Evaluation of the Genotoxicity of Endodontic Materials for Deciduous Teeth Using the Comet Assay

Nilton Cesar Nogueira  dos Santos; Bruno Marques  Soares; Cláudia do Ó  Pessoa; Livia Maria Andrade  de Freitas; João Pedro Pedrosa  Cruz; Maria Emília Santos Pereira  Ramos; Matheus Melo  Pithon; Eneida Moraes de Marcílio  Cerqueira

Authors

Nilton Cesar Nogueira dos Santos
Bruno Marques Soares
Cláudia do Ó Pessoa
Livia Maria Andrade de Freitas
João Pedro Pedrosa Cruz
Maria Emília Santos Pereira Ramos
Matheus Melo Pithon
Eneida Moraes de Marcílio Cerqueira

Keywords:

Endodontics, Tooth, Deciduous, Mutagenicity Tests, Cells, Cultured

Abstract

Objective: To evaluate genotoxicity of zinc oxide, P. A. calcium hydroxide, mineral trioxide aggregate and an iodoform paste using comet assay on human lymphocytes. Material and Methods: Two positive controls were used: methyl-methanesulfonate for the P.A. calcium hydroxide and mineral trioxide aggregate; and doxorubicin for the iodoform paste and zinc oxide. There were also two negative controls: distilled water for the P.A. calcium hydroxide and mineral trioxide aggregate; and DMSO for the iodoform paste and zinc oxide. Comets were identified using fluorescence microscopy and 100 of them were counted on each of the three slides analyzed per drug test. A damage index was established, taking into consideration the score pattern that had previously been determined from the size and intensity of the comet tail. Analysis of variance, followed by Tukey’s test, was used to compare the means of the DNA damage indices. Results: The DNA damage index observed for mineral trioxide aggregate (7.08 to 8.58) and P.A. calcium hydroxide (6.50 to 8.33), which were similar to negative control index. On the other hand, damage index for zinc oxide (104.7 to 218.50) and iodoform paste (115.7 to 210.7) were similar to positive control index. Conclusion: Iodoform paste and zinc oxide showed genotoxicity at all concentrations used.

References

Pinheiro HHC, Assunção LRS, Torres DKB, Miyahra LAN, Arantes DC. Endodontic therapy in primary teeth by pediatric dentists. Pesqui Bras Odontopediatria Clin Integr 2013; 13(4):351-60. https://doi.org/10.4034/pboci.2013.134.08

Tannure PN, Fidalgo TKS, Barcellos R, Primo LG, Maia LC. Analysis of root canal treated primary incisor after trauma: two-year outcomes. J Clin Pediatr Dent 2012; 36(3):257-62. https://doi.org/10.17796/jcpd.36.3.f8nv08266257v6g4

Massara MLA, Tavares WLF, Noronha JC, Henriques LCF, Ribeiro-Sobrinho AP. Efficacy of calcium hydroxide in the endodontic treatment of primary teeth: six years of follow-up. Pesqui Bras Odontopediatria Clin Integr 2012; 12(2):155-9. https://doi.org/10.4034/PBOCI.2012.122.01

Fuks AB. Vital Pulp Therapy with new materials for primary teeth: new directions and treatment perspectives. J Endod 2008; 34(7s):19s-24s. https://doi.org/10.1016/j.joen.2008.02.031

American Academy of Pediatric Dentistry (AAPD). Guideline on Pulp Therapy for Primary and Immature Permanent Teeth. Pediatr Dent 2016; 38(6):280-8. https://doi.org/10.1016/j.joen.2008.02.031

da Silva GN, Braz MG, de Camargo EA, Salvadori DM, Ribeiro DA. Genotoxicity in primary human peripheral lymphocytes after exposure to regular and white mineral trioxide aggregate. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102(5):e50-e54. https://doi.org/10.1016/j.tripleo.2006.02.032

Ramos MESP, Cavalcanti BC, Lotufo LVC, Moraes MO, Cerqueira EMM, Pessoa C. Evaluation of mutagenic effects of formocresol: detection of DNA-protein cross-links and micronucleus in mouse bone marrow. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008; 105(3):398-404. https://doi.org/10.1016/j.tripleo.2007.08.009

Camargo CHR, Camargo SEA, Valera MC, Hiller K-A, Schmalz G, Schweikl H. The induction of cytotoxicity, oxidative stress, and genotoxicity by root canal sealers in mammalian cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009; 108(6):952-60. https://doi.org/10.1016/j.tripleo.2009.07.015

Zeferino EG, Bueno CE, Oyama LM, Ribeiro DA. Ex vivo assessment of genotoxicity and cytotoxicity in murine fibroblasts exposed to white MTA or white Portland cement with 15% bismuth oxide. Int Endod J 2010; 43(10):843-8. https://doi.org/10.1111/j.1365-2591.2010.01747.x

