Alterações epigenéticas do gene p16 em ratos tratados com altas doses de I-metionina / Epigenetics changes of the p16 gene in rats treated with high doses of lmethionine

AUTOR(ES)
DATA DE PUBLICAÇÃO

2009

RESUMO

Several evidences suggest that to diet is a relevant factor in modifying the risks of developing cancer and the nutrient-genome interaction has a significant influence for the maintenance of a good health condition. Nutrigenomic establishes a relation between to diet and the investigation of epigenetic DNA modifications, which may play an important role in the etiology of various degenerative diseases. DNA methylation is an important epigenetic event modification of gene expression and there been reports of cancers associated with promoter methylation region of tumor suppressor genes such as p16 gene. Methionine (Met) is an essential aminoacid to maintain methionine cycle, very important mechanism in methylation reactions. Another factor that can change the methylation pattern is the chemotherapeutic drugs. DNA methylation alteration can also modify chromosome structure and lead to a genomic instability related to the development of neoplasia. The aim of this study was to analyze the effect of methionine supplementation on changes in the methylation pattern of the promoter region of the p16 gene in the liver and kidneys of rats and the possible changes induced by interaction with the antitumoral doxorubicin (DXR), besides the possible genomic instability in bone marrow cells. Six Wistar rats groups (n=60) received commercial diet or commercial diet plus Met 2% for six weeks. At third week and 24 hours before euthanasia they received saline or DXR (1 or 10 mg/Kg) intraperitoneally. DNA extraction of the kidneys and liver was used for the study of the methylation pattern of promoter region on the p16 gene by COBRA method, after DNA sodium bisulfite modification and PCR amplification. EcoRI, TaqI and HphI enzymes were used to determine the methylation pattern of promoter region of the p16 gene and TasI enzyme was used to validate bisulfite conversion. All animals had digestion with TasI and HphI enzymes, however no restriction with EcoRI and TaqI enzymes. Kidneys and liver DNA methylation pattern promoter region of p16 gene did not change by Met supplementation or DXR administration, when comparing the controled and the treated groups. The justification is that the p16 gene is very important in cycle cellular regulation, apoptosis and senescence and its regulation follows a well controlled mechanism. To study Met supplementation in genomic instability the micronucleus (MN) test was realized in the rat bone marrow. By Micronuclei analysis, met supplementation did not alter MN frequency, but DXR treatment (1 or 10 mg/Kg) induced MN formation when compared to Control group. The association of Met+DXR did not decrease MN frequency by induced DXR. In conclusion, Met on the diet or DXR administration did not change methylation pattern of promoter region of the p16 gene. The supplemented Met did not show mutagenic effect in MN test. There was no antimutagenic effect of Met when associated to DXR.

ASSUNTO(S)

doxorubicin metilação dna micronucleus gene p16 micronúcleo methionine gene p16 dna methylation doxorrubicina metionina

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