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

Abstract

Activation of ras proto-oncogenes occurs frequently in vivo in chemically induced rodent tumours, including rat hepatomas induced by aflatoxin B1. This study examines the in vitro activation of a human ras gene by this mycotoxin. A plasmid containing the human Ha-ras proto-oncogene, together with a neomycin resistance gene (pECneo), was incubated in vitro with a microsomal system generating aflatoxin B1 8,9-epoxide. Subsequent transfection of the plasmid into mouse NIH 3T3 fibroblasts, followed by G418 selection and s.c. injection of surviving cells into immunodeficient mice demonstrated that the proto-oncogene had acquired transforming capacity. Although a single tumour resulted from similar treatment of incubated unconjugated plasmid, no tumours were produced by a secondary round of transfections using DNA from this tumour. Selective PCR amplification of the human Ha-ras gene in extracted tumour DNA followed by sequencing demonstrated the presence of G→T transversions either at the first or middle base of codon 12 in tumours resulting from transfection with the aflatoxin-B1-modified pECneo plasmid, but this was not detected in the single tumour resulting from transfection with the unmodified plasmid. Thus, although a mutation in the Ha-ras gene has not been reported for human primary hepatomas occurring in aflatoxin-exposed populations, metabolically activated aflatoxin B1 is capable of mutating this proto-oncogene to its oncogenic form in vitro. No mutations were observed in codon 61. It appears that, in contrast to the frequently reported G→T transversions in codon 249 of the p53 gene in primary hepatomas in aflatoxin-exposed humans, the failure to detect Ha-ras mutations in these tumours is not due to an inability of aflatoxin B1 to activate this protooncogene. The G→T transversions observed in this study contrast with the most frequent aflatoxin B1 in vivo induced mutations, G→A transitions in the rat Ki-ras gene. Possible mechanisms for these differences are discussed.