KINDS OF MUTATIONS INDUCED BY AFLATOXIN-B1 IN A SHUTTLE VECTOR REPLICATING IN HUMAN-CELLS TRANSIENTLY EXPRESSING CYTOCHROME-P450IA2 CDNA

Transient expression of rat liver cytochrome P450IA2 cDNA was combined with the use of a shuttle vector as a mutational target to determine the frequency and types of mutation caused by the conversion of aflatoxin B1 into genotoxic metabolites within human cells. Ad293 cells were first transfected with p91-IA2, a rat liver P450IA2 cDNA expression vector, or with p91-IA2(i) (a control vector that has the P450 CDNA in the inverted orientation) and incubated for 24 h to permit P45-IA2 accumulation. Cells were then transfected with the pS189 shuttle-vector plasmid, which carries the Escherichia coli supF gene as a mutational target, and incubated for a further 24 h in the presence of aflatoxin B1 to permit promutagen activation and pS189 replication. In shuttle vectors replicated in p91-IA2-transfected cells, the supF point-mutation frequency increased with increasing concentration of aflatoxin B1. This frequency was nine to 23 times greater than the background point-mutation frequency obtained with aflatoxin B1-treated control (p91-IA2(i)-transfected) cells. The large majority of the aflatoxin B1-induced supF point mutations were base substitutions, mostly G:C --> T:A transversions. This mutagenesis system permits the molecular analysis of mutations induced by specific P450/promutagen pairs in a shuttle vector replicating in human cells and will permit the investigation of host cell mechanisms involved in the generation of these mutations.