DETECTION OF DIBENZO[A,L]PYRENE-INDUCED H-RAS CODON-61 MUTANT-GENES IN PRENEOPLASTIC SENCAR MOUSE SKIN USING A NEW PCR-RFLP METHOD

One of the key events in tumor initiation in mouse skin is mutational activation of the H-ras gene. Papillomas induced by the most carcinoge nic environmental polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]p yrene (DB[a,l]P), in SENCAR mouse skin contain a specific H-ras codon 61 (CAA-->CTA) mutation. We describe here detection of these mutations in preneoplastic skin by measuring the frequency of an induced XbaI R FLP, created by the mutation. Development of the PCR-XbaI RFLP method, sensitive enough to detect 1 codon 61 mutant allele among 10 000 wild -type genes, is described. The results indicate that codon 61 mutation s are induced 1 day (0.1%) after DB[a,l]P treatment on mouse skin, rea ch a high value (5%) by day 3, rapidly decline between days 7-9 and in crease again during the clonal expansion of pre-papillomas into tumors . The detection of codon 61 mutations 1 day after DB[a,l]P exposure su ggests that mutations occurred by pre-replication misrepair.