Genotoxic effects in oral mucosal cells caused by the use of orthodontic fixed appliances in patients after short and long periods of treatment

  • María Gabriela Flores-Bracho
  • Catarina Satie Takahashi
  • Willian Orlando Castillo
  • Maria Conceição Pereira Saraiva
  • Erika Calvano Küchler
  • Mírian Aiko Nakane Matsumoto
  • José Tarcísio Lima Ferreira
  • Paulo Nelson-Filho
  • Fabio Lourenço RomanoEmail author
Original Article



This study aimed to evaluate the genotoxic effects in the oral epithelial cells of patients undergoing fixed orthodontic treatment and to compare these to a control group without treatment. The null hypothesis to be tested is that corrective orthodontic treatment at different periods does not cause genotoxic effects in patients.

Material and methods

An observational cross-sectional study including 74 patients enrolled in corrective orthodontic treatment and 21 control patients, between 11 and 35 years of age, of both genders, participated in the research. Patients undergoing treatment were divided into four treatment groups differentiated by treatment periods: G1, n = 21 (1 month to 12 months); G2, n = 21 (13 to 24 months); G3, n = 23 (25 to 48 months); and G4, n = 9 (over 48 months). Cells were collected by scraping the internal side of the cheek and subsequently placed in tubes containing 0.9% sodium chloride solution. The sample underwent evaluation for genotoxic effects by means of the micronucleus test (MNT). Bivariate analyses were performed using parametric tests (t test or ANOVA) and nonparametric tests (Chi-square test, Kruskal-Wallis test, Dunn post-test). The adopted level of significance was 5%.


Statistically significant differences for any of the genotoxic abnormalities (binucleated, trinucleated, karyolysis, piknosis, nuclear buds) were not found except for karyolysis, which was higher in the control group than in G4 (p < 0.05).


This study did not demonstrate evidence of genotoxic effects even after long periods of corrective orthodontic treatment.

Clinical relevance

This study explores genotoxic effects in fixed orthodontic patients.


Orthodontic appliances Genetic Cytogenetic Test of micronucleus 


Authors’ contributions

María Gabriela Flores Bracho conducted a review of literature, wrote the manuscript, collected the data, and did the experimental procedures of the project.

Catarina Satie Takahashi supervised all the experimental procedures of the project.

Willian Orlando Castillo supervised all the experimental procedures of the project.

Maria Conceição Pereira Saraiva designed the study and performed the statistical analyses.

Erika Calvano Küchler organized the data and wrote the manuscript.

Mírian Aiko Nakane Matsumoto collaborated in the selection of experimental subjects.

José Tarcísio Lima Ferreira collaborated in the selection of experimental subjects. Fabio Lourenço Romano and Paulo Nelson-Filho conceived the study and supervised all aspects of its implementation.

All authors contributed to the interpretation of results and manuscript review.


The work was supported by CAPES-Coordination for the Improvement of Higher Education Personnel-Brazilian federal government.

Compliance with ethical standards

The Research Ethics Committee (CAAE 35905914.1.0000.5419) approved this project. The patients agreed to participate in the study and they, or their guardians, signed an informed consent.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The Research Ethics Committee (CAAE 35905914.1.0000.5419) approved this project. All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

Informed consent

Informed consent was obtained from all individual participants that were included in the study.


