In vitro activation of the human Harvey-ras proto-oncogene by aflatoxin B1

Activation of ras proto-oncogenes occurs frequently in vivo in chemicallyinduced rodent tumours, including rat hepatomas induced by aflatoxin B1.This study examines the in vitro activation of a human ras gene by thismycotoxin. A plasmid containing the human Ha-ras proto- oncogene, togetherwith a neomycin resistance gene (pECneo), was incubated in vitro with amicrosomal system generating aflatoxin B1 8,9- epoxide. Subsequenttransfection of the plasmid into mouse NIH 3T3 fibroblasts, followed byG418 selection and s.c. injection of surviving cells into immunodeficientmice demonstrated that the proto-oncogene had acquired transformingcapacity. Although a single tumour resulted from similar treatment ofincubated unconjugated plasmid, no tumours were produced by a secondaryround of transfections using DNA from this tumour. Selective PCRamplification of the human Ha-ras gene in extracted tumour DNA followed bysequencing demonstrated the presence of G-->T transversions either atthe first or middle base of codon 12 in tumours resulting from transfectionwith the aflatoxin-B1-modified pECneo plasmid, but this was not detected inthe single tumour resulting from transfection with the unmodified plasmid.Thus, although a mutation in the Ha-ras gene has not been reported forhuman primary hepatomas occurring in aflatoxin-exposed populations,metabolically activated aflatoxin B1 is capable of mutating thisproto-oncogene to its oncogenic form in vitro. No mutations were observedin codon 61. It appears that, in contrast to the frequently reportedG-->T transversions in codon 249 of the p53 gene in primary hepatomas inaflatoxin-exposed humans, the failure to detect Ha-ras mutations in thesetumours is not due to an inability of aflatoxin B1 to activate thisproto-oncogene. The G-->T transversions observed in this study contrastwith the most frequent aflatoxin B1 in vivo induced mutations, G-->Atransitions in the rat Ki-ras gene. Possible mechanisms for thesedifferences are discussed.