Submitted by Ricardo Cedraz Duque Moliterno (ricardo.moliterno@uefs.br) on 2015-09-10T00:55:01Z No. of bitstreams: 1 Nilton C N Santos Tese Geno Cito Endo 10 ago 20h.pdf: 2096544 bytes, checksum: fd3a63f3f5235dae758262d72d8a3bb2 (MD5) / Made available in DSpace on 2015-09-10T00:55:01Z (GMT). No. of bitstreams: 1 Nilton C N Santos Tese Geno Cito Endo 10 ago 20h.pdf: 2096544 bytes, checksum: fd3a63f3f5235dae758262d72d8a3bb2 (MD5) Previous issue date: 2015-06-29 / Pulp therapy for deciduous teeth is the last resort for preventing tooth loss, but among the products used for this, i.e. so-called filling pastes, none is considered to be ideal. The objective of this study, using the micronucleus test on the bone marrow of mice and the comet assay on human lymphocytes, was to evaluate the genotoxic and cytotoxic effects of four filling pastes that are used in this therapy: zinc oxide, calcium hydroxide P.A., mineral trioxide aggregate and an iodoform paste (Guedes-Pinto paste). To perform the micronucleus test, male Swiss mice (Mus musculus) were divided into groups of ten animals each: four groups were each exposed to one of the filling pastes, administered intraperitoneally at dilutions of 1/10, 1/50, 1/500 and 1/1000. Cyclophosphamide was used as the positive control. The negative controls used were the dilution vehicles: dimethylsulfoxide (DMSO) for the Guedes paste and zinc oxide; and phosphate-buffered saline solution (PBS) for the calcium hydroxide P.A. and mineral trioxide aggregate. The animals were sacrificed 24 h and 48 h after the treatment. The bone marrow was extracted, in order to calculate the micronucleus occurrence rate in 1000 polychromatic erythrocytes (PCE) in each of the animals, under an optical microscope (1000 X), in a blinded test. Cytotoxicity was evaluated by determining the PCE/NCE (normochromatic erythrocyte) ratio in 200 erythrocytes/animal. For the comet assay, human lymphocytes were cultured in different dilutions of each of the filling pastes (1:500, 1:750, 1:1000 and 1:2000), for 3 h at 37 ?C, under an atmosphere containing 5% CO2. Two positive controls were used: methyl-methanesulfonate (0.4 ?M) for the calcium hydroxide P.A. and mineral trioxide aggregate; and doxorubicin (0.6 ?M) for the Guedes paste and zinc oxide. Two negative controls were also used here: distilled water for the calcium hydroxide P.A. and mineral trioxide aggregate; and DMSO for the Guedes paste and zinc oxide. Comets were identified by means of fluorescence microscopy (400 X), and 100 of them were counted on each of the three slides that were analyzed for each drug test. The statistical analysis on the results from the micronucleus test was performed using the conditional test for comparing proportions in situations of rare events. Analysis of variance, followed by the Tukey test, was used to assess the PCE/NCE ratio obtained from the different treatments and also to compare the means from the DNA damage indices that were obtained through the comet test, using the Prisma software, version 4.0. The micronucleus occurrence rate was significantly higher among the animals treated with Guedes paste, at all the dilutions tested and at both sacrifice times and also among the animals treated with zinc oxide and sacrificed 48 h after treatment at the dilutions of 1:50, 1:500 and 1:1000. Cytotoxic effects from these pastes were detected in the animals sacrificed at both times. Calcium hydroxide P.A. and mineral trioxide aggregate did not present any cytotoxic or genotoxic effects. The results obtained from the comet assay also showed that zinc oxide and Guedes paste presented genotoxicity, whereas calcium hydroxide P.A. and mineral