MUTAGENESIS OF RAS PROTOONCOGENES IN RAT-LIVER TUMORS INDUCED BY VINYL-CHLORIDE

Vinyl chloride is a DNA-damaging carcinogen which induces liver angios arcomas in humans and animals. Activation of the Ki-ras 2 gene by a GC --> AT transition at the second base of codon 13 in human liver angio sarcomas associated with occupational exposure to vinyl chloride has b een reported recently. In order to compare the molecular pathways of c arcinogenesis in humans and animals, Sprague-Dawley rats were exposed to vinyl chloride and hepatic tumors, including two hepatocellular car cinomas and five liver angiosarcomas, were investigated for mutations at codons 12, 13 and 61 of the Ha-ras, Ki-ras and N-ras genes. High mo lecular weight DNA was amplified by the polymerase chain reaction and point mutations were analyzed by allele specific oligonucleotide hybri dization, direct sequencing of polymerase chain reaction products and sequencing after cloning. None of the tumors exhibited a mutation in c odons 12, 13 and 61 of the Ki-ras gene, nor in codons 12 of the Ha-ros gene or 61 of the N-ras gene. However, an activating AT --> TA transv ersion at base 2 of codon 61 of the Ha-ras gene was detected in the tw o hepatocellular carcinomas. Mutations involving codon 13 (GGC --> GAC ) and codon 36 (ATA --> CTA) of the N-ras A gene were detected in two liver angiosarcomas, suggesting that the nature of the ras gene affect ed by a given carcinogen depends on host factors specific to cell type s. Several additional base pair substitutions were found in exon 1 of the N-ras B and C sequences. NIH 3T3 transfection assays and Southern blot analysis of DNA from transformed NIH 3T3 cells confirmed the pres ence of a dominant activated N-ras gene. These results emphasize the d ifferences in the molecular pathways leading to tumors in humans and r ats and within a given species between different cell types.