Rat liver homogenate (S9)-mediated binding of benzo[ a]pyren to DNA in V79 cells, V79 cell nuclei and aqueous solution

Abstract

The role of the target cell in determining the structures and the amounts of hydrocarbon-DNA adducts formed after hydrocarbon activation by an exogenous metabolic ativation system was investigated by exposing intact cells of the Chinese hamster lung cell line V79, V79 cell nuclei and calf thymus DNA to benzo[ a]pyrene (B[ a]P) in the presenceof a rat liver homogenate activation system (S9). The DNA was isolated, enzymatically degraded to deoxyribonucleosides and the B[ a]P-deoxyribonucleoside adducts analyzed by high-performance liquid chromatography. Two major adducts were present in all samples; one formed by reaction of r-7, t-8-dihydroxy- t-9, 10-epoxy-7, 8, 9, 10-tetrahydro-B[ a]P ( anti-B[ a]PDE) with the 2-amino group of deoxyguanosine, the other formed by reaction of a metabolite of 9-hydroxybenzo[ a]pyrene (9-OH-B[ a]P) with an unidentified deoxyribonucleoside. The ratios of the anti-B[ a]PDE-DNA adduct to the 9-OH-B[ a]P-DNA adduct were: calf thymus DNA, 3 to 1: DNA from V79 nuclei, 8 to 1; DNA from intact V79 cells, 11 to 1. Similar several-fold increases in the proportion of anti-B[ a]PDE-DNA adducts in V79 cells over those in calf thymus DNA were observed for a dose range of 1–10 μg B[ a]P per ml. The relative extent of binding of the activated metabolite of 9-OH-B[ a]P to DNA was also much lower in intact V79 cells than in calf thymus DNA after exposure to 9-OH-B[ a]P in the presence of the S9 activation system. These results demonstrate that the relative abilities of various reactive bbenzo[ a]pyrene metabolites formed by an exogenous activation system to reach DNA differ substantially. Therefore, assessment of the biological activity of hydrocarbons in mutation assays using exogenous activation systems must take into account not only the amounts of different reactive hydrocarbon metabolites formed but also the relative abilities of these metabolites to reach the DNA of the target cell.