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Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro
ORIGINAL ARTICLE
Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro Fernanda Angelieri,a Joao Paulo C. Marcondes,b Danielle Cristina de Almeida,c Daisy M. F. Salvadori,d and Daniel A. Ribeiroe S~ao Paulo, Brazil
Introduction: The purpose of this study was to evaluate whether corrosion eluates obtained from commercially available orthodontic brackets are able to induce genetic damage in vitro. Material and Methods: Genotoxicity was assessed by the single cell gel (comet) assay using Chinese hamster ovary (CHO) cells. The following or~o Jose do Rio Preto, Brazil); Dentauthodontic metallic brackets were used: Morelli (Sorocaba, Brazil); Abzil (Sa rum (Pforzheim, Germany); and 3M Unitek (Puchheim, Germany). Each dental bracket was submitted to a corrosion process in a solution containing equal amounts of acetic acid and sodium chloride at 0.1 M concentration for 1, 3, 7, 14, 21, 35, and 70 days. CHO cells were exposed to eluates for 30 minutes at 37 C. The negative control was treated with the same solution used for corrosion process for 30 minutes at 37 C. Independent positive control was performed with methyl methanesulfonate (MMS) (Sigma Aldrich, St. Louis, Mo) at 1 ug/mL for 1 hour. Results: None of the eluates was found to exhibit genotoxicity, regardless of the different commercial brands of orthodontic appliance used. Conclusions: In summary, our results indicate corrosion eluates obtained from orthodontic brackets do not induce genetic damage as assessed by single cell gel (comet) assay. (Am J Orthod Dentofacial Orthop 2011;139:504-9)
B
iocompatibility is the ability of a material to perform an appropriate host response in a specific application.1 This means that the tissue of the patient that comes into contact with the material does not suffer from any toxic, irritating, inflammatory, allergic, genotoxic, or carcinogenic action.2 Accumulating evidence suggests that the oral environment is suitable for the biodegradation of metals as a result of its thermal, microbiologic, and enzymatic properties.3 Intraoral fixed orthodontic appliances
a Associate professor, Department of Orthodontics, S~ao Paulo Methodist University, S~ao Bernardo do Campo, S~ao Paulo, Brazil. b Graduate student, Department of Pathology, Botucatu Medical School, S~ao Paulo State University, UNESP, S~ao Paulo, Brazil. c Graduate student, Department of Pathology, Botucatu Medical School, S~ao Paulo State University, UNESP, S~ao Paulo, Brazil. d Professor, Department of Pathology, Botucatu Medical School, S~ao Paulo State University, UNESP, S~ao Paulo, Brazil. e Professor, Department of Biosciences, Federal University of S~ao Paulo, UNIFESP, Santos, S~ao Paulo, Brazil. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. This work was supported by grants from FAPESP (Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo, Grant number 07/00345-7). D.A.R. is a recipient of a CNPq fellowship. Reprint requests to: Daniel A. Ribeiro, Departamento de Bioci^encias, Universidade Federal de S~ao Paulo–UNIFESP, Av. Ana Costa, 95, Vila Mathias, Santos–SP, Brazil, 11060-001; e-mail, [email protected]; [email protected]. Submitted, February 2009; revised and accepted, March 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.03.058
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include brackets, bands, and arch wires that are made of alloys containing nickel, cobalt, and chromium in different percentages. Actually, different types of orthodontic brackets are available in the global market. The number of bracket systems for orthodontic therapy has increased significantly. Although they possess undeniable efficiency and local biocompatibility, there is concern about metal release from orthodontic devices to adjacent tissues such as oral mucosa cells and/or periodontal tissues. Thus, further biocompatibility data are needed in order to evaluate all risks of these components. Indeed, the limited data existing on the biocompatibility of these compounds appear to be insufficient. Genotoxicity tests can be defined as in vitro and in vivo tests designed to detect compounds that induce genetic damage, including DNA damage, gene mutation, chromosomal breakage, altered DNA repair capacity, and cellular transformation. In the last decades, genotoxicity assays have gained widespread acceptance as an important and useful indicator of carcinogenicity.4 As the incidence of head and neck cancer has increased in recent years—particularly in developing countries such as India, Vietnam, and Brazil, where it constitutes up to 25% of all types of cancer— risk factors other than tobacco smoke and the abuse of alcohol are of special concern.5 Particularly, little information is available on the genotoxicity of orthodontic brackets so far.