Leite ACGL, Rosenblatt A, Calixto MS, Santos CM, Santos N. Genotoxic effect of formocresol pulp therapy of deciduous teeth. Mutat Res 2012; 747(1):93-7. https://doi.org/10.1016/j.mrgentox.2012.04.006

Naghavi N, Ghoddusi J, Sadeghnia HR, Asadpour E, Asgary S. Genotoxicity and cytotoxicity of mineral trioxide aggregate and calcium enriched mixture cements on L929 mouse fibroblast cells. Dent Mater J 2014; 33(1):64-9. https://doi.org/10.4012/dmj.2013-123

Santos NC, Ramos ME, Ramos AF, Cerqueira AB, Cerqueira EM. Evaluation of the genotoxicity and cytotoxicity of filling pastes used for pulp therapy on deciduous teeth using the micronucleus test on bone marrow from mice (Mus musculus). Mutagenesis 2016; 31(5):589-95. https://doi.org/10.1093/mutage/gew026

Pires CW, Botton G, Cadoná FC, Machado AK, Azzolin VF, da Cruz IB, et al. Induction of cytotoxicity, oxidative stress and genotoxicity by root filling pastes used in primary teeth. Int Endod J 2016; 49(8):737-45. https://doi.org/10.1111/iej.12502

Mohammadi Z, Shalavi S, Jafarzadeh H, Bhandi S, Patil S. Genotoxicity of endodontic materials: a critical review. J Contemp Dent Pract 2015; 16(8):692-6. https://doi.org/10.5005/jp-journals-10024-1742

Breivik J. The evolutionary origin of genetic instability in cancer development. Sem Cancer Biol 2005; 15(1):51-60. https://doi.org/10.1016/j.semcancer.2004.09.008

Ribeiro DA, Yujra VQ, DE Moura CFG, Handan BA, DE Barros Viana M, Yamauchi LY, et al. Genotoxicity Induced by Dental Materials: A Comprehensive Review. Anticancer Res 2017; 37(8):4017-24. https://doi.org/10.21873/anticanres.11786

Lou J, He J, Sheng W, Jin L, Chen Z, Chen S, et al. Investigating the genetic instability in the peripheral lymphocytes of 36 untreated lung cancer patients with comet assay and micronucleus assay. Mutat Res 2007; 617(1-2):104-10. https://doi.org/10.1016/j.mrfmmm.2007.01.004

Ribeiro DA, Scolastici C, De Lima PL, Marques ME, Salvadori DM. Genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in Chinese hamster ovary (CHO) cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99(5):637-40. https://doi.org/10.1016/j.tripleo.2004.07.010

Pires CW, Botton G, Cadoná FC, Machado AK, Azzolin VF, da Cruz IB, et al. Induction of cytotoxicity, oxidative stress and genotoxicity by root filling pastes used in primary teeth. Int Endod J 2016; 49(8):737-45. https://doi.org/10.1111/iej.12502

Singh MP, McCoy M, Tice RR, Schineider P. A simple technique for quantitation of low levels of DNA in individual cells. Exp Cell Res 1988; 175(1):184-91. https://doi.org/10.1016/0014-4827(88)90265-0

Olive PG, Banáth JP. The comet assay: a method to measure DNA damage in individual cells. Nat Protoc 2006; 1(1):23-9. https://doi.org/10.1038/nprot.2006.5

Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, et al. Fourth International Workgroup on Genotoxicity testing: Results of the in vivo Comet assay workgroup. Mutat Res 2007; 627(1):31-5. https://doi.org/10.1016/j.mrgentox.2006.08.011

Ribeiro DA, Marques MEA, Salvadori DMF. Antimicrobial endodontic compounds do not modulate alkylation-induced genotoxicity and oxidative stress in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2006; 102(2):e32-e36. https://doi.org/10.1016/j.tripleo.2005.11.026

Braz MG, Camargo MG, Salvadori DMF, Marques MEA, Ribeiro DA. Evaluation of genetic damage in human peripheral lymphocytes exposed to mineral trioxide agreggate and Portland cements. J Oral Rehabil 2006; 33(3):234-9. https://doi.org/10.1111/j.1365-2842.2005.01559.x

Brzovic V, Miletic I, Zeljezic D, Mladinic M, Kasuba V, Ramic S, et al. In vitro genotoxicity of root canal sealers. Int Endod J 2009; 42(3):253-63. https://doi.org/10.1111/j.1365-2591.2008.01510.x

Guedes-Pinto AC, De Paiva JG, Bozzola JR. Endodontic treatment of deciduous teeth with pulp necrosis. Rev Assoc Paul Cir Dent 1981; 35(3):240-4.

