Biomonitoring of mutagenicity and cytotoxicity in patients undergoing fixed orthodontic therapy
Fernanda Angelieri,a Viviane Carlin,b Renato A. Martins,c and Daniel A. Ribeirob,d S~o Paulo, Brazil a

Introduction: The aim of the present study was to evaluate DNA damage (micronucleus) and cellular death (pyknosis, karyolysis, and karyorrhexis) in exfoliated buccal mucosa cells from adults after fixed orthodontic therapy. Material and Methods: A total of 23 healthy adults (10 men and 13 women) undergoing orthodontic therapy were included in this setting. Results: The results pointed out no significant statistically differences (P .0.05) of micronucleated oral mucosa cells. In the same way, orthodontic therapy was not able to increase other nuclear alterations closely related to cytotoxicity such as karyorrhexis, pyknosis and karyolysis (P .0.05). Conclusion: In summary, these data indicate that orthodontic therapy may not be a factor that induces chromosomal damage, nor it is able to promote cytotoxicity. Since DNA damage and cellular death are important events during carcinogenic processes, especially in early phases, this study represents a correct evaluation with respect to real health risks induced by orthodontic devices. (Am J Orthod Dentofacial Orthop 2011;139:e399-e404)

nvironmental health sciences focus on the link between the presence of contaminants in the environment and their relation to possible adverse health effects. Within this context, human biomonitoring data have proved to be a valuable addition to, or have even surpassed, estimates of exposure based on environmental measures.1 Particularly, biomonitoring of human populations exposed to potential mutagens, cytotoxins, or even carcinogens can provide an early detection system for the initiation of cell deregulation in the development of cancer.2 As a result, several markers have been identified to monitor the exposure of humans to mutagens and carcinogens. Classically, mutagenicity tests evaluate chromosomal aberration and sister chromatid exchange. Data are accumulating that support
a Associate professor, Department of Orthodontics, S~o Paulo Methodist Univera sity, S~o Bernardo do Campo, SP, Brazil. a b Graduate student, Department of Pathology, Paulista Medical School, Federal University of S~o Paulo, SP, Brazil. a c Undergraduate student, Department of Biosciences, Federal University of S~o a Paulo, Santos, SP, Brazil. d Professor, Department of Pathology, Paulista Medical School, Federal University of S~o Paulo, SP, Brazil; Department of Biosciences, Federal University of S~o a a Paulo, Santos, SP, Brazil. The authors report no commercial, proprietary, or financial interest in the products or companies described in this article. This study was supported by grants from S~o Paulo Research Foundation (Grant a number: 07/00345-7). R.A.M. is a recipient of the S~o Paulo Research Foundation’s a student fellowship and D.A.R. is a recipient of the CNPq fellowship. Reprint requests to: Daniel Araki Ribeiro, Departamento de Biociencias Universidade Federal de S~o Paulo – UNIFESP, Av. Ana Costa 95, 11060-001, Santos – SP, Brazil; a e-mail,; Submitted, May 2009; revised and accepted, June 2009. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2009.06.029


the hypothesis that mutagenicity end points are predictors of human cancer risk. However, the relationship among DNA damage, persistence and repair, and mutagenic end points remains complex.2 Intraoral fixed orthodontic appliances 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. In a previous study conducted by Faccioni et al3, it was found that, as assessed by the single-cell gel (comet) assay in human patients, orthodontic therapy can induce DNA damage in oral mucosa cells as a result of nickel and cobalt released from fixed orthodontic appliances. The alkaline version of the single-cell gel (comet) assay is sensitive for a wide variety of DNA lesions. Among them are single strand 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.4 However, the single-cell gel (comet) assay does not necessarily predict the mutagenic potential of metals; moreover, the genotoxicity can be modulated in combination with other DNA-damaging agents that are present in the environment.5 This is because the absence of a close relationship between DNA migration in the comet assay and mutagenesis may be explained by the fact that some effects seen in the comet assay occur as a consequence of an error-free DNA repair process.6 Herein, further evaluation of orthodontic therapy and

