Estudos moleculares de tecidos normais e tumores malignos de roedores tratados com samambaia (Pteridium aquilinum)

Abstract

Bracken fern (Pteridium aquilinum) toxicity and carcinogenicity properties have been extensively described in domestic and experimental animals. Some epidemiologic studies have associated the human exposition to the bracken fern and /or to its toxins with a higher risk for the occurrence of esophagus cancer in Venezuela and stomach cancer in Japan, Brazil, Costa Rica, United Kingdom ad Venezuela. However, these epidemiologic studies are not enough to unequivocally establish a causal relationship between bracken fern and cancer development in human beings. In the present work we proposed to investigate the presence of molecular markers of the bracken fern toxins on the animals genome which, if found, would be used in the assessment of the risk to the bracken exposure for humans. Initially, we investigated DNA adducts formation in normal targets tissues of female rats treated by gavage with a single dose of alcoholic extracts or other different preparations obtained from young fronds of freshly collected bracken for medium and long term. We used 32P-postlabeling method to identify the DNA adducts. Our results indicate that DNA adducts detectable by 32P-postlabeling are not formed in stomach or ileum of rats treated with bracken fern via digestive tube. We evaluate the presence of adducts in DNA samples obtained from upper digestive tube of mice treated with an acute single dose of bracken extract and spores and we found three adducts that had been previously described and also other four adducts which were not characterized. None of these adducts presented chromatography mobility similar to the single adduct obtained from in vitro modified DNA with activated ptaquiloside (a carcinogenic glycoside extracted from bracken). We conclude that other substances, besides ptaquiloside, are able to induce DNA adducts formation, which might contribute to the bracken carcinogenicity in mice. Following the strategy to identify molecular markers of the bracken action in the rat genome, and considering colorectal tumors carcinogenesis in humans as model, we examined eight malignant tumors induced by bracken treatment in rats for the presence of mutations in genes related to the classical pathway for the colorectal cancer, p53 and ras and also evaluated the mutator pathway by studying microsatellites stability. The exons 5 9 of the p53 and exons 1 and 2 of the K-ras and H-ras genes were sequenced and none mutation was found in the eight tumors. The amplification of five microsatellites loci previously validated (one mononucleotide repeat, three dinucleotide repeat and one tetranucleotide repeat) in the malignant tumors and in normal adjacent tissue did not reveal any microsatellite instability. The envolvement of mutations occurring in other tumor suppressor genes or oncogenes of the classical pathway as well as in genes required for chromosomal segregation and epigenetic events still must be investigated in the search for molecular markers of the bracken fern induced tumors.