A simple and sensitive method for in vitro quantitation of abasic sites in DNA.
Chem Res Toxicol
; 12(6): 476-82, 1999 Jun.
Article
em En
| MEDLINE
| ID: mdl-10368309
ABSTRACT
A novel method for the quantitation of abasic sites (AP sites) in DNA is described. As abasic sites can be generated by controlled thermal treatment of base-modified DNA, this method can be used for estimation of the extent of DNA damage resulting from exposure to genotoxic agents. The method involves use of probe molecules 1 and 2 that contain a fluorescent label linked to an aminooxy group which reacts specifically with the aldehydic function of the ring-opened form of abasic sites. The two fluorescent probes 1 and 2 were found to react with 2-deoxyribose, a model substrate, at the optimum of pH 4.0. As spontaneous depurination occurs at low pH, the reactions with abasic DNA were carried out at neutral pH with an excess concentration of the probes. Studies with alkylated, depurinated calf thymus DNA showed that the method is selective and quantitative. Good correlations were found between the level of 7-methylguanine (7-MeGua), generated in vitro in DNA by the methylating agent dimethyl sulfate, and the amount of AP sites as determined by the method presented here. In addition, similar correlations were found when the assay was used to detect abasic sites in DNA isolated from rats treated with carcinogenic alkylating agents. In each case, the level of abasic sites, as expected, is slightly higher than the level of 7-MeGua which is known to represent about 70% of the total modifications of DNA following exposure to the methylating agent. This method may be useful not only in experimental settings but also in studies of DNA damage in humans resulting from chemotherapy or exposure to environmental agents.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Dano ao DNA
/
DNA
Tipo de estudo:
Diagnostic_studies
Limite:
Animals
Idioma:
En
Revista:
Chem Res Toxicol
Assunto da revista:
TOXICOLOGIA
Ano de publicação:
1999
Tipo de documento:
Article
País de afiliação:
França