  1. 1.
    Wataha JC (2000) Biocompatibility of dental casting alloys: a review. J Prosthet Dent 83:223–234CrossRefGoogle Scholar
  2. 2.
    House K, Sernetz F, Dymock D, Sandy JR, Ireland AJ (2008) Corrosion of orthodontic appliances--should we care? Am J Orthod Dentofac Orthop 133:584–592CrossRefGoogle Scholar
  3. 3.
    Ristic L, Vucevic D, Radovic L, Djordjevic S, Nikacevic M, Colic M (2014) Corrosive and cytotoxic properties of compact specimens and microparticles of Ni-Cr dental alloy. J Prosthodont 23:221–226CrossRefGoogle Scholar
  4. 4.
    Petoumeno E, Kislyuk M, Hoederath H, Keilig L, Bourauel C, Jäger A (2008) Corrosion susceptibility and nickel release of nickel titanium wires during clinical application. J Orofac Orthop 69:411–423CrossRefGoogle Scholar
  5. 5.
    Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F et al (2000) IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. International Programme on Chemical Safety. Mutat Res 463:111–172CrossRefGoogle Scholar
  6. 6.
    Toy E, Ozturk F, Karatas OH, Yalcin M (2014) Evaluation of the genotoxicity and cytotoxicity in the buccal epithelial cells of patients undergoing orthodontic treatment with three light-cured bonding composites by using micronucleus testing. Korean J Orthod 44:128–135CrossRefGoogle Scholar
  7. 7.
    Tolbert PE, Shy CM, Allen JW (1992) Micronuclei and other nuclear anomalies in buccal smears: methods development. Mutat Res 271:69–77CrossRefGoogle Scholar
  8. 8.
    Zalacain M, Sierrasesúmaga L, Patiño A (2005) The cytogenetic assay as a measure of genetic instability induced by genotoxic agents. An Sist Sanit Navar 28:227–236CrossRefGoogle Scholar
  9. 9.
    Fenech M, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmüller S, Holland N (2007) Harmonisation of the micronucleus assay in human buccal cells--a human micronucleus (HUMN) project ( initiative commencing in 2007. Mutagenesis 22:3–4CrossRefGoogle Scholar
  10. 10.
    Thomas P, Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmueller S, Fenech M (2009) Buccal micronucleus cytome assay. Nat Protoc 4:825–837CrossRefGoogle Scholar
  11. 11.
    Nersesyan A, Kundi M, Atefie K, Schulte-Hermann R, Knasmüller S (2006) Effect of staining procedures on the results of micronucleus assays with exfoliated buccal mucosa cells. Cancer Epidemiol Biomark Prev 15:1835–1840CrossRefGoogle Scholar
  12. 12.
    Saran R, Tiwari RK, Reddy PP, Ahuja YR (2008) Risk assessment of buccal cancer in patients with pre-cancerous states of the buccal cavity using micronucleus test and challenge assay. Buccal Oncol 44:354–360Google Scholar
  13. 13.
    Holland N, Bolognesi C, Kirsch-Volders M, Bonassi S, Zeiger E, Knasmueller S, Fenech M (2008) The micronucleus assay in human buccal cells as a tool for biomonitoring DNA damage: the HUMN project perspective on current status and knowledge gaps. Mutat Res 659:93–108CrossRefGoogle Scholar
  14. 14.
    Martino-Roth MG, Viégas J, Roth DM (2003) Occupational genotoxicity risk evaluation through the comet assay and the micronucleus test. Genet Mol Res 2:410–417Google Scholar
  15. 15.
    Migliore L, Guidotti P, Favre C, Nardi M, Sessa MR, Brunori E (1991) Micronuclei in lymphocytes of young patients under antileukemic therapy. Mutat Res 263:243–248CrossRefGoogle Scholar
  16. 16.
    Xue KX, Wang S, Ma GJ, Zhou P, Wu PQ, Zhang RF, Xu Z, Chen WS, Wang YQ (1992) Micronucleus formation in peripheral-blood lymphocytes from smokers and the influence of alcohol- and tea-drinking habits. Int J Cancer 50:702–705CrossRefGoogle Scholar
  17. 17.
    Fenech M (1993) The cytokinesis-block micronucleus technique: a detailed description of the method and its application to genotoxicity studies in human populations. Mutat Res 285:35–44CrossRefGoogle Scholar
  18. 18.
    Landi S, Lazzolino E, Barale R (2000) Are baseline frequencies of SCEs, CAs, and MN in human lymphocytes related to hematological values? Mutat Res 469:159–166CrossRefGoogle Scholar
  19. 19.
    Bukvic N, Gentile M, Susca F, Fanelli M, Serio G, Buonadonna L, Capurso A, Guanti G (2001) Sex chromosome loss, micronuclei, sister chromatid exchange and aging: a study including 16 centenarians. Mutat Res 498:159–167CrossRefGoogle Scholar
  20. 20.
    Lee TK, Allison RR, O'Brien KF, Khazanie PG, Johnke RM, Brown R et al (2004) Ginseng reduces the micronuclei yield in lymphocytes after irradiation. Mutat Res 557:75–84CrossRefGoogle Scholar
  21. 21.
