title: 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 proto-oncogene. 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.
authors.affiliation: Medical Research Council Toxicology Unit, University of Leicester, UK.
authors.forename: J
authors.initials: J
authors.lastname: Riley
authors.forename: H G
authors.initials: HG
authors.lastname: Mandel
authors.forename: S
authors.initials: S
authors.lastname: Sinha
authors.forename: D J
authors.initials: DJ
authors.lastname: Judah
authors.forename: G E
authors.initials: GE
authors.lastname: Neal
mesh_terms: D016475:3T3 Cells; D016604:Aflatoxin B1 / Q000494:pharmacology*; D000818:Animals; D011905:Genes, ras; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D016296:Mutagenesis / Q000187:drug effects*; D017354:Point Mutation; D000090063:Proto-Oncogene Mas; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics*; D011519:Proto-Oncogenes; D014162:Transfection
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D016604:Aflatoxin B1; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)