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

✍ Scribed by Yvon Trottier; William I. Waithe; Alan Anderson

• Publisher: John Wiley and Sons
• Year: 1992
• Tongue: English
• Weight: 820 KB
• Volume: 6
• Category: Article
• ISSN: 0899-1987
• DOI: 10.1002/mc.2940060209

✦ Synopsis

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 β~1~. This frequency was nine to 23 times greater than the background point‐mutation frequency obtained with aflatoxin B~1~‐treated control (p91‐IA2(i)‐transfected) cells. The large majority of the aflatoxin B~1~–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. © 1992 wiley‐Liss, Inc.