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Previous issue date: 2008-04-28 / The decoction of Brazilian pepper tree barks (Schinus terebinthifolius, Raddi), is used in medicine as wound healing and antiinflamatory. Once extracts from this plant are used
for acceleration of scar s process, it is important to study their mutagenic and genotoxic potential. In previous works in our laboratory, it was observed mutagenicity caused by the decoction when in high concentrations. Among the chemical compounds of this plant that could be able to induce mutation, the flavonoids were the only group that was referred to have either an oxidant or antioxidant potential. The flavonoids were isolated, purified and quantified by adsorptive column chromatography under silica gel, bacterial and in vitro genotoxic tests were realized to determine if the flavonoids were the responsible agents for this mutagenicity found. The tests realized with plasmidial DNA were indicative that the flavonoids are probably genotoxic, due to the presence of correlation between increase of the flavonoid concentration and in plasmidial DNA double strand breakage visualized in agarose gel, as well as they were capable to generated abasic sites shown by the in vitro treatment with exonuclease III. The same tests with plasmidial DNA in the presence of copper [10 ?M] and of a Tris-HCl pH 7.5 [10 ?M] buffer were realized with the isolated flavonoids to determine if there would be
or not participation of reactive oxygen species (ROS). The transformation of plasmidial DNA in different bacterial strains proficient and deficient in DNA repair enzymes in the
presence or not of a Tris-HCl buffer, suggests that the enzymes that repair oxidative lesions are necessary to repair the lesions generated by the flavonoids and that ROS
are generated and are necessary to promote the lesions. Bacterial tests with Escherichia coli strains of the CC collection (deficient or not for DNA repair enzymes),
showed that the flavonoids are able to increase the frequency of mutations, mainly in strains mutated in repair enzymes (MutM, MutY-glicosylases and double mutant),
suggesting that these agents are responsible for the enhancement in the mutation rate. In order to determine the mutation spectrum caused by the flavonoids of the Brazilian pepper tree stem bark, plasmidial DNA previously treated with the flavonoids were transformed in bacterial strains deficient and proficient in the DNA repair enzymes,
followed by a blue-white selection with X-gal, DNA amplification by PCR and sequencing the positive mutant clones. Analysis of the mutants obtained from strains
CC104, CC104mutM, CC104mutY, CC104mutMmutY, BW9101, BW9109 indicated a predominance of some mutations like G:C to C:G that can be correlated with the origin of 8-oxoG, due to oxidative lesions caused by the flavonoids. So it can concluded that the flavonoid isolated or in fractions enriched on them are genotoxic and mutagenic, and their mutations are predominantly oxidative, mediated by ROS, and the lesions are recognized by the BER system. In this way it is proposed that the flavonoids can act in two different ways to generate the DNA lesion: 1. in a Fenton-like reaction, when the flavonoid are in the presence of metal ions and that together with the water generate ROS that promotes the DNA lesions; 2. in another way the lesions can be generated by the formation of ROS due to the internal chemical structure of the flavonoid molecule due to the quantity and location of hydroxyl groups, and so producing the DNA lesions, those lesions can be directly (suggested by the in vitro experiments) or indirectly done (supported by the experiments using the CC bacterial strains) / O decocto feito de cascas da Aroeira (Schinus terebinthifolius, Raddi) ? usado na medicina popular como cicatrizante e anti-inflamat?rio, mas, apesar de seu efeito medicinal, tamb?m foi demonstrado um efeito mutag?nico. O objetivo deste trabalho foi determinar se os flavon?ides da aroeira s?o os respons?veis pela mutagenicidade bem como propor um mecanismo de a??o. Para tanto, diferentes fra??es enriquecidas em flavon?ides foram preparadas e os flavon?ides foram isolados, purificados e quantificados via cromatografia de adsor??o em gel de s?lica. Testes de genotoxicidade, in vitro e em sistemas bacterianos foram realizados a fim de determinar o mecanismo de a??o. Testes realizados com DNA plasmidial In vitro foram indicativos de que as fra??es enriquecidas
