Kinds of mutations induced by aflatoxin B1 in a shuttle vector replicating in human cells transiently expressing cytochrome P450IA2 cDNA

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Abstract
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 B, into genotoxic metabolites within human cells. Ad293 cells were first transfected with p91‐IA2, a rat liver P450lA2 cDNA expression vector, or with p91‐lA2(i) (a control vector that has the P450 cDNA in the inverted orientation) and incubated for 24 h to permit P450IA2 accumulation. Cells were then transfected with the pS189 shuttlevector plasmid, which carries the Escherichia coli supF gene as a mutational target, and incubated for a further 24 h in the presence of aflatoxin B, to permit promutagen activation and pS189 replication. In shuttle vectors replicated in p91‐lA2–transfected cells, the supF point‐mutation frequency increased with increasing concentration of aflatoxin β1. 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.

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