and karyolysis. karyolysis. The level of statistical significance was set at 5%. and at final orthodontic therapy.04%. the air-dried slides were stained.2% during the treatment or stainless steel (Ormco Corporation. and karyolysis.5 6 7 years who had submitted to orthodontic therapy at the Department of Orthodontics. Calif): nickel. such data will contribute to a better understanding of orthodontic therapy on the cellular system. karyolysis and karyorrhexis were also evaluated in this setting. A total of 3 individuals included in this trial used oral antiseptic solutions (chlorhexidine.11. The arch wires used in this study were a nickel-titanium alloy (Morelli Dental Company. fixed in 3:1 methanol/acetic acid. Sorocaba. 49. cells were obtained by scraping the right/left cheek mucosa with a moist wooden spatula. 72. Brazil): nickel. In the same way. San Rafael. For this purpose. Calif). pyknosis. iron. Brackets were provided by Abzil (S~o Paulo. centrifuged (800 rpm) over 5 minutes. as depicted by the frequency of karyorrhexis. Brazil): iron.15 Statistical methods The Friedman test for dependent samples was used to compare the frequencies of micronuclei and other cytotoxic alterations among the samples before. MATERIAL AND METHODS Subjects Feulgen/Fast Green method. karyorrhexis. 170 days after beginning the treatment). version 1.05) were noticed either during or after orthodontic therapy. S~o Bernardo do Campo. during. After rinsing the mouth with tap water. Later. and after therapy (ie.10 or with malignant tumors undergoing radio. at least 6 months after the insertion of stainless steel arches).13 Two thousand cells were scored from each patient for each sampling time (before. 50. S~o Paulo a Methodist University.or chemotherapy. For this purpose. For cytotoxicity. pyknosis. Brazil. the present study was to investigate the frequency of micronucleated cells in oral mucosa from individuals who had submitted to fixed orthodontic therapy. These data are summarized in Figure 2. we used SigmaStat software. Daily alcohol consumption was not considered in this study because a recall bias phenomenon has occurred. 71%. To monitor cytotoxic effects. nickel. April 2011  Vol 139  Issue 4  Supplement 1 American Journal of Orthodontics and Dentofacial Orthopedics .8%. 8%.e400 Angelieri et al mutagenicity and/or cytotoxicity is a necessary step to better establish the real health risks. during orthodontic therapy (defined as the time after the alignment and leveling with nickel titanium arches. Cells were transferred to a tube containing saline solution. Before the beginning of orthodontic device exposure. exposure to known genotoxins was not related to any of the study participants. Data analysis Micronuclei were scored according to the criteria described by Sarto et al14 as a parameter for DNA damage (mutagenicity). a great deal of enthusiasm was raised by the application of the micronucleus test to uncultured exfoliated cells. a The fixed appliances consisted of an average of 4 to 8 bands and 20 bonded brackets. using the Figure 1 shows the frequencies of micronucleated cells in individuals undergoing orthodontic therapy.0. during. Figure 3 shows a micronucleated cell. and examined under a light microscope at 4003 magnification to determine the frequency of micronucleated cells as described elsewhere. the following nuclear alterations were considered: pyknosis. DISCUSSION The aim of this study was to employ the micronucleus test to assess chromosome damage and/or cellular death in adults who had submitted to orthodontic therapy. Results were expressed as a percentage together. pyknosis. No statistically significant differences (P . To avoid putative confounding factors.8 Recently. titanium. and at final orthodontic treatment).12 As a result and because of inappropriate in-vivo evidence. Micronucleus test in oral mucosa cells Exfoliated oral mucosa cells were collected before orthodontic therapy. ie. 20% at the end of the orthodontic therapy. all participants of this study were nonsmokers. 19%. we have applied this methodology with success in individuals (adults and children) exposed to dental radiographs. and Figure 4 displays abnormalities closely related to cytotoxicity. and karyorrhexis. Orange. In addition.6%.7 Micronucleus arises from acentric fragments or whole chromosomes that are not included in the main nuclei of the daughter cells.6%. RESULTS The subjects of this study comprised a total of 23 healthy adults (10 men and 13 women) with a mean age of 18. Listerine) regularly. Certainly. chromium. Such analysis was established in a previous study conducted by our research group. 8. an increase of other nuclear alterations were not observed throughout the experimental design. the mean frequency of micronucleated cells was 0. and a chromium.0 (Jandel Scientific. The formation of micronuclei can be induced by substances that cause chromosome breakage (clastogens) as well as by agents that affect the spindle apparatus (aneugens).9. and dropped onto precleaned slides.

D. inherited defects in DNA metabolism and/ or repair). Micronuclei frequencies after orthodontic therapy. alcohol. American Journal of Orthodontics and Dentofacial Orthopedics April 2011  Vol 139  Issue 4  Supplement 1 . P \0. It has been well established that genomic damage is produced by environmental exposure to genotoxins. Feulgen/Fast Green stain.18 Micronucleated cell indexes may reflect genomic instability.21 Buccal cells have been shown to have limited DNA repair capacity relative to peripheral blood lymphocytes and therefore may more accurately reflect genomic instability events in epithelial tissues. Smaller micronuclei are the result of structural aberrations and consist of chromosomal fragments. The measurement of the frequency of micronuclei induced in cells by mutagenic agents is widely used for analysis of cytogenetic damage.23-25 It has been postulated absence of primary DNA damage in oral mucosa cells from patients undergoing orthodontic therapy. there will arise bigger micronuclei from whole chromosomes as a follow up to damage of the spindle apparatus of the cell (aneugen). and the precision obtained from scoring larger numbers of cells. titanium miniplates do not exert DNA injury in vivo. drugs.19 The detection of an elevated frequency of micronuclei in a given population indicates increased risk of cancer. To the best of our knowledge. Faccioni et al. 3100 magnification. and genetic factors (eg. medical procedures (eg.Angelieri et al e401 Fig 1. Results are expressed as percentage (mean 6 S.0. folate). where cells undergo mitosis.05. The key advantage of the micronucleus assay is the relative ease of scoring. Figure 3.05. Genomic damage is probably the most important fundamental cause of developmental and degenerative diseases. cell types that repair DNA damage efficiently are likely to show lower levels of residual damage than cells less proficient in DNA repair. during. pyknosis. and karyolysis) after orthodontic therapy. the limited costs and persontime required. By comparison. In addition. Fig 2.8 Hence.8 Micronuclei contain genetic material that is lost from the genome during mitosis as a result of a clastogen or aneugen occurrence. Cellular death parameters (karyorrhexis. previous studies conducted by our group demonstrated that corrosion eluates obtained from orthodontic devices or endosseous dental implants were not genotoxic in vitro. radiation and chemicals).22 Our results demonstrated that the micronucleus frequencies were not significantly different before. the approach has not been addressed in the literature so far. Rapid turnover of epithelial tissues brings the cells to the surface where they exfoliate.26 Nevertheless. micronutrient deficiency (eg. Results are expressed as percentage (mean 6 SD) P . and even after orthodontic therapy in this trial.17.16 Damage that leads to the formation of micronuclei takes place in the basal layer of the epithelial tissue.20 However. Micronucleated cell (arrow). lifestyle factors (eg. and stress). smoking.).

