Shuttle-vector mutagenesis by aflatoxin B 1 in human cells: effects of sequence context on the supF mutational spectrum

Abstract

Rat-liver microsomes were used to activate aflatoxine B 1 for in vitro modification of the pS189 shuttle vector and the related signature vector pSP189, both of which carry the Escherichia coli supF gene as a mutational target. Plasmid degradation was minimized by carrying out the in vitro incubations in the absence of Mg 2+ ions. Modified plasmids were transfected into human Ad293 cells, then recovered and electroporated into E. coli MBM7070 for mutant identification. Point mutation frequencies for in vitro modified plasmids were dramatically increased over the spontaneous background level. Mutant plasmids were characterized by DNA-sequence analysis. The vast majority of aflatoxin B 1-induced mutations were base substitutions, mostly G:C to T:A transversions. The spectrum of aflatoxin B 1-induced mutations in the pSP189 supF gene was very similar to that observed previously for the pSP189 supF gene with aflatoxin B 1 activated by cytochrome P450 1A2 (CYP1A2) synthesized from a cDNA expression vector within transfected Ad293 cells. However, the spectrum for pSP189, which carries the same supF gene as pSP189, but with different surrounding sequences, exhibited some notable differences from that of pSP189; this suggests that sequence context effects on mutagenic specificity can operate over distances of tens of base pairs.