Covalent Binding of Benzo[a]pyrene to Free and Membrane-Bound DNA in Isolated Liver Nuclei from 3-Methylcholanthrene-Induced Rats

Abstract

Benzo[a]pyrene (BP) is bound to DNA by two major pathways: one-electron oxidation and monooxygenation. One-electron oxidation requires that the highly reactive radical cation be formed in close proximity to the DNA. Thus, in intact cell nuclei, binding of BP radical cation would be expected to occur predominantly in DNA associated with the membrane. To examine this, nuclei from the livers of 3-methylcholanthrene-induced male MRC Wistar rats were incubated with [³H]BP and NADPH. After the incubation, the “free” DNA was separated from “membrane-bound” DNA by extraction in low-Mg buffer. Binding of BP to membrane-bound DNA was 9.5 ± 2.0 μmol/mol DNA-P, whereas binding to free DNA was 2.2 ± 0.7 μmol/mol DNA-P. BP adducts formed by one-electron oxidation and lost from the nuclear DNA by depurination were also examined. The three depurination adducts previously found with rat liver microsomes and in mouse skin were also obtained from nuclei. These results suggest that binding of BP to DNA in intact nuclei occurs preferentially to DNA associated with the nuclear membrane.

Key Words:

• benzo[a]pyrene
• rat liver nuclei
• DNA adducts
• radical cations
• one-electron oxidation