Angelieri et al
As a result of inappropriate evidence, the aim of this study was to evaluate whether corrosion eluates obtained from commercially available orthodontic brackets are able to induce genetic damage in vitro in order to predict the real risks when the corrosion process occurs during orthodontic therapy in vivo. MATERIAL AND METHODS Cell culture
In vitro test systems for genotoxicity evaluation can be differentiated into prokaryotic and/or eukaryotic tests. Studies conducted with the eukaryotic systems are thought to provide more reliable information with respect to the genotoxicity of chemicals.6 Therefore, we aimed to investigate the genotoxic potential of corrosion eluates obtained from orthodontic brackets. For this we were able to use Chinese hamster ovary (CHO) cells. Our choice of CHO cells (a continuous cell line) provided an accurate evaluation of the changes independent from confounding factors such as age and metabolic and hormonal states of the donor. This cell line has a small number of relatively large chromosomes; they grow fast and reproducible results can be obtained from the same cell source.7 For this purpose, CHO cells (lineage CHO K-1) were growth to confluence in 75-cm2 culture flasks (Corning, Incorporated, New York, NY) using Ham's F-10 medium (Invitrogen Corporation, Grand Island, NY) supplemented with 10% fetal calf serum and 100 U/mL penicillin (Life Technologies, Carlsbad, Calif) and 100 mg/ mL streptomycin (Invitrogen Corporation) at 37 C with 5% CO2. Cells were cultured for 5 days prior to treatment with test substances. Confluent cells were detached with 0.15% trypsin (Invitrogen Corporation) for 5 minutes; after that, 2 mL complete medium was added and cells were centrifuged at 1000 rpm (180 g) for 5 minutes. Cell suspension was counted using a Neubauer chamber and seeded in 96-well microtiter plates (Corning) at a density of 1 3 104 cells per well (at a concentration of 1 3 106/mL). Cell treatment
For this study, the following commercially available orthodontic brackets were used: Morelli, Abzil, Dentaurum, and 3M Unitek. There are no differences regarding metal composition among them according to the manufacturer’s instructions. Each dental bracket was submitted to a corrosion process in a solution containing equal amounts of acetic acid and sodium chloride (Merck & Co., Inc., Whitehouse Station, NJ), at 0.1 M concentration, for 1, 3, 7, 14, 21, 35, and 70 days. A volume of 10 uL of cells (approximately 10,000 cells) was
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then added individually to each final solution of eluate maintained for 30 minutes at 37 C. After exposure, cells were washed in phosphate buffered solution (PBS). The negative control was treated with the same solution used for the corrosion process for 30 minutes at 37 C. As cytotoxicity is a confounding factor in genotoxicity studies, it is not recommended to perform the single cell gel (comet) assay on samples with more than 30% cytotoxicity.8 Thus, the exposure period as well as the final concentrations used here were established in a previous study conducted in our laboratory for evaluating the genotoxicity of these compounds only.9 An independent positive control was performed with MMS at 1 mg/mL for 1 hour in order to ensure the reproducibility and sensitivity of the assay. In addition, each treatment was performed consecutively 3 times to ensure reproducibility. Single cell gel (comet) assay
To evaluate the magnitude of DNA damage, we used the alkaline version of the single cell gel (comet) assay. The comet assay is a relatively new, rapid, simple, and reliable biochemical technique for evaluating DNA damage in mammalian cells.8 This technique includes embedding cells in agarose gel on microscope slides, incubating them with the test compound, and then lysing the cells with detergent and high salts.10 During electrophoresis under alkaline conditions, cells with damaged DNA display increased rates of DNA migration to the anode. The increase in DNA migration rate results from the formation of smaller fragments of DNA caused by double-strand breaks, single-strand breaks, and alkalilabile sites. Smaller fragments of DNA migrate further in the electric field compared with intact DNA, and the cellular lysates thus resemble a “comet” with brightly fluorescent head and a tail region. Our own recent studies have demonstrated that the single cell gel (comet) assay is a suitable tool to investigate genotoxicity of dental compounds used in clinical practice.11-14 The protocol used for the single cell gel (comet) assay followed the guidelines purposed by Tice et al.8 Slides were prepared in duplicate per treatment. Thus, a volume of 10 mL of treated or control cells (1 3 104 cells) was added to 120 mL of 0.5% low-melting-point agarose at 37 C, layered onto a precoated slide with 1.5% regular agarose, and covered with a coverslip. After brief agarose solidification in a refrigerator, the coverslip was removed and the slides were immersed in the lysis solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid [EDTA] [Merck & Co., Inc.]; 10 mM Tris-HCl buffer pH 5 10 [Sigma Aldrich, St. Louis, Mo]; 1% sodium sarcosinate [Sigma Aldrich]; with 1% Triton X-100 [Sigma Aldrich]; and 10% DMSO [Merck & Co. Inc.]) for about 1 hour. Prior to electrophoresis, the slides were left in an alkaline
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buffer (0.3 mM NaOH [Merck & Co., Inc.] and 1 mM EDTA [Merck & Co., Inc., pH . 13]) for 20 minutes and electrophoresed for another 20 minutes, at 25 V (0.86 V/cm) and 300 mA. After electrophoresis, the slides were neutralized in 0.4 M Tris-HCl (pH 5 7.5) for 15 minutes, fixed in absolute ethanol, and stored at room temperature until analysis. All of the steps described above were conducted in the dark to prevent additional DNA damage. Throughout this study, some diluted and treated aliquots were tested for viability by trypan blue exclusion,10 and greater than 75% of cells constantly excluded trypan.
able to detect the significant increase in tail moment of the positive control (MMS-treated cells) compared with the negative controls. However, corrosion eluates obtained from brackets of Morelli did not induce genetic damage at any experimental period established, as verified by no statistically significant differences (P .0.05) when compared with negative controls. Such findings are presented in Table I. The same picture occurred with the other brackets commercially tested (ie, Abzil, Dentaurum, and 3M Unitek; (Table I). Figure 1 shows an undamaged CHO cell from a negative control, a cell exposed to corrosion eluate for 70 days, and an MMS-induced comet cell (positive control).
Comet capture and analysis
DISCUSSION
A total of 50 randomly captured comets per treatment (25 cells from each slide)15 were examined blindly by 1 expertise observer (D.A.R.) at 4003 magnification using a fluorescence microscope (Olympus, Center Valley, Pa) connected through a black and white camera to an image analysis system (Comet Assay II, Perceptive Instruments Ltd, Haverhill, UK). For all experiments, we evaluated 2 image analysis parameters: tail intensity (% migrated DNA) and tail moment. Tail moment was calculated by the image analysis system as the product of the tail length (DNA migration) and the fraction of DNA in the comet tail (% DNA in the tail). In none of the experiments there was a significant difference between these parameters. Therefore, we chose tail moment for the presentation of the results.
