Odontology (2005) 93:2429 DOI 10.

1007/s10266-005-0055-8

The Society of The Nippon Dental University 2005

ORIGINAL ARTICLE

Takashi Miyachi Takeki Tsutsui

Ability of 13 chemical agents used in dental practice to induce sisterchromatid exchanges in Syrian hamster embryo cells

Received: August 25, 2004 / Accepted: June 15, 2005

Abstract To evaluate the genotoxic potential of 13 chemical agents used in dental practice, the abilities of these agents to induce sister-chromatid exchanges (SCEs) were examined using Syrian hamster embryo (SHE) cells. Statistically signicant increases in the frequencies of SCEs were observed in SHE cells treated with all seven of the chemical agents used as endodontic medicaments: pchlorophenol, m-cresol, formaldehyde, guaiacol, hydrogen peroxide, p-phenolsulfonic acid, and sodium hypochlorite (P < 0.01; Student t test). Assessment of two chemical agents that are applied to the oral mucosa as antiseptics showed that SCEs were induced by iodine (P < 0.01), but not by chlorhexidine. Of three chemical agents that are used as dyes for disclosing dental plaque, erythrosine B had no effect on SCE induction, while acid fuchsin and basic fuchsin increased the SCE frequencies in SHE cells (P < 0.01). Glutaraldehyde, which is used as a disinfectant for dental instruments and impressions, also induced SCEs (P < 0.01). Because SCE assays are used as a sensitive indicator for evaluating genetic toxicity of chemicals, the chemical agents that had a positive response in the present study are potentially genotoxic to mammalian cells. Key words Chemicals used in dentistry Sister-chromatid exchanges Syrian hamster embryo cells

Introduction
Numerous and varied chemical agents are used as locally applied drugs in dental practice. It has been demonstrated that some of the agents, such as arsenics, chloroform, and formaldehyde, are carcinogenic or genotoxic.1 As chemical agents used topically in dental practice are administered
T. Miyachi T. Tsutsui (*) Department of Pharmacology, The Nippon Dental University, School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan Tel. +81-3-3261-8771; Fax +81-3-3263-5452 e-mail: takeki@tokyo.ndu.ac.jp

directly to the oral cavity, it is important to assess carcinogenicity or genetic effects related to the carcinogenicity of the agents. One approach to assessing these effects is to quantitatively assay the cell-transforming and genotoxic activities of chemical agents using cultured mammalian cells. The cell transformation assay system using Syrian hamster embryo (SHE) cells is one of a range of in vitro carcinogenicity tests for carcinogen screening.24 This system is able to differentiate carcinogens from noncarcinogens with a fairly high degree of accuracy, that is, with an overall concordance with rodent carcinogenicity of 80%.4 SHE cells have also been utilized to evaluate the genotoxic activities of chemicals by determining their abilities to induce chromosome aberrations, gene mutations, unscheduled DNA synthesis (UDS), and sister-chromatid exchanges (SCEs).2,3,5,6 By using SHE cells, we have examined the celltransforming and genotoxic activities of 30 chemical agents used in dental practice, including 19 endodontic medicaments, three oral mucosal antiseptics, three dyes for disclosing dental plaque, one anticaries agent, three local anesthetics, and one disinfectant for dental instruments and impressions.514 In the present study, 13 chemical agents previously tested in the SHE-cell assays were tested for induction of SCEs in SHE cells as part of a large analysis of the correlation between results of cell-transformation assays and those of genetic toxicity assays. SCEs represent the interchange of DNA replication products at apparently homologous loci and involve DNA breakage and reunion. Although the biological signicance of SCEs is not known because the exact mechanism of their formation is unclear, SCE assays remain widely used as a sensitive indicator to assess genotoxic activities of chemicals.15 Seven of the 13 chemical agents tested are used as endodontic medicaments: p-chlorophenol and guaiacol are used as pulp sedatives and antiseptics for carious cavities and root canals; m-cresol and formaldehyde are used as root canal antiseptics; hydrogen peroxide is used as a root canal irrigant; pphenolsulfonic acid is used as a root canal enlarging agent; and sodium hypochlorite is used as a root canal clearing

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agent. Of the other six chemical agents, two, chlorhexidine and iodine, are used as oral mucosal antiseptics; three, acid fuchsin, basic fuchsin, and erythrosine B, are used as dyes for disclosing dental plaque; and the remaining chemical agent, glutaraldehyde, is used as a disinfectant for dental instruments and impressions.