Calif).30 Interestingly. an age-related increase of micronuclei has been postulated. therefore. whereas acute toxicity was observed for Transbond XT primer (3M Unitek. and MTT test values were significantly reduced at both 2. Moreover. Based on our results. the toxicity of orthodontic adhesives was assessed in vitro. B.32 It is important to stress that repeated exposure to cytotoxicants can result in chronic cell injury.30 Human peripheral blood mononuclear cell death rates were highest at nickel concentrations of 29 ppm when the cells were cultured without a cell growth promoter. since comparison between the different wires revealed that stainless steel induced less toxicity/loss of viability when compared with titanium-molybdenum alloy (TMA) and nickel-titanium wire. some confounding factors must be considered. Feulgen/Fast Green stain. we postulated the lack of clastogenic and/or aneugenic effects related to orthodontic therapy exposure on buccal mucosa cells of healthy individuals. karyorrhexis. Cytotoxicity parameters evaluated in this study: A. C.3 Nevertheless. For example.29 Significant increased cell proliferation was not observed for any nickel concentration. the mutagenic potential of alcohol is controversial and quite complicated to interpret using micronucleus assay in exfoliated cells. Also. the frequency of karyorrhexis. Some authors have shown high frequencies of apoptotic cells in regard to a significant decrease in cellular viability in patients undergoing orthodontic therapy.14. such discrepancies are due to differences in studies design. almost all participants consumed alcohol and tobacco. our results demonstrated that orthodontic treatment was not able to induce cellular death. a correlation between cell proliferation and induction of cancer is assumed. no genotoxic effect of alcohol was found. orthodontic wires made of different materials caused toxicity and loss of viability. 3100 magnification.35 Furthermore. in 2 reports.36 Thus. karyolysis. the influence of the individual factors could not be elucidated. and titanium-molybdenum alloys were not neurotoxic.05) between values before vs after or before vs during orthodontic therapy. pyknosis. as depicted by no significant differences (P . failures in the DNA repair system. a significant increase in micronuclei frequency 30 days after the beginning of the treatment has been demonstrated.35 All participants of this study were of a similar age. the influence of tobacco smoke has also been usually considered as a relevant confounding factor. and ultimately tumor development. Elgin. Viruses. have noticed genotoxic damage induced by orthodontic therapy in human buccal mucosa cells as assessed by the single-cell gel (comet) assay in vivo. proliferation may increase the risk of mutations within target cells and also be important in selective clonal expansion of (exogenously or endogenously) initiated cells from preneoplastic foci and eventually tumors. hyperplasia. Ill) were significantly toxic in cortical cell cultures. In human cytogenetic studies. April 2011  Vol 139  Issue 4  Supplement 1 American Journal of Orthodontics and Dentofacial Orthopedics .34 Probably. all adults recruited to participate in this study were nonsmokers. 0.9 and 29 ppm when a growth promoter was included. while neither of the 2 drugs alone caused an elevation of micronuclei frequencies. demmonstrating no toxic reactions. copper-nickel-titanium. and a combination of the 2 were examined. which correlated with the concentration of released ions in osteblastic-like cells in vitro. and interindividual variations have already been associated with increased frequency of chromosome aberrations.34 Further research will be required to fully elucidate this relationship.37 In the other 2 studies. a synergistic effect of alcohol and nicotine was observed. To monitor cytotoxic effects. compensatory cell proliferation.e402 Angelieri et al Figure 4.33 In fact. while stainless steel and Elgiloy metals (Elgiloy Specialty Metals.28 Copper and silver ions showed a timedependent low biocompatibility. alterations in the immune system.31 In the same way. In particular. and pyknosis were evaluated in this experimental design.39 in which the effects of alcohol consumption.38 In a study by Stich and Rosin. other studies have indicated that nickel-titanium. karyolysis. Monrovia.27 Probably. cigarette smoking.3 Furthermore.

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