The aim of this study was to evaluate the genotoxic damage induced by corrosion eluates obtained from orthodontic appliances using CHO cells in vitro. The investigation was conducted using the single cell gel (comet) assay. To the best of our knowledge, the approach has not been addressed so far. The alkaline version of the single cell gel (comet) assay used here is sensitive for a wide variety of DNA lesions. Among them are single- and double-strand breaks; oxidative DNA base damage; alkali-labile sites, including abasic and incomplete repair sites; and DNADNA/DNA-protein/DNA-drug cross-linking in any eukaryotic cell.8 Tail moment is a virtual measure calculated by the computerized image analysis system, considering both the length of DNA migration in the comet tail and the tail intensity. This parameter is one of the best indices of induced DNA damage among the various parameters calculated by this method.8 In this study, a cell culture technique was employed in order to evaluate the putative noxious activities for corrosion products from dental brackets in vitro. In vitro studies are simple, inexpensive to perform, provide a significant amount of information, can be conducted under controlled conditions, and may clarify the mechanisms of cellular toxicity.16 The results obtained from in vitro assays may be indicative of the effects observed in vivo. The most frequently used cells for in vitro studies when screening for potential genotoxicity are L5178Y mouse lymphoma cells or CHO cells because these cellular types are easy to handle and well characterized with regard to, for example, their growth pattern.17 Thus, CHO cells, a continuous cell line, were used as the target cell in this setting. On the basis of tail moment data, the results of this study pointed out that the alkaline single cell gel (comet) assay in the experimental conditions used failed to detect the presence of DNA damage after treatment by corrosion eluates from four different commercial brands of
Statistical methods
The parameter from the comet assay (tail moment) was assessed by 1-way analysis of variance (ANOVA) followed by post hoc analysis and the Tukey test using SigmaStat software, version 1.0 (Jandel Scientific, Rafael, Calif). A P value less than 0.05 was considered statistically significant. RESULTS
The cytotoxicity of eluates from brackets was measured in CHO cells through a trypan blue assay in range-finding experiment prior to the determination of chemically induced genotoxicity. The dose-response relationships on cell viability of all solutions tested at the following periods (1, 3, 7, 14, 21, 35 and 70 days) were assessed by trypan blue assay. The mean cell viability for CHO cells was approximately 83% for all solutions tested, without any statistical differences (P .0.05) among them (data not shown). The effect of exposure to the test materials on DNA damage in CHO cells is given in Table 1. The assay was
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Table I. DNA damage (tail moment) in Chinese hamster ovary cells exposed to corrosion eluates from orthodontic brackets* Brackets Commercially Available Days 0 1 3 7 14 21 35 70 Positive control*,y
Morelli 0.67 6 0.22 0.98 6 0.45 1.20 6 0.67 1.22 6 0.54 0.67 6 0.45 0.98 6 0.57 0.57 6 0.32 1.20 6 0.57 5.72 6 0.72z
Abzil 0.67 6 0.22 0.54 6 0.43 0.63 6 0.62 0.76 6 0.44 0.58 6 0.40 0.54 6 0.34 0.87 6 0.45 0.65 6 0.72 5.72 6 0.72z
Dentaurum 0.67 6 0.22 0.54 6 0.22 0.67 6 0.34 0.76 6 0.64 0.96 6 0.54 0.53 6 0.37 0.98 6 0.56 1.04 6 0.54 5.72 6 0.72z
3M Unitek 0.67 6 0.22 0.58 6 0.16 0.99 6 0.54 0.89 6 0.43 1.02 6 0.47 0.45 6 0.33 0.76 6 0.32 0.67 6 0.43 5.72 6 0.72z
*Data of the 3 different experiments. Values are expressed as mean and 6 standard deviation; yMethyl methanesulfonate, 1 mg/mL; zP \0.05 when compared with the negative control (day zero).
Fig 1. Representative comet images of a CHO cell from a negative control (A), a cell exposed to corrosion eluates for 70 days (B), and a MMS-treated cell (positive control) (C). DNA was stained with ethidium bromide; 403 magnification.
brackets at all experimental periods established in this setting. By comparison, previous studies conducted by our group have demonstrated that corrosion eluates obtained from endosseous dental implants were not genotoxic in vitro, and titanium miniplates do not exert DNA injury in vivo.14,18 Such findings are the same magnitude as reported by others.19 It has been postulated that there is an absence of primary DNA damage in oral mucosa cells from patients undergoing orthodontic therapy.20 Nevertheless, some authors have noticed genotoxic damage induced by orthodontic therapy in human buccal mucosa cells as assessed by the single cell gel (comet) assay in vivo.3 Herein, the application of the single cell gel (comet) assay using epithelial cells merits discussion. In the last few years, our research group has standardized the single cell (comet) assay in human epithelial cells, such as urothelial cells21,22 and gastric cells.23,24 In particular, oral cells are a type of stratified squamous epithelium that undergoes a terminal differentiation to form a protective barrier
that covers the cells. This cell envelope, which is rich in a small proline-rich protein, provides a barrier against what can be a very hostile environment in the mouth, and accounts for the high resistance of oral cells to lysis.25 In this regard, lysis using a trypsin- or proteinase K–assisted procedure is required in order for the single cell gel (comet) assay to be performed. Our preliminary results using the single cell gel (comet) assay in humans indicated that only a few cells from oral mucosa cell samples yielded comets that can be analyzed by current methods because the oral cells sustained massive DNA damage with subsequent disintegration.26 Therefore, the single cell gel (comet) assay, as it is commonly performed, may not be useful for genotoxicity monitoring in human oral mucosa cells. Furthermore, our data generated after enzymatic enrichment of viable cells and immunomagnetic separation of epithelial cells suggested that most of the oral mucosa cells that do form comets are probably leukocytes.26 These results were confirmed by others.27
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In a recent study conducted by Luft et al,28 all brackets tested seemed to be biocompatible and applicable for orthodontic therapy. No significant differences in the nickel, chromium, and iron levels were noticed in orthodontic appliances at all study periods in vitro.29,30 By comparison, none of the bracket extracts altered L929 cell viability or morphology, as assessed by crystal violet and MTT assays.31 The same findings were reported by others.32 It is important to stress that no single test is capable of detecting all mechanisms by which compounds induce genetic damage. Therefore, for a more detailed judgment of a compound, a battery of tests—including those that assess metaphase chromosomal aberrations, micronuclei, sister chromatid exchanges, and host cell activation— would be significant. In the present study, as well as in all of our previous investigations using the single cell gel (comet) assay, we have always excluded comets without clearly identifiable heads during the image analysis. Although it should be emphasized that it is still not completely understood what these hedgehogs actually represent, this type of comet was excluded on the basis of the assumption that these cells represent dead cells, resulting from putative cytotoxic effects of corrosion eluates rather than primary DNA damage after a direct interaction between DNA and a genotoxic agent.33
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CONCLUSIONS
The results of this study suggest that corrosion eluates obtained from orthodontic appliances did not induce DNA breakage as depicted by the single cell gel (comet) assay. Since DNA damage is an important step in events leading from carcinogen exposure to cancer, the results of the present study represent a potential alert to the correct evaluation of the potential health risks associated with exposure to these compounds.
