, 2-ethoxybenzoic acid probably acts as a free-radical scavenger. Since 2-ethoxybenzoic acid significantly, but not completely, scavenged radicals, the combination of 2-ethoxybenzoic acid and eugenol may be beneficial to make 2-ethoxybenzoic acid cements biocompatible. However, further studies should be performed to clarify the mechanism of cytotoxicity induction by 2-ethoxybenzoic acid or acetylsalicylic acid and eugenol.
ESR spectra of eugenol radical at pH 6.9–12.7. Eugenol, 100 mM:
Effects of 2-ethoxybenzoic acid The cytotoxicity of 2-ethoxybenzoic acid (EBA), acetylsalicylic acid (ASA), and
calcium hydroxide [Ca(OH)2] with or without the addition of eugenol
(EUG) (0.1 or 0.3 mM) against human submandibular gland (HSG) cells
Footnotes
Acknowledgements
This study was supported in part by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan (to S. Fujisawa, No.10671842; T. Atsumi, No.13671948; H. Sakagami, No.14370607).