trioxide aggregate did not. These results show that there is a need to reassess the use of zinc oxide and Guedes paste and provide encouragement for conducting additional studies to evaluate the genotoxicity and cytotoxicity of these pastes. / A terapia pulpar de dentes dec?duos se constitui no ?ltimo recurso de preven??o da perda dent?ria, mas, dentre os produtos empregados para tal, as denominadas pastas obturadoras, nenhum ? considerado como ideal. O objetivo deste trabalho foi avaliar, com o uso do teste de micron?cleo em medula ?ssea de camundongo e do ensaio cometa em linf?citos humanos, os efeitos citot?xicos e genot?xicos de quatro pastas obturadoras utilizadas nesta terapia: ?xido de zinco, hidr?xido de c?lcio P.A., agregado tri?xido mineral e uma pasta iodoformada (pasta Guedes-Pinto). Para realiza??o do teste de micron?cleo, camundongos Swiss (Mus musculus), machos, foram divididos em grupos de dez animais, que foram expostos ?s pastas obturadoras, administradas via intraperitoneal nas dilui??es de 1/10, 1/50, 1/500 e 1/1000. Ciclofosfamida foi utilizada como controle positivo. Os controles negativos foram: dimetilsulf?xido (DMSO) para a pasta Guedes-Pinto e ?xido de zinco; e solu??o salina tamponada (PBS) para o hidr?xico de c?lcio P.A. e agregado trioxido mineral. Os animais foram sacrificados 24h e 48h ap?s tratamento, a medula ?ssea foi extra?da e foram analisados 1000 eritr?citos policrom?ticos (PCE) de cada um dos animais, sob microscopia ?ptica (1000X) e em teste cego. A citotoxicidade foi avaliada pela rela??o PCE (eritr?cito policrom?tico) / NCE (eritr?cito normocrom?tico) em 200 eritr?citos/animal. Para o ensaio cometa, linf?citos humanos foram cultivados nas dilui??es de 1:500, 1:750, 1:1000 e 1:2000 das pastas obturadoras, durante 3h, a 37?C, em atmosfera de 5% de CO2. Foram utilizados dois controles positivos: metil-metanosulfonato (0,4?M) para o hidr?xido de c?lcio P.A. e agregado tri?xido mineral, e doxorrubicina (0,6 ?M) para a pasta Guedes-Pinto e ?xido de zinco. Foram tamb?m dois os controles negativos utilizados: ?gua destilada para o hidr?xido de c?lcio P.A. e agregado tri?xido mineral, e DMSO para pasta Guedes-Pinto e ?xido de zinco. A identifica??o do cometa foi realizada sob microscopia de fluoresc?ncia (400X), sendo computados 100 deles em cada uma das tr?s l?minas analisadas para cada droga teste. A an?lise estat?stica dos resultados do teste de micron?cleo foi realizada com o uso do teste condicional para compara??o de propor??es em situa??o de eventos raros. An?lise de vari?ncia, seguida do teste de Tukey, foram utilizados para avalia??o da rela??o PCE/NCE obtida com os diferentes tratamentos e tamb?m para compara??o das m?dias dos ?ndices de danos ao DNA obtidos no ensaio cometa com o software Prisma vers?o 4.0. A ocorr?ncia de micron?cleos foi significativamente maior nos animais tratados com a pasta Guedes-Pinto em todas as dilui??es testadas, nos dois tempos de sacrif?cio e tamb?m para os animais tratados com ?xido de zinco e sacrificados 48h ap?s tratamento, nas dilui??es 1:50; 1:500 e 1:1000. Efeitos citot?xicos destas pastas foram detectados nos animais sacrificados nos dois tempos. O hidr?xido de c?lcio P.A. e o agregado tri?xido mineral n?o apresentaram efeitos citot?xicos nem genot?xicos. Os resultados obtidos com o ensaio cometa tamb?m apontaram para a genotoxicidade do ?xido de zinco e pasta Guedes-Pinto, e n?o do hidr?xido de c?lcio P.A. e agregado tri?xido mineral. Estes resultados mostram a necessidade de reavalia??o do uso do ?xido de zinco e pasta Guedes-Pinto e suscitam a realiza??o de estudos adicionais avaliando a genotoxicidade e citotoxicidade destas pastas.