16. 17.
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REFERENCES 20. 1. Gartner AH, Dom SO. Advances in endodontic therapy. Dent Clin N Am 1992;36:357-78. 2. Valey JW, Simonian PT, Conrad EU. Carcinogenicity and metallic implants. Am J Orthod Dentofacial Orthop 1995;24:319-24. 3. Faccioni F, Franceschetti P, Cerpelloni M, Fracasso ME. In vivo study on metal release from fixed orthodontic appliances and DNA damage in oral mucosa cells. Am J Orthod Dentofacial Orthop 2003;124:687-93. 4. Auletta A, Ashby J. Workshop on the relationship between short-term information and carcinogenicity: Williamsburg, Virginia, January 20-23, 1987. Environ Mol Mutagen 1988;11: 135-45. 5. Ribeiro DA, Favero Salvadori DM, da Silva RN, Ribeiro Darros B, Alencar Marques ME. Genomic instability in non-neoplastic oral mucosa cells can predict risk during 4-nitroquinoline 1-oxide-
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induced rat tongue carcinogenesis. Oral Oncol 2004;40: 910-5. Ribeiro DA. Do endodontic compounds induce genetic damage? A comprehensive review. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:251-6. Yang HW, Chou LSS, Chou MY, Chang YC. Assessment of genetic damage by methyl methacrylate employing in vitro mammalian test system. Biomaterials 2003;24:2909-14. Tice RR, Agurell E, Anderson D, et al. Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 2000;35:206-21. Ribeiro DA, Matsumoto MA, Padovan LE, Marques ME, Salvadori DM. Genotoxicity of corrosion eluates obtained from endosseous implants. Implant Dent 2007;16:101-9. McKelvey-Martin VJ, Green MHL, Schmezer P, Pool-Zobel BL, De Meo MP, Collins A. The single cell gel electrophoresis assay (comet assay): a European review. Mutat Res 1993;288:47-63. Ribeiro DA, Marques ME, Salvadori DM. Biocompatibility of glass-ionomer cements using mouse lymphoma cells in vitro. J Oral Rehabil 2006;33:912-7. Braz MG, Camargo EA, Salvadori DM, Marques ME, Ribeiro DA. Evaluation of genetic damage in human peripheral lymphocytes exposed to mineral trioxide aggregate and Portland cements. J Oral Rehabil 2006;33:234-9. Grassi TF, Camargo EA, Salvadori DM, Marques ME, Ribeiro DA. DNA damage in multiple organs after exposure to chlorhexidine in Wistar rats. Int J Hyg Environ Health 2007;210:163-7. Braz MG, de Castro Marcondes JP, Matsumoto MA, Duarte MA, Salvadori DM, Ribeiro DA. Genotoxicity in primary human peripheral lymphocytes after exposure to radiopacifiers in vitro. J Mater Sci Mater Med 2008;19:601-5. Hartmann A, Agurell E, Beevers C, et al. Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis 2000; 18:45-51. Geurtsen W. Substances released from dental resin composites and glass ionomer cements. Eur J Oral Sci 1998;106:687-95. Andersson M, Agurell E, Vaghef H, Bolesfoldi G, Hellman B. Extended-term of human T-lymphocytes and the comet assay: a useful combination when testing for genotoxicity in vitro? Mutat Res 2003;540:43-55. Piozzi R, Ribeiro DA, Padovan LE, Nary Filho H, Matsumoto MA. Genotoxicity and cytotoxicity in multiple organs induced by titanium miniplates in Wistar rats. J Biomed Mater Res A 2009;88:342-7. Tomakidi P, Koke U, Kern R, et al. Assessment of acute cyto- and genotoxicity of corrosion eluates obtained from orthodontic materials using monolayer cultures of immortalized human gingival keratinocytes. J Orofac Orthop 2000;61:2-19. Westphalen GH, Menezes LM, Pra D, et al. In vivo determination of genotoxicity induced by metals from orthodontic appliances using micronucleus and comet assays. Genet Mol Res 2008;7: 1259-66. Gontijo AM, Elias FN, Salvadori DM, et al. Single-cell gel (comet) assay detects primary DNA damage in non-neoplastic urothelial cells of smokers and ex-smokers. Cancer Epidemiol Biomarkers Prev 2001;10:987-93. Gontijo AM, Marcondes JP, Elias FN, et al. DNA damage in cytologically normal urothelial cells of patients with a history of urothelial cell carcinoma. Environ Mol Mutagen 2002;40:190-9. Ladeira MS, Rodrigues MA, Salvadori DM, Queiroz DM, Freire-Maia DV. DNA damage in patients infected by Helicobacter pylori. Cancer Epidemiol Biomarkers Prev 2004;13:631-7. Ladeira MS, Rodrigues MA, Freire-Maia DV, Salvadori DM. Use of Comet assay to assess DNA damage in patients infected by
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Helicobacter pylori: comparisons between visual and image analyses. Mutat Res 2005;586:76-86. Lee CH, Marckov S, Kim S, Brahim JS, Park MH, Steinert PM. Small protein-rich protein 1 is the major component of the cell envelope of normal human oral keratinocytes. FEBS Lett 2000;477:268-72. Pinhal D, Gontijo AM, Reyes VA, Salvadori DM. Viable human buccal mucosa cells do not yield typical nucleoids: impacts on the single-cell gel electrophoresis/comet assay. Environ Mol Mutagen 2006;47:117-26. Osswald K, Mittas A, Glei M, Pool-Zobel BL. New revival of an old biomarker: characterisation of buccal cells and determination of genetic damage in the isolated fraction of viable leucocytes. Mutat Res 2003;544:321-9. Luft S, Keilig L, J€ager A, Bourauel C. In-vitro evaluation of the corrosion behavior of orthodontic brackets. Orthod Craniofac Res 2009;12:43-51.
