DNA-damaging effects of genotoxins in mixture: Modulation of covalent binding to DNA

Modulation of DNA adduct formation by pre-existing adducts was examined in synthetic oligonucleotides and genomic DNA (calf thymus); genotoxins studie d were N-acetoxy-acetylaminofluorene (N-AcO-AAF), aminofluorene (AF), aflat oxin B-1-8,9-epoxide (AFB(1)-8,9-epoxide), and dimethylsulfate (DMS). Oligo deoxynucleotides containing either guanine-C8-AAF (Gua-C8-AAF) or Gua-C8-AF adducts and a neighboring unadducted guanine (G) (target G), located 1, 2, or 4 nucleotides from the adduct, were reacted, as single- (ss) or double- stranded (ds) substrates, with dimethylsulfate (DMS) or AFB(1)-8,9-epoxide. A modified Maxam-Gilbert technique showed that the presence of the AAF add uct lowered the extent to which AFB(1)-8,9-epoxide, but not DMS, reacted wi th target G. Binding of AFB(1)-8,9-epoxide to the target G was attenuated ( greater than or equal to 5-fold) when the target was located immediately ad jacent to an AAF, but not AF, adduct in ds-DNA, Reaction with AFB(1)-8,9-ep oxide increased when the target G was located 2 or 4 nucleotides from the A AF adduct. Pretreatment of calf thymus DNA with AAF (0-1.8% nucleotides mod ified) reduced levels of Gua-N7-AFB(1) adducts formed after subsequent trea tment with AFB(1)-8,9-epoxide. Pretreatment of calf thymus DNA with AFB(1) did not alter levels of adducts formed after subsequent treatment with N-Ac O-AAF. The supposition that aflatoxin B-1-binding to DNA may be altered by conformational changes in the helix, due to the presence of a pre-existing AAF adduct, is supported by the absence of an effect by AF and confirmation of local denaturation of the oligomer helix by use of chemical probes hydr oxylamine and diethylpyrocarbonate. Nonetheless, the importance of changes in the nucleophilicity of neighboring nucleotides and local steric effects cannot be ruled out.