Gene-specific oxidative lesions in aged rat brain detected by polymerase chain reaction inhibition assay.

ABSTRACT

An exposure of isolated rat brain genomic DNA to oxidative stress in the form of iron salts (Fe2+) and ascorbate results in gene-specific DNA lesions detectable by a quantitative polymerase chain reaction (PCR) based assay in which PCR amplification efficiency of the affected genes (e.g. beta-actin and p53) is grossly impaired. Such oxidative DNA lesions are prevented by hydroxyl radical scavengers like mannitol (20 mM) and sodium benzoate (20 mM) or by the antioxidant enzyme catalase (50 microg/ml) present in the incubation mixture during exposure to Fe2+ and ascorbate. When brain DNA isolated from young (4-6 months of age) and aged (20-24 months of age) rats are analyzed similarly by the PCR based method, the amplification levels of beta-actin and p53 genes are noticeably decreased in the case of aged rat indicating an accumulation of gene-specific DNA lesions during brain aging.