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29. Iijima M, Endo K, Yuasa T, et al. Galvanic corrosion behavior of orthodontic archwire alloys coupled to bracket alloys. Angle Orthod 2006;76:705-11. 30. Matos de Souza R, Macedo de Menezes L. Nickel, chromium and iron levels in the saliva of patients with simulated fixed orthodontic appliances. Angle Orthod 2008;78: 345-50. 31. Costa MT, Lenza MA, Gosch CS, Costa I, Ribeiro-Dias F. In vitro evaluation of corrosion and cytotoxicity of orthodontic brackets. J Dent Res 2007;86:441-5. 32. Kao CT, Ding SJ, Min Y, Hsu TC, Chou MY, Huang TH. The cytotoxicity of orthodontic metal bracket immersion media. Eur J Orthod 2007;29:198-203. 33. Ribeiro DA, Pereira PC, Machado JM, Silva SB, Pessoa AW, Salvadori DM. Does toxoplasmosis cause DNA damage? An evaluation in isogenic mice under normal diet or dietary restriction. Mutat Res 2004;559:169-76.
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Genotoxicity of corrosion eluates obtained from orthodontic brackets in vitro Fernanda Angelieri,a Joao Paulo C. Marcondes,b Danielle Cristina de Almeida,c Daisy M. F. Salvadori,d and Daniel A. Ribeiroe S~ao Paulo, Brazil
Introduction: The purpose of this study was to evaluate whether corrosion eluates obtained from commercially available orthodontic brackets are able to induce genetic damage in vitro. Material and Methods: Genotoxicity was assessed by the single cell gel (comet) assay using Chinese hamster ovary (CHO) cells. The following or~o Jose do Rio Preto, Brazil); Dentauthodontic metallic brackets were used: Morelli (Sorocaba, Brazil); Abzil (Sa rum (Pforzheim, Germany); and 3M Unitek (Puchheim, Germany). Each dental bracket was submitted to a corrosion process in a solution containing equal amounts of acetic acid and sodium chloride at 0.1 M concentration for 1, 3, 7, 14, 21, 35, and 70 days. CHO cells were exposed to eluates for 30 minutes at 37 C. The negative control was treated with the same solution used for corrosion process for 30 minutes at 37 C. Independent positive control was performed with methyl methanesulfonate (MMS) (Sigma Aldrich, St. Louis, Mo) at 1 ug/mL for 1 hour. Results: None of the eluates was found to exhibit genotoxicity, regardless of the different commercial brands of orthodontic appliance used. Conclusions: In summary, our results indicate corrosion eluates obtained from orthodontic brackets do not induce genetic damage as assessed by single cell gel (comet) assay. (Am J Orthod Dentofacial Orthop 2011;139:504-9)
B
iocompatibility is the ability of a material to perform an appropriate host response in a specific application.1 This means that the tissue of the patient that comes into contact with the material does not suffer from any toxic, irritating, inflammatory, allergic, genotoxic, or carcinogenic action.2 Accumulating evidence suggests that the oral environment is suitable for the biodegradation of metals as a result of its thermal, microbiologic, and enzymatic properties.3 Intraoral fixed orthodontic appliances
a Associate professor, Department of Orthodontics, S~ao Paulo Methodist University, S~ao Bernardo do Campo, S~ao Paulo, Brazil. b Graduate student, Department of Pathology, Botucatu Medical School, S~ao Paulo State University, UNESP, S~ao Paulo, Brazil. c Graduate student, Department of Pathology, Botucatu Medical School, S~ao Paulo State University, UNESP, S~ao Paulo, Brazil. d Professor, Department of Pathology, Botucatu Medical School, S~ao Paulo State University, UNESP, S~ao Paulo, Brazil. e Professor, Department of Biosciences, Federal University of S~ao Paulo, UNIFESP, Santos, S~ao Paulo, Brazil. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. This work was supported by grants from FAPESP (Fundac¸~ao de Amparo a Pesquisa do Estado de S~ao Paulo, Grant number 07/00345-7). D.A.R. is a recipient of a CNPq fellowship. Reprint requests to: Daniel A. Ribeiro, Departamento de Bioci^encias, Universidade Federal de S~ao Paulo–UNIFESP, Av. Ana Costa, 95, Vila Mathias, Santos–SP, Brazil, 11060-001; e-mail, [email protected]; [email protected]. Submitted, February 2009; revised and accepted, March 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.03.058
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include brackets, bands, and arch wires that are made of alloys containing nickel, cobalt, and chromium in different percentages. Actually, different types of orthodontic brackets are available in the global market. The number of bracket systems for orthodontic therapy has increased significantly. Although they possess undeniable efficiency and local biocompatibility, there is concern about metal release from orthodontic devices to adjacent tissues such as oral mucosa cells and/or periodontal tissues. Thus, further biocompatibility data are needed in order to evaluate all risks of these components. Indeed, the limited data existing on the biocompatibility of these compounds appear to be insufficient. Genotoxicity tests can be defined as in vitro and in vivo tests designed to detect compounds that induce genetic damage, including DNA damage, gene mutation, chromosomal breakage, altered DNA repair capacity, and cellular transformation. In the last decades, genotoxicity assays have gained widespread acceptance as an important and useful indicator of carcinogenicity.4 As the incidence of head and neck cancer has increased in recent years—particularly in developing countries such as India, Vietnam, and Brazil, where it constitutes up to 25% of all types of cancer— risk factors other than tobacco smoke and the abuse of alcohol are of special concern.5 Particularly, little information is available on the genotoxicity of orthodontic brackets so far.
