Unrepaired fjord region polycyclic aromatic hydrocarbon-DNA adducts in rascodon 61 mutational hot spots

The fjord region diol-epoxide metabolites of polycyclic aromatic hydrocarbo ns display stronger tumorigenic activities in rodent studies than comparabl e bay region diol-epoxides, but the molecular basis for this difference bet ween fjord and bay region derivatives is not understood. Here we tested whe ther the variable effects of these genotoxic metabolites of polycyclic arom atic hydrocarbons may result from different DNA repair reactions. In partic ular, we compared the repairability of DNA adducts formed by bay region ben zo[a]pyrene (B[a]P) diol-epoxides and the structurally similar but signific antly more tumorigenic fjord region diol-epoxide metabolites of benzo[c]phe nanthrene (B[c]Ph). For that purpose, we incorporated both types of polycyc lic aromatic hydrocarbon adducts into known hot spot sites for carcinogen-i nduced proto-oncogene activation. Synthetic DNA substrates were assembled u sing a portion of human N-ras or H-ras that includes codon 61, and stereosp ecific B[a]P or B[c]Ph adducts were synthesized on adenine N-6 at the secon d position of these two ras codon 61 sequences. DNA repair was determined b y incubating the site-directed substrates in human cell extracts, followed by electrophoretic visualization of radiolabeled oligonucleotide excision p roducts, These cell-free assays showed that all tested bay region B[a]P-N-6 -dA adducts are removed by the human nucleotide excision repair system, alt hough excision efficiency varied with the particular stereochemical configu ration of each B[a]P residue. In contrast, all fjord region B[c]Ph-N-6-dA a dducts located in the identical sequence context and with exactly the same stereochemical properties as the corresponding B[a]P lesions were refractor y to the nucleotide excision repair process. These findings indicate that t he exceptional tumorigenic potency of B[c]Ph or related fjord region diol-e poxides may be attributed, at least in part, to slow repair of the stable b ase adducts deriving from the reaction of these compounds with DNA.