SCEs Cells (5 105) were plated into 75-cm2 asks, incubated overnight, and treated with each of the 13 chemical agents at varying concentrations for 24 h in the presence of 5bromodeoxyuridine (10 mg/ml) under dark conditions. When chemical agents exhibited low cytotoxicity, SCEs were examined further in cells treated with the agents at increased concentrations up to the maximum solubility, not to exceed 10 mM. Three hours before the end of treatment, colcemid was added to give a nal concentration of 0.2 mg/ml, and metaphase chromosomes were spread on glass slides as described previously.5 The differential staining of sister chromatids was performed as described previously.5 In brief, the slides were stained for 15 min in a solution of Hoechst 33258 (50 mg/ml in water), washed, dipped in PBS(-), and exposed to near UV from a row of 6 F15TB/BLB uorescent bulbs (33 J/m2/s; Sylvania, Springeld, VA, USA) for 1 h at 55C. The slides were then stained with 3% Giemsa solution for 10 min. Thirty second-division metaphases with the diploid number of chromosomes were analyzed for SCE frequency.

Materials and methods

Cells and chemical agents SHE cell cultures were grown as described previously.5 m-Cresol (>98% pure), formaldehyde solution (37% formaldehyde with 7%13% methanol), guaiacol (>99% pure), hydrogen peroxide solution (30% w/v), p-phenolsulfonic acid (80%), sodium hypochlorite solution (13.03%), iodine (>99.9% pure), and erythrosine B were purchased from Wako Pure Chemical (Osaka, Japan). p-Chlorophenol (>95% pure; Daiichi Pure Chemicals, Tokyo, Japan), glutaraldehyde solution (24.8% w/v; Taab Laboratories, Reading, UK), chlorhexidine gluconate solution (chlorhexidine, 5% w/v; Sumitomo Seiyaku, Osaka, Japan), acid fuchsin (Koso Chemical, Tokyo, Japan), and basic fuchsin (Daiichi Pure Chemicals) were obtained from the indicated sources. p-Chlorophenol, p-phenolsulfonic acid, and sodium hypochlorite were diluted with Ca2+- and Mg2+-free phosphate-buffered saline [PBS(-), pH 7.4] to a nal concentration of 100 mM. m-Cresol and guaiacol were diluted with culture medium to 10 mM. Formaldehyde, glutaraldehyde, and hydrogen peroxide were diluted with PBS(-) to 300 mM and lter-sterilized. Chlorhexidine was diluted with deionized water to 11 mM and lter-sterilized. Iodine was dissolved in dimethyl sulfoxide for a nal concentration of 473 mM. Acid fuchsin, basic fuchsin, and erythrosine B were dissolved in culture medium for a nal concentration of 2 mM and lter-sterilized. All these solutions were diluted with culture medium to the desired concentrations and applied to SHE cells. All experiments were carried out under yellow lights.

Results
The abilities of 13 chemical agents to induce SCEs in SHE cells were examined (Table 1). Cytotoxicities determined by the colony-forming efciencies of SHE cells treated with these chemical agents were increased with increasing concentrations of the agents. Treatment with each of seven chemical agents used as endodontic medicaments, pchlorophenol, m-cresol, formaldehyde, guaiacol, hydrogen peroxide, p-phenolsulfonic acid, and sodium hypochlorite, induced a statistically signicant increase in the frequencies of SCEs in SHE cells. Assessment of two chemical agents used as oral mucosal antiseptics showed that chlorhexidine had no effect on SCE induction, while iodine resulted in a signicant increase in SCEs in a concentration-dependent manner. SCEs were induced by two of the dental plaque disclosing agents, acid fuchsin and basic fuchsin, but not by erythrosine B. Glutaraldehyde, which is used as a disinfectant for dental instruments and impressions, also induced SCEs.

Cytotoxicity Cytotoxicity of the chemical agents tested was determined by the colony-forming efciencies of SHE cells treated with these agents. SHE cells (5 105) in tertiary culture were plated into 75-cm2 asks (Costar, Cambridge, MA, USA), incubated overnight, and treated with each of the 13 chemical agents at varying concentrations for 24 h. After harvesting with 0.1% trypsin, the cells were replated in triplicate onto 100-mm dishes (Costar) at 2000 cells/dish and incubated for 7 days for colony formation. The relative colony-forming efciency was expressed as the number of colonies in the treated dishes divided by the number in the control dishes 100.