Angelieri et al
As a result of inappropriate evidence, the aim of this study was to evaluate whether corrosion eluates obtained from commercially available orthodontic brackets are able to induce genetic damage in vitro in order to predict the real risks when the corrosion process occurs during orthodontic therapy in vivo. MATERIAL AND METHODS Cell culture
In vitro test systems for genotoxicity evaluation can be differentiated into prokaryotic and/or eukaryotic tests. Studies conducted with the eukaryotic systems are thought to provide more reliable information with respect to the genotoxicity of chemicals.6 Therefore, we aimed to investigate the genotoxic potential of corrosion eluates obtained from orthodontic brackets. For this we were able to use Chinese hamster ovary (CHO) cells. Our choice of CHO cells (a continuous cell line) provided an accurate evaluation of the changes independent from confounding factors such as age and metabolic and hormonal states of the donor. This cell line has a small number of relatively large chromosomes; they grow fast and reproducible results can be obtained from the same cell source.7 For this purpose, CHO cells (lineage CHO K-1) were growth to confluence in 75-cm2 culture flasks (Corning, Incorporated, New York, NY) using Ham's F-10 medium (Invitrogen Corporation, Grand Island, NY) supplemented with 10% fetal calf serum and 100 U/mL penicillin (Life Technologies, Carlsbad, Calif) and 100 mg/ mL streptomycin (Invitrogen Corporation) at 37 C with 5% CO2. Cells were cultured for 5 days prior to treatment with test substances. Confluent cells were detached with 0.15% trypsin (Invitrogen Corporation) for 5 minutes; after that, 2 mL complete medium was added and cells were centrifuged at 1000 rpm (180 g) for 5 minutes. Cell suspension was counted using a Neubauer chamber and seeded in 96-well microtiter plates (Corning) at a density of 1 3 104 cells per well (at a concentration of 1 3 106/mL). Cell treatment
For this study, the following commercially available orthodontic brackets were used: Morelli, Abzil, Dentaurum, and 3M Unitek. There are no differences regarding metal composition among them according to the manufacturer’s instructions. Each dental bracket was submitted to a corrosion process in a solution containing equal amounts of acetic acid and sodium chloride (Merck & Co., Inc., Whitehouse Station, NJ), at 0.1 M concentration, for 1, 3, 7, 14, 21, 35, and 70 days. A volume of 10 uL of cells (approximately 10,000 cells) was
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then added individually to each final solution of eluate maintained for 30 minutes at 37 C. After exposure, cells were washed in phosphate buffered solution (PBS). The negative control was treated with the same solution used for the corrosion process for 30 minutes at 37 C. As cytotoxicity is a confounding factor in genotoxicity studies, it is not recommended to perform the single cell gel (comet) assay on samples with more than 30% cytotoxicity.8 Thus, the exposure period as well as the final concentrations used here were established in a previous study conducted in our laboratory for evaluating the genotoxicity of these compounds only.9 An independent positive control was performed with MMS at 1 mg/mL for 1 hour in order to ensure the reproducibility and sensitivity of the assay. In addition, each treatment was performed consecutively 3 times to ensure reproducibility. Single cell gel (comet) assay
To evaluate the magnitude of DNA damage, we used the alkaline version of the single cell gel (comet) assay. The comet assay is a relatively new, rapid, simple, and reliable biochemical technique for evaluating DNA damage in mammalian cells.8 This technique includes embedding cells in agarose gel on microscope slides, incubating them with the test compound, and then lysing the cells with detergent and high salts.10 During electrophoresis under alkaline conditions, cells with damaged DNA display increased rates of DNA migration to the anode. The increase in DNA migration rate results from the formation of smaller fragments of DNA caused by double-strand breaks, single-strand breaks, and alkalilabile sites. Smaller fragments of DNA migrate further in the electric field compared with intact DNA, and the cellular lysates thus resemble a “comet” with brightly fluorescent head and a tail region. Our own recent studies have demonstrated that the single cell gel (comet) assay is a suitable tool to investigate genotoxicity of dental compounds used in clinical practice.11-14 The protocol used for the single cell gel (comet) assay followed the guidelines purposed by Tice et al.8 Slides were prepared in duplicate per treatment. Thus, a volume of 10 mL of treated or control cells (1 3 104 cells) was added to 120 mL of 0.5% low-melting-point agarose at 37 C, layered onto a precoated slide with 1.5% regular agarose, and covered with a coverslip. After brief agarose solidification in a refrigerator, the coverslip was removed and the slides were immersed in the lysis solution (2.5 M NaCl, 100 mM ethylenediaminetetraacetic acid [EDTA] [Merck & Co., Inc.]; 10 mM Tris-HCl buffer pH 5 10 [Sigma Aldrich, St. Louis, Mo]; 1% sodium sarcosinate [Sigma Aldrich]; with 1% Triton X-100 [Sigma Aldrich]; and 10% DMSO [Merck & Co. Inc.]) for about 1 hour. Prior to electrophoresis, the slides were left in an alkaline
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buffer (0.3 mM NaOH [Merck & Co., Inc.] and 1 mM EDTA [Merck & Co., Inc., pH . 13]) for 20 minutes and electrophoresed for another 20 minutes, at 25 V (0.86 V/cm) and 300 mA. After electrophoresis, the slides were neutralized in 0.4 M Tris-HCl (pH 5 7.5) for 15 minutes, fixed in absolute ethanol, and stored at room temperature until analysis. All of the steps described above were conducted in the dark to prevent additional DNA damage. Throughout this study, some diluted and treated aliquots were tested for viability by trypan blue exclusion,10 and greater than 75% of cells constantly excluded trypan.
