IN-VITRO MUTATIONAL SPECTRUM OF AFLATOXIN B-1 IN THE HUMAN HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE GENE

The in vitro mutational spectrum of aflatoxin B-1 (AFB1) in exon 3 of the human hypoxanthine guanine phosphoribosyltransferase gene in B-lym phoblasts was examined by a combination of polymerase chain reaction a nd denaturing gradient gel electrophoresis. The cell line used in this study contained an expression vector that produced high levels of hum an cytochrome P450 CYP1A1. CYP1A1 metabolizes AFB1 to form an epoxide intermediate which can react with DNA. About 1200 independent mutants were induced at the hypoxanthine guanine phosphoribosyltransferase loc us by AFB1 and were selected en masse by addition of 6-thioguanine to the bulk culture. Two independent cultures were treated with AFB1. Pol ymerase chain reaction was used to amplify exon 3 from the complex mut ant population, and denaturing gradient gel electrophoresis was used t o separate wild-type DNA sequences from mutant sequences. Mutational h otspots were visible as discrete bands on the denaturing gradient gel. Scanning densitometry was used to determine the fraction of the compl ex population that was represented in each non-wild-type band. The ban ds containing the mutations were excised from the denaturing gradient gel and sequenced. In this way, the nature and frequency of mutational hotspots in a population of >1000 mutants were determined. AFB1 produ ced one strong mutational hotspot in exon 3. Between 10 and 17% of the AFB1-induced mutants contained a single GC-->TA base substitution at base pair 209. This hotspot occurred in a GGGGGG sequence (the mutated base is underlined). This mutation was observed reproducibly in two i ndependently treated cultures. Several other mutations were observed i n only one culture but at a lower frequency. Our results are the first report of the mutational spectrum of AFB1 in a native human gene.