Discussion
The ability of 13 chemical agents used in dental practice to induce SCEs was examined by using SHE cells. Eleven of the 13 chemical agents described below induced SCEs in our results. p-Chlorophenol is known to induce chromosome aberrations in SHE cells with exogenous metabolic activation (Hagiwara et al. unpublished data). However, it does not elicit cell transformation or unscheduled DNA synthesis (UDS) in SHE cells.10,13 m-Cresol induces cell transformation13 and chromosome aberrations14 in SHE cells. The inducibility of chromosome

26 Table 1. The ability of 13 chemical agents used in dental practice to induce sister-chromatid exchanges (SCEs) in Syrian hamster embryo cells Classication Chemical agent Concentration (mM) 0 100 300 0 100 300 1000 0 3.3 10 33 0 100 300 0 90 300 0 100 300 1000 0 130 400 1300 0 0.3 1 3 10 0 79 236 473 0 2 5 20 0 3 9 30 0 11 33 110 0 1 3 10 Relative colony-forming efciency (%) 100 a 67 61 100 105 89 84 100 98 94 68 100 90 76 100 79 75 100 85 82 80 100 85 79 58 100 98 99 89 6 100 91 90 47 100 90 87 83 100 101 98 81 100 77 72 77 100 80 76 58 SCEs/cell

Endodontic medicament

p-Chlorophenol

m-Cresol

Formaldehyde

Guaiacol

Hydrogen peroxide

p-Phenolsulfonic acid

Sodium hypochlorite

8.03 3.54b 13.23 3.61** 16.53 3.59** 8.03 3.54 11.67 4.89** 13.97 4.54** 16.03 5.14** 9.27 3.26 9.30 3.34 12.27 4.08** 18.13 7.51** 8.03 3.54 12.50 4.99** 16.80 6.00** 8.03 3.54 9.53 3.88 19.23 9.58** 8.03 3.54 9.97 4.51 10.00 3.46* 12.27 4.08** 8.03 3.54 12.43 3.16** 13.00 3.65** 16.17 5.32** 9.27 3.26 9.50 4.07 9.37 3.58 10.57 3.43 Few metaphases 8.03 3.54 11.63 4.60** 12.67 4.24** 13.80 3.81** 8.03 3.54 9.90 3.13* 10.47 3.17** 10.50 3.46** 8.03 3.54 9.17 3.29 9.97 3.32* 12.00 3.55** 9.27 3.26 10.20 2.72 9.17 2.80 10.53 3.59 8.63 3.22 9.93 3.42 11.17 5.00* 15.47 6.31**

Oral mucosal antiseptic

Chlorhexidine

Iodine

Dental plaque disclosing agent

Acid fuchsin

Basic fuchsin

Erythrosine B

Disinfectant

Glutaraldehyde

The actual mean colony-forming efciency of control cultures was 12.7 0.6 (SD)% Means SD * Signicantly different from control (P < 0.05; Student t test) (n = 30) ** Signicantly different from control (P < 0.01; Student t test) (n = 30)

aberrations by m-cresol is enhanced in the presence of exogenous metabolic activation.14 m-Cresol also induces UDS in SHE cells with exogenous metabolic activation,10 and it induces SCEs in normal human broblasts.16 The positive responses in both assays with SHE cells for chromosome aberrations and UDS indicate that a combination of

SHE cells and rat liver postmitochondrial supernatant may be sufcient for converting m-cresol to one or more genotoxic metabolites, because SHE cells themselves retain endogenous metabolizing enzymes that exhibit oxidative and peroxidative activities.17 m-Cresol does not elicit mutations in Salmonella typhimurium in assays with or without

27 Table 2. Comparison of exposure concentrations of the seven principal chemical agents used in the present study with those used in dental practice Chemical agent Concentrations used Present study (A) p-Chlorophenola Formaldehydeb Guaiacol Hydrogen peroxide Sodium hypochlorite Iodine Basic fuchsin 300 mM 33 mM 300 mM 300 mM 1300 mM 473 mM 30 mM Dental practice (B) 2.8 M 6.2 M 9.1 M 2.5%3.5% solution (735.11029.1 mM) 3.0%6.0% solution (403.0806.0 mM) 5.7%6.3% solution (449.2496.5 mM) 1.5% solution (44.4 mM) 9333.3 187 878.8 30333.3 2450.3 310.0 949.6 1480.0 Ratio (B/A)

a A constituent of camphorated p-monochlorophenol (CMCP) used as a root canal antiseptic. CMCP is a mixture of p-chlorophenol and dlcamphor at the weight ratio 7 : 13. The concentration of p-chlorophenol was expressed at the molar concentration of p-chlorophenol in CMCP b A constituent of formocresol used as a root canal antiseptic. Formocresol is a mixture of formalin (37% formaldehyde solution) and tricresol at the weight ratio 1 : 1. The concentration of formaldehyde is expressed as the molar concentration of formaldehyde in formocresol