able to detect the significant increase in tail moment of the positive control (MMS-treated cells) compared with the negative controls. However, corrosion eluates obtained from brackets of Morelli did not induce genetic damage at any experimental period established, as verified by no statistically significant differences (P .0.05) when compared with negative controls. Such findings are presented in Table I. The same picture occurred with the other brackets commercially tested (ie, Abzil, Dentaurum, and 3M Unitek; (Table I). Figure 1 shows an undamaged CHO cell from a negative control, a cell exposed to corrosion eluate for 70 days, and an MMS-induced comet cell (positive control).
Comet capture and analysis
DISCUSSION
A total of 50 randomly captured comets per treatment (25 cells from each slide)15 were examined blindly by 1 expertise observer (D.A.R.) at 4003 magnification using a fluorescence microscope (Olympus, Center Valley, Pa) connected through a black and white camera to an image analysis system (Comet Assay II, Perceptive Instruments Ltd, Haverhill, UK). For all experiments, we evaluated 2 image analysis parameters: tail intensity (% migrated DNA) and tail moment. Tail moment was calculated by the image analysis system as the product of the tail length (DNA migration) and the fraction of DNA in the comet tail (% DNA in the tail). In none of the experiments there was a significant difference between these parameters. Therefore, we chose tail moment for the presentation of the results.
The aim of this study was to evaluate the genotoxic damage induced by corrosion eluates obtained from orthodontic appliances using CHO cells in vitro. The investigation was conducted using the single cell gel (comet) assay. To the best of our knowledge, the approach has not been addressed so far. The alkaline version of the single cell gel (comet) assay used here is sensitive for a wide variety of DNA lesions. Among them are single- and double-strand breaks; oxidative DNA base damage; alkali-labile sites, including abasic and incomplete repair sites; and DNADNA/DNA-protein/DNA-drug cross-linking in any eukaryotic cell.8 Tail moment is a virtual measure calculated by the computerized image analysis system, considering both the length of DNA migration in the comet tail and the tail intensity. This parameter is one of the best indices of induced DNA damage among the various parameters calculated by this method.8 In this study, a cell culture technique was employed in order to evaluate the putative noxious activities for corrosion products from dental brackets in vitro. In vitro studies are simple, inexpensive to perform, provide a significant amount of information, can be conducted under controlled conditions, and may clarify the mechanisms of cellular toxicity.16 The results obtained from in vitro assays may be indicative of the effects observed in vivo. The most frequently used cells for in vitro studies when screening for potential genotoxicity are L5178Y mouse lymphoma cells or CHO cells because these cellular types are easy to handle and well characterized with regard to, for example, their growth pattern.17 Thus, CHO cells, a continuous cell line, were used as the target cell in this setting. On the basis of tail moment data, the results of this study pointed out that the alkaline single cell gel (comet) assay in the experimental conditions used failed to detect the presence of DNA damage after treatment by corrosion eluates from four different commercial brands of
Statistical methods
The parameter from the comet assay (tail moment) was assessed by 1-way analysis of variance (ANOVA) followed by post hoc analysis and the Tukey test using SigmaStat software, version 1.0 (Jandel Scientific, Rafael, Calif). A P value less than 0.05 was considered statistically significant. RESULTS
The cytotoxicity of eluates from brackets was measured in CHO cells through a trypan blue assay in range-finding experiment prior to the determination of chemically induced genotoxicity. The dose-response relationships on cell viability of all solutions tested at the following periods (1, 3, 7, 14, 21, 35 and 70 days) were assessed by trypan blue assay. The mean cell viability for CHO cells was approximately 83% for all solutions tested, without any statistical differences (P .0.05) among them (data not shown). The effect of exposure to the test materials on DNA damage in CHO cells is given in Table 1. The assay was
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Table I. DNA damage (tail moment) in Chinese hamster ovary cells exposed to corrosion eluates from orthodontic brackets* Brackets Commercially Available Days 0 1 3 7 14 21 35 70 Positive control*,y
Morelli 0.67 6 0.22 0.98 6 0.45 1.20 6 0.67 1.22 6 0.54 0.67 6 0.45 0.98 6 0.57 0.57 6 0.32 1.20 6 0.57 5.72 6 0.72z
Abzil 0.67 6 0.22 0.54 6 0.43 0.63 6 0.62 0.76 6 0.44 0.58 6 0.40 0.54 6 0.34 0.87 6 0.45 0.65 6 0.72 5.72 6 0.72z
Dentaurum 0.67 6 0.22 0.54 6 0.22 0.67 6 0.34 0.76 6 0.64 0.96 6 0.54 0.53 6 0.37 0.98 6 0.56 1.04 6 0.54 5.72 6 0.72z
3M Unitek 0.67 6 0.22 0.58 6 0.16 0.99 6 0.54 0.89 6 0.43 1.02 6 0.47 0.45 6 0.33 0.76 6 0.32 0.67 6 0.43 5.72 6 0.72z
*Data of the 3 different experiments. Values are expressed as mean and 6 standard deviation; yMethyl methanesulfonate, 1 mg/mL; zP \0.05 when compared with the negative control (day zero).
