Mutagenic specificity of the food mutagen 2-amino-3-methylimidazo(4,5-f)quinoline in Escherichia coli using the yeast URA3 gene as a target

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a strong mutagen/carcinogen, belongs to a group of heterocyclic amines that are formed (ng/g amounts) during the cooking of protein containing food. The mutational specificity of IQ in Escherichia coli was determined in a forward mutation assay using the yeast URA3 gene as a target. The plasmid pTU-AC, containing the target URA3, was randomly modified in vitro using N-hydroxy-IQ, and subsequently transformed into an E.coli pyrF strain (DB6656). Mutant clones were directly selected by their ability to grow on medium containing 5-fluoro-orotic acid which is toxic to URA3 + clones and thereby selects for URA3-mutants. Single Strand Conformation Polymorphism (SSCP) was used to map the mutation-containing regions of URA3, so that it was necessary to sequence only the relevant, mutation-containing fragment and not the entire gene. At a modification level of 7 IQ-lesions/URA3 gene, the predominant mutations were base substitutions (∼70 %), followed by complex gene rearrangements (∼ 20%) and frameshifts (∼ 10%). More than 96% of the base substitutions occurred at G:C base pairs and were predominantly G:C→A:T transitions, followed by G:C→T:A and G:C→C:G transversions. Next neighbour analysis revealed that deoxyguanosines situated within the sequence 5'-TGC were more susceptible to mutations induced by IQ. With one exception, all frameshift mutations were -1 deletions at runs of three consecutive dGs. At higher IQ-modification levels, predominantly complex sequence rearrangements were observed.