exogenous metabolic activation.18 Some additional factors may be necessary for metabolic activation of m-cresol in the bacterial system. Formaldehyde is carcinogenic to animals, mutagenic to bacteria, yeast, and Drosophila melanogaster, and clastogenic to mammalian cells and plants.19 In addition, it induces SCEs in mammalian cells.19 Cell transformation and UDS are induced in SHE cells by this agent.10,13 Guaiacol induces SCEs in human lymphocytes.20 It also elicits cell transformation,13 chromosome aberrations,14 and UDS10 in SHE cells. Guaiacol is a major constituent of creosote, which induces mutations in Salmonella typhimurium TA1537, TA1538, TA98, and TA100 with exogenous metabolic activation.21 These ndings suggest that the SCE induction by guaiacol shown in the present study may be related to the DNA-damaging or mutagenic activities of creosote. When hydrogen peroxide is applied to root canals to cleanse the area, the gaseous oxygen released by interaction with the ubiquitous tissue enzyme catalase mechanically loosens and removes debris in the canals. The genetic activities of hydrogen peroxide have been well demonstrated in many assay systems with bacterial or mammalian cells.22 Hydrogen peroxide induces DNA damage, SCEs, and chromosome aberrations in mammalian cells.22 Cell transformation,13 chromosome aberrations,14 and UDS10 in SHE cells are also induced by hydrogen peroxide. p-Phenolsulfonic acid shows negative responses to cell transformation,13 chromosome aberrations (Hagiwara et al. unpublished data), and UDS10 in SHE cells. To our knowledge, there is no report on SCE induction by p-phenolsulfonic acid. Sodium hypochlorite is negative for carcinogenic activity in animals.23 Sodium hypochlorite is mutagenic in Salmonella typhimurium but not in Bacillus subtilis.23 This agent induces chromosome aberrations in Chinese hamster lung cells but not in human broblasts.23 No induction of micronuclei, aneuploidy, or chromosome aberrations has been observed in the bone marrow of mice.23 SCEs are induced in normal human broblasts by this agent.24 Sodium hypochlorite induces cell transformation13 and

chromosome aberrations (Hagiwara et al. unpublished data) but not UDS10 in SHE cells. There are a few reports of SCE induction in lymphocytes of patients receiving radioiodine therapy.25 Iodine induces cell transformation13 and chromosome aberrations,14 but not UDS11 in SHE cells. In the UDS assay with SHE cells, sodium hypochlorite and iodine show a negative response.10,11 These halogen compounds were administered for 1 h in the UDS assay. The other halogen compound, sodium uoride, induces UDS in SHE cells treated with the agent for more than 12 h5, suggesting that a prolonged treatment period may be necessary for UDS induction. Both acid fuchsin and basic fuchsin induce cell transformation13 and UDS11 in SHE cells. Chromosome aberrations in SHE cells are induced by basic fuchsin but not by acid fuchsin (Hagiwara et al. unpublished data). Basic fuchsin induces frameshift mutations in Salmonella typhimurium with exogenous metabolic activation.26 Glutaraldehyde exhibits a weak mutagenic response in a Salmonella mutagenicity test with TA100 in the presence of metabolic activation.27 It induces SCEs in Chinese hamster ovary cells,28 but no chromosome aberrations are elicited in either the presence or absence of metabolic activation.27 Cell transformation,13 UDS,11 and chromosome aberrations14 are not induced in SHE cells treated with this agent. There are few reports of SCE induction by the two chemical agents, chlorhexidine and erythrosine B, which failed to induce SCEs in SHE cells in our study. Chlorhexidine induces cell transformation13 and UDS11 in SHE cells, but does not induce chromosome aberrations14 in the cells. Erythrosine B is not carcinogenic in mice and rats.29,30 Oral administration of erythrosine B results in DNA damage in specic organs of mice, as detected by an in vivo comet assay.31 However, at the concentrations used in the present study, erythrosine B exhibits negative responses for cell transformation,13 UDS,11 and chromosome aberrations (Hagiwara et al. unpublished data) in SHE cells. Eleven of the 13 chemical agents examined induced SCEs in SHE cells in this study. A positive SCE response does not mean that a compound is mutagenic, because some

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compounds, such as NaCl and KCl, which are not expected to be genotoxic, induce SCEs.15 However, SCEs are still used as an indicator of genotoxic activity. Nine of the chemical agents that induced SCEs in the present study have been shown to exhibit positive responses to either chromosome aberrations or UDS or both in SHE cells.10,11,14 In addition, eight of the nine chemical agents induce cell transformation in SHE cells.13 Although additional celltransformation and genetic toxicity assays should be carried out to investigate carcinogenic potential and its related effects of these chemical agents and to clarify their mechanisms of action, these ndings suggest that most of the chemical agents that induced SCEs in the present study may have transforming and genotoxic activities in mammalian cells. High concentrations of chemical agents are topically applied to the oral cavity. Comparisons of the concentrations used in dental practice of the seven principal chemical agents that induced SCEs with the concentrations used in the present study indicate that the concentrations to which patients are exposed in the oral cavity are at least 310.0 to 187 878.8 times as high as the concentrations used in this study (Table 2). Therefore, care must be taken when using these chemical agents in dental practice to minimize their possible adverse effects on human health.

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