Fig 1. Representative comet images of a CHO cell from a negative control (A), a cell exposed to corrosion eluates for 70 days (B), and a MMS-treated cell (positive control) (C). DNA was stained with ethidium bromide; 403 magnification.
brackets at all experimental periods established in this setting. By comparison, previous studies conducted by our group have demonstrated that corrosion eluates obtained from endosseous dental implants were not genotoxic in vitro, and titanium miniplates do not exert DNA injury in vivo.14,18 Such findings are the same magnitude as reported by others.19 It has been postulated that there is an absence of primary DNA damage in oral mucosa cells from patients undergoing orthodontic therapy.20 Nevertheless, some authors have noticed genotoxic damage induced by orthodontic therapy in human buccal mucosa cells as assessed by the single cell gel (comet) assay in vivo.3 Herein, the application of the single cell gel (comet) assay using epithelial cells merits discussion. In the last few years, our research group has standardized the single cell (comet) assay in human epithelial cells, such as urothelial cells21,22 and gastric cells.23,24 In particular, oral cells are a type of stratified squamous epithelium that undergoes a terminal differentiation to form a protective barrier
that covers the cells. This cell envelope, which is rich in a small proline-rich protein, provides a barrier against what can be a very hostile environment in the mouth, and accounts for the high resistance of oral cells to lysis.25 In this regard, lysis using a trypsin- or proteinase K–assisted procedure is required in order for the single cell gel (comet) assay to be performed. Our preliminary results using the single cell gel (comet) assay in humans indicated that only a few cells from oral mucosa cell samples yielded comets that can be analyzed by current methods because the oral cells sustained massive DNA damage with subsequent disintegration.26 Therefore, the single cell gel (comet) assay, as it is commonly performed, may not be useful for genotoxicity monitoring in human oral mucosa cells. Furthermore, our data generated after enzymatic enrichment of viable cells and immunomagnetic separation of epithelial cells suggested that most of the oral mucosa cells that do form comets are probably leukocytes.26 These results were confirmed by others.27
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In a recent study conducted by Luft et al,28 all brackets tested seemed to be biocompatible and applicable for orthodontic therapy. No significant differences in the nickel, chromium, and iron levels were noticed in orthodontic appliances at all study periods in vitro.29,30 By comparison, none of the bracket extracts altered L929 cell viability or morphology, as assessed by crystal violet and MTT assays.31 The same findings were reported by others.32 It is important to stress that no single test is capable of detecting all mechanisms by which compounds induce genetic damage. Therefore, for a more detailed judgment of a compound, a battery of tests—including those that assess metaphase chromosomal aberrations, micronuclei, sister chromatid exchanges, and host cell activation— would be significant. In the present study, as well as in all of our previous investigations using the single cell gel (comet) assay, we have always excluded comets without clearly identifiable heads during the image analysis. Although it should be emphasized that it is still not completely understood what these hedgehogs actually represent, this type of comet was excluded on the basis of the assumption that these cells represent dead cells, resulting from putative cytotoxic effects of corrosion eluates rather than primary DNA damage after a direct interaction between DNA and a genotoxic agent.33
6.
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11.
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CONCLUSIONS
The results of this study suggest that corrosion eluates obtained from orthodontic appliances did not induce DNA breakage as depicted by the single cell gel (comet) assay. Since DNA damage is an important step in events leading from carcinogen exposure to cancer, the results of the present study represent a potential alert to the correct evaluation of the potential health risks associated with exposure to these compounds.
16. 17.
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29. Iijima M, Endo K, Yuasa T, et al. Galvanic corrosion behavior of orthodontic archwire alloys coupled to bracket alloys. Angle Orthod 2006;76:705-11. 30. Matos de Souza R, Macedo de Menezes L. Nickel, chromium and iron levels in the saliva of patients with simulated fixed orthodontic appliances. Angle Orthod 2008;78: 345-50. 31. Costa MT, Lenza MA, Gosch CS, Costa I, Ribeiro-Dias F. In vitro evaluation of corrosion and cytotoxicity of orthodontic brackets. J Dent Res 2007;86:441-5. 32. Kao CT, Ding SJ, Min Y, Hsu TC, Chou MY, Huang TH. The cytotoxicity of orthodontic metal bracket immersion media. Eur J Orthod 2007;29:198-203. 33. Ribeiro DA, Pereira PC, Machado JM, Silva SB, Pessoa AW, Salvadori DM. Does toxoplasmosis cause DNA damage? An evaluation in isogenic mice under normal diet or dietary restriction. Mutat Res 2004;559:169-76.
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