MAPPING OF (+/-)-ANTI-BENZO[A]PYRENE DIOL EPOXIDE ADDUCTS TO HUMAN C-HA-RAS1 PROTOONCOGENE

The relative reactivity of the chemical carcinogen (+/-)-7-beta,8-alpha-dihydroxy-9-alpha,10-alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] to the guanine bases of the first two coding exons of the human c-Ha-ras1 protooncogene is determined to test if (+/-)-anti-BPDE reactivity is correlated with mutations reported for human c-Ha-ras1 protooncogene activation. Plasmid DNA containing the sequence for the human c-Ha-ras1 gene is modified with (+/-)-anti-BPDE to provide approximately 1 covalent adduct per 250 bp. High-resolution mapping of the covalent adducts is achieved by laser-induced photolysis of P-32-labeled restriction fragments of the BPDE-modified plasmid DNA. The (+/-)-anti-BPDE binding profiles to exons 1 and 2 of the human c-Ha-ras1 protooncogene show enhanced reactivity to guanine-rich regions. The guanine bases of oncogene-activating codons 12 (GGC) and 13 (GGT) are 5 times more reactive than the least reactive guanine analyzed within this region of the gene. The guanine base of oncogene-activating codon 61 (CAG) exhibits intermediate reactivity relative to the guanines analyzed within this region of the gene. Although preferential chemical reactivity plays a role in the activation of the c-Ha-ras1 protooncogene, the in vivo activation of the c-Ha-ras1 protooncogene by (+/-)-anti-BPDE is a complex process, with other important factors involved in the chemically induced activation.