Activating mutations in human c-Ha-ras-1 protooncogene induced by stereoisomeric fjord-region benzo[c]chrysene diol-epoxides.

The mutagenicity of fjord-region benzo[c]chrysene diol-epoxide (BcCDE) stereoisomers((+) anti-BcCDE, (-)anti-BcCDE, (+)syn-BcCDE, and (-)syn-BcCDE) was studied in a forward-mutation system. pEC plasmid containing the human c-Ha-ras-1 proto-oncogene was reacted in vitro with each optically active isomer separately and transfected into NIH/3T3 cells. Morphologically transformed foci were cloned, and DNA obtained from these foci was tested for the presence of Ha-ras-1 sequence by Southern blot analysis. A total of 50 transformed foci (11-14 for each diastereomer) were generated. To determine the nature of mutations responsible for activating the proto-oncogene, regions of the gene likely to contain the activating mutations were amplified by polymerase chain reaction and then subjected to hybridization with specific oligonucleotides. Gene mutations in 42 of 50 transformed foci were characterized by these methods, and most were found at codon 61 (27), followed by codons 12 (13) and 13 (two). All mutations observed were either G --> T or A --> A --> T transversions. Thirty-six were G --> T transversion mutations occurring at codons 61, 12, and 13. The remaining six were A --> T transversions at codon 61.BcCDE stereoisomers may specifically attack guanine and adenine and result in the mutations observed. Some differences in codon preference but not in the types of mutations were found among these optically active isomers.