Ellagic Acid Inhibits DNA Binding of Benzo(a)pyrene Activated by Different Modes.

Abstract

The possible mechanisms of inhibition of benzo(a)pyrene (BaP) binding to DNA in vivo and in vitro by ellagic acid (EA) were investigated. In the in vitro studies BaP was activated by cytochrome P-450-dependent mixed function oxidase system, ascorbate-dependent lipid peroxidation, and peroxidation of linoleic acid. Reactions were performed in the dark at 37°C for 20 to 30min in buffered aqueous solutions with 2mg DNA and 40nmol tritiated BaP. The levels of BaP-DNA adducts were determined by assay of the radioactivity. BaP-DNA adduct levels ranged from 146 to 170fmol/mg DNA in ascorbate-and NADPH-dependent reactions and the level was 8.25±0.425pmol/mg DNA in the hematin-mediated linoleic acid reaction. Addition of EA to the reaction mixtures resulted in a significant inhibition in BaP-DNA adduct formation, depending upon the concentration of EA; e.g., 500μM EA resulted in 42 to 53% inhibition of binding in the three systems of carcinogen activation. Similarly, EA feeding to male NMRI mice at a concentration of 12μg/ml drinking water significantly decreased the levels of carcinogen-DNA adducts in the lungs. Reactions performed in vitro, in which DNA was preincubated overnight at 4°C with EA, revealed a decrease of 20% in adduct formation. MDA formation during the process of lipid peroxidation, stimulated by NADPH and ascorbate in the liver microsomes of mouse and by hematin in linoleic acid, was taken as an index for the free radical reactions. Addition of EA to in vitro systems inhibited the MDA formation with an IC50 (concentration for 50% inhibition) of 480, 400, and 400μM for NADPH, ascorbate, and hematin-mediated free radical reactions, respectively.