de flavon?ides s?o capazes de gerar dupla quebra na fita do DNA, bem como s?o capazes de gerar s?tios ab?sicos, evidenciados na presen?a da exonuclease III. O mesmo
teste com DNA plasmidial, mas na presen?a de cobre [10 ?M] e de tamp?o Tris-HCl pH 7.5 [10 ?M], foi realizado, para determinar se haveria ou n?o participa??o de esp?cies
reativas de oxig?nio na indu??o de danos. A transforma??o do DNA plasmidial em diferentes cepas bacterianas, proficientes e deficientes em diferentes vias de reparo de DNA, na aus?ncia e presen?a de tamp?o Tris-HCl pH 7.5 [10 ?M], sugerem que enzimas de reparo de les?es oxidativas s?o necess?rias para reparar as les?es geradas pelos flavon?ides e que esp?cies reativas de oxig?nio s?o geradas e necess?rias para promover as les?es. Teste de mutagenicidade direta com diferentes cepas de Escherichia coli derivadas da linhagem CC104 sugeriram que os flavon?ides aumentam a freq??ncia
de muta??o em cepas deficientes nas enzimas MutM e MutY glicosilases, principalmente na duplo mutante, sugerindo que as les?es predominantemente oxidativas, s?o substratos destas enzimas no DNA. A fim de determinar o espectro de muta??o causado pelos flavon?ides da aroeira, DNA plasmidial foi transformado previamente tratado com as fra??es enriquecidas em flavon?ides em cepas deficientes em enzimas de reparo de
DNA, seguido de sele??o por colora??o com x-gal (sele??o azul-branco), amplifica??o de DNA por PCR e sequenciamento dos clones mutantes. An?lise dos mutantes obtidos nas
cepas CC104, CC104mutM, CC104mutY e CC104mutMmutY indicaram a predomin?ncia de muta??es do tipo G:C para C:G, que pode estar correlacionada a origem de 8-oxoG, devido a les?es oxidativas causadas pelos flavon?ides. Assim, pode-se concluir que os flavon?ides isolados ou em fra??es enriquecidas nos mesmos s?o respons?veis pelo potencial mutag?nico dos extratos da aroeira, que as les?es geradas s?o oxidativas,
predominando a forma??o de 8-oxoG, devido ? forma??o de EROs, e que s?o reconhecidas por enzimas do sistema de reparo por excis?o de base. Desta forma, prop?e-se que os flavon?ides possam gerar esp?cies reativas de oxig?nio de duas
maneiras diferentes: i) em uma rea??o Fenton-like, quando os flavon?ides est?o na presen?a de metais e ?gua ou ii) devido ? estrutura interna da mol?cula de flavon?ide pela disposi??o e quantidade de grupamento hidroxilas na pr?pria mol?cula. As les?es podem ser geradas diretamente (sugerido pelos experimentos de tratamento in vitro) ou indiretamente (pelos experimentos obtidos com as linhagens CC)
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/13128 |
| Date | 28 April 2008 |
| Creators | Barca, Francisco Napole?o Tulio Varela |
| Contributors | CPF:32398336468, http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4781004Y8, Cavalcanti J?nior, Geraldo Barroso, CPF:10861203453, http://lattes.cnpq.br/0091662650633339, Lima, Lucymara Fassarela Agnez, CPF:00297997750, http://lattes.cnpq.br/1083882171718362, Barbosa Filho, Jos? Maria, CPF:09857400434, lattes.cnpq.br/8892459126928726, Felzenszwalb, Israel, CPF:37589806749, lattes.cnpq.br/8132847165466920, Medeiros, S?lvia Regina Batistuzzo de |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Ci?ncias da Sa?de, UFRN, BR, Ci?ncias da Sa?de |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/embargoedAccess |
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