Klaude M, Erikson S, Nygren J, Ahnstrom G. The comet assay: Mechanisms and technical considerations. Mutat Res 1996; 363(2):363-89. https://doi.org/10.1016/0921-8777(95)00063-1

Miyamae Y, Yamamoto M, Sasaki YF, Kobayashi H, Igarashi-Soga M, Shimoi K, et al. Evaluation of a tissue homogenization that isolates nuclei for the in vivo single cell gel electropho-resis (Comet) assay: A collaborative study by five laboratories. Mutat Res 1998; 418(2-3):131-40. https://doi.org/10.1016/S1383-5718(98)00112-0

Lourenço-Neto N, Fernandes AP, Marques NCT, Sakai VT, Moretti ABS, Machado MAAM, et al. Terapia pulpar em dentes decíduos: possibilidades terapêuticas baseadas em evidências. Rev Odontol UNESP 2013; 42(2):130-7. https://doi.org/10.1590/S1807-25772013000200011

Barja-Fidalgo F, Mourinho-Ribeiro M, Oliveira MAA, Oliveira BH. A systematic review of root canal filling materials for deciduous teeth: is there an alternative for zinc oxide-eugenol? ISRN Dent 2011; 1:1-7. https://doi.org/10.5402/2011/367318

Wong VWC, Szeto YT, Collins AR, Benzie IFF. The comet assay: a biomonitoring tool for nutraceutical research. Curr Top Nutraceutical Res 2005; 3(1):1-14.

Vijayalaxmi V, Tice RR, Strauss GHS. Assessment of radiation-induced DNA damage in human blood lymphocytes using the single-cell gel electrophoresis technique. Mutat Res 1992; 271(3):243-52. https://doi.org/10.1016/0165-1161(92)90019-i

Hikiba H, Watanabe E, Barret JC, Tsutui T. Ability of fourteen chemical agents used in dental practice to indice chromosome aberrations in syrian hamster embryo cells. J Pharmacol Sci 2005; 97(1):146-52. https://doi.org/10.1254/jphs.FPJ04044X

Hagiwara M, Watanabe E, Barret JC, Tsutsui T. Assessment of genotoxicity of 14 chemical agents used in dental practice: ability to induce chromosome aberrations in Syrian hamster embryo cells. Mutat Res 2006; 603(2):111-20. https://doi.org/10.1016/j.mrgentox.2005.08.011

Ribeiro DA, Marques MEA, Salvadori DMF. Lack of genotoxicity of formocresol, paramonochlorofenol, and calcium hydroxide on mammalian cells by comet assay. J Endod 2004; 30(8):593-6. https://doi.org/10.1097/01.don.0000121614.10075.a3

Ribeiro DA, Sugui MM, Matsumoto MA, Duarte MAH, Marques MEA, Salvadori DMF. Genotoxicity and cytotoxicity of mineral trioxide aggregate and regular and white Portland cements on Chinese hamster ovary (CHO) cells in vitro. Oral Surg Oral Med Oral Pathol Oral Radiol Oral Endod 2006; 101(2):258-61. https://doi.org/10.1016/j.tripleo.2005.02.080

Dhawan A, Bajpayee M, Parmar D. Comet assay; a reliable tool for the assessment of DNA damage in diferente models. Cell Biol Toxicol 2009; 25(1):5-32. https://doi.org/10.1007/s10565-008-9072-z

Collins AR. The comet assay for DNA damage and repair: principles, applications, and limitations. Mol Biotechnol 2004; 26(3):249-61. https://doi.org/10.1385/mb:26:3:249.

Logrado LPL, Santos CO, Romeiro LS, Costa AM, Ferreira JR, Cavalcanti BC, et al. Synthesis and cytotoxicity screening of substituted isobenzofuranones designed from anacardic acids. Eur J Med Chem 2010; 45(8):3480-9. https://doi.org/10.1016/j.ejmech.2010.05.015

Paiva JCG, Cabral IO, Soares BM, Sombra CML, Ferreira JRO, Moraes MO, et al. Biomonitoring of ruralworkers exposed to a complex mixture of pesticides in the municipalities of Tianguá and Ubajara (Ceará State, Brazil): Genotoxic and cytogenetic studies. Environ Mol Mutagen 2011; 52(6):492-501. https://doi.org/10.1002/em.20647

Hartmann A, Agurell E, Beevers C, Brendler-Scwaad S, Burlinson B, Clay P, et al. Recommendations for conducting the in vivo alkaline comet assay. Mutagenesis 2003; 18(1):45-51. https://doi.org/10.1093/mutage/18.1.45