6533b85cfe1ef96bd12bc190

RESEARCH PRODUCT

Interaction of antimutagenic 1,4-dihydropyridine AV-153-Na with DNA and DNA-damaging molecules and its impact on DNA repair activity

subject

description

1,4-dihydropyridines (1,4-DHP) possess important biochemical and pharmacological properties, including antioxidant and antimutagenic activities. Interaction of some 1,4-DHP with DNA was recently reported. AV-153-Na, an antimutagenic and DNA-repair-enhancing compound appeared to be able to interact with DNA by intercalation. The aim of the current study was to characterize DNA’s capacity for the binding of AV-153-Na, and using different approaches, to test intracellular distribution of the compound, to test the ability of the compound to scavenge peroxynitrite and hydroxyl radical and to assess the ability of the compound to modify the activity of DNA repair enzymes. The DNA binding activity of AV-153-Na was determined by means of fluorescence assay. Titration of the AV-153-Na solutions with DNA gradually increased fluorescence of the solution, indicating direct interactions of the molecule with DNA. AV-153-Na quenched the fluorescence of ethidium bromide and DNA complex, which points to intercalation binding mode. Binding via intercalation was confirmed by means of cyclic voltammetry and circular dichroism spectroscopy. The compound could interact with the four DNA bases in vitro, manifesting a higher affinity to guanine. Some ability to scavenge hydroxyl radical by AV-153-Na was detected by the EPR method. AV-153-Na turned out to be incapable of reacting chemically with peroxynitrite. However, AV-153-Na effectively decreased DNA damage produced by peroxynitrite in cultured HeLa cells. The effects of AV-153-Na on the activity of DNA repair enzymes were tested using Glyco-SPOT and ExSy-SPOT assays. The Glyco-SPOT test essentially revealed an inhibition by AV-153-Na of the enzymes involved thymine glycol repair. Results with ExSy-SPOT chip indicate that AV-153-Na significantly stimulates excision/synthesis repair of 8-oxoguanine (8-oxoG), abasic sites (AP sites) and alkylated bases. Laser confocal scanning fluorescence microscopy demonstrated that within the cells AV-153-Na was found mostly in the cytoplasm; however, a stain in nucleolus was also detected. Binding to cytoplasmic structures might occur due to high affinity of the compound to protein, revealed by fluorescence spectroscopy titration and circular dichroism. Activation of DNA repair enzymes after binding to DNA appears to be the basis for the antimutagenic effects of AV-153-Na.