    Bonassi S, Znaor A, Ceppi M, Lando C, Chang WP, Holland N et al (2007) An increased micronucleus frequency in peripheral blood lymphocytes predicts the risk of cancer in humans. Carcinogenesis 28:625–631CrossRefGoogle Scholar
  22. 22.
    Bloching M, Reich W, Schubert J, Grummt T, Sandner A (2008) Micronucleus rate of buccal mucosal epithelial cells in relation to buccal hygiene and dental factors. Bucal Oncol 44:220–226Google Scholar
  23. 23.
    Konopacka M (2003) Effect of smoking and aging on micronucleus frequencies in human exfoliated buccal cells. Neoplasma 50:380–382Google Scholar
  24. 24.
    Bonassi S, Coskun E, Ceppi M, Lando C, Bolognesi C, Burgaz S (2011) The human MicroNucleus project on exfoliated buccal cells (HUMN(XL)): the role of life-style, host factors, occupational exposures, health status, and assay protocol. Mutat Res 728:88–97CrossRefGoogle Scholar
  25. 25.
    Fenech M, Holland N, Zeiger E, Chang WP, Burgaz S, Thomas P, Bolognesi C, Knasmueller S, Kirsch-Volders M, Bonassi S (2011) The HUMN and HUMNxL international collaboration projects on human micronucleus assays in lymphocytes and buccal cells-past, present and future. Mutagenesis 26:239–245CrossRefGoogle Scholar
  26. 26.
    Katarkar A, Mukherjee S, Khan MH, Ray JG, Chaudhuri K (2014) Comparative evaluation of genotoxicity by micronucleus assay in the buccal mucosa over comet assay in peripheral blood in buccal precancer and cancer patients. Mutagenesis 29:325–334CrossRefGoogle Scholar
  27. 27.
    Westphalen GH, Menezes LM, Prá D, Garcia GG, Schmitt VM, Henriques JA, Medina-Silva R (2008) In vivo determination of genotoxicity induced by metals from orthodontic appliances using micronucleus and comet assays. Genet Mol Res 7:1259–1266CrossRefGoogle Scholar
  28. 28.
    Hafez HS, Selim EM, Kamel Eid FH, Tawfik WA, Al-Ashkar EA, Mostafa YA (2011) Cytotoxicity, genotoxicity, and metal release in patients with fixed orthodontic appliances: a longitudinal in-vivo study. Am J Orthod Dentofac Orthop 140:298–308CrossRefGoogle Scholar
  29. 29.
    Natarajan M, Padmanabhan S, Chitharanjan A, Narasimhan M (2011) Evaluation of the genotoxic effects of fixed appliances on buccal mucosal cells and the relationship to nickel and chromium concentrations: an in-vivo study. Am J Orthod Dentofac Orthop 140:383–388CrossRefGoogle Scholar
  30. 30.
    Heravi F, Abbaszadegan MR, Merati M, Hasanzadeh N, Dadkhah E, Ahrari F (2013) DNA damage in buccal mucosa cells of patients with fixed orthodontic appliances. J Dent 10:494–500Google Scholar
  31. 31.
    Martín-Cameán A, Jos A, Mellano-García P, Iglesias-Linares A, Solano E, Cameán AM (2015) In vitro and in vivo evidence of cytotoxic and genotoxic effects of metal ions released by orthodontic appliances: a review. Environ Toxicol Pharmacol 40:86–113CrossRefGoogle Scholar
  32. 32.
    von Elm E, Altman DG, Egger M, Pocock SJ, Gøtzsche PC, Vandenbroucke JP, STROBE Initiative (2007) The strengthening the reporting of observational studies in epidemiology (STROBE) statement: guidelines for reporting observational studies. Lancet 370(9596):1453–1457CrossRefGoogle Scholar
  33. 33.
    Titenko-Holland N, Moore LE, Smith MT (1994) Measurement and characterization of micronuclei in exfoliated human cells by fluorescence in situ hybridization with a centromeric probe. Mutat Res 312:39–50CrossRefGoogle Scholar
  34. 34.
    Ramirez A, Saldanha PH (2002) Micronucleus investigation of alcoholic patients with buccal carcinomas. Genet Mol Res 1:246–260Google Scholar
  35. 35.
    Thomas P, Hecker J, Faunt J, Fenech M (2007) Buccal micronucleus cytome biomarkers may be associated with Alzheimer's disease. Mutagenesis 22:371–379CrossRefGoogle Scholar
  36. 36.
    Fenech M (2007) Cytokinesis-block micronucleus cytome assay. Nat Protoc 2:1084–1104CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • María Gabriela Flores-Bracho
    • 1
  • Catarina Satie Takahashi
    • 2
  • Willian Orlando Castillo
    • 2
  • Maria Conceição Pereira Saraiva
    • 1
  • Erika Calvano Küchler
    • 1
  • Mírian Aiko Nakane Matsumoto
    • 1
  • José Tarcísio Lima Ferreira
    • 1
  • Paulo Nelson-Filho
    • 1
  • Fabio Lourenço Romano
    • 1
    Email author
  1. 1.Department of Pediatric Clinic, School of Dentistry of Ribeirão PretoUniversity of São PauloRibeirão PretoBrazil
  2. 2.Mutagenesis and Cytogenetics Laboratory, Department of Biology, Faculty of Philosophy, Science and Letters of Ribeirão PretoUniversity of São PauloRibeirão PretoBrazil

Personalised recommendations