IDENTIFICATION OF APRT GENE-MUTATIONS INDUCED IN REPAIR-DEFICIENT ANDP450-EXPRESSING CHO CELLS BY THE FOOD-RELATED MUTAGEN CARCINOGEN, PHIP/

We investigated the specific sequence changes produced by the dietary mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in UV5P 3 cells [a Chinese hamster ovary (CHO) cell line]. Sequence analysis o f the PhIP-induced mutations in the adenine phosphoribosyltransferase (aprt) gene, which is heterozygous in the UV5P3 cells, can provide ins ight into the mutagenic mechanism in these repair-deficient cells expr essing P4501A2. Two allele-specific 20 mer oligonucleotide primer pair s were used in the polymerase chain reaction and the allele of interes t was amplified. Single-base transversions occurred in 31/32 PhIP-indu ced mutants; of these, 6 were A . T-->T . A, 18 were C . G-->A . T and 6 were G . C-->T . A. Twenty of the 30 changes altered specific amino acid sequences and the other 10 resulted in a stop codon. One mutant had a change from C . G-->G . C at the 3' splice site of intron 4, the reby creating a new AG splice acceptor site. Another mutant had an ins ertion of T within a run of repeated sequences and resulted in a frame shift mutation. There were three 'hot-spots', two at the 3' end of exo n 2 and one at the beginning of exon 3; 6 (19%) mutants showed a chang e from A . T-->T . A (exon 2, amino acid residue 57), 11 (34%) mutants from C . G-->A . T (exon 2, amino acid residue 62), and 7 (22%) mutan ts from C . G-->A . T (exon 3, amino acid residue 66). Consequently, 7 5 % of the mutations were observed at these three sites. In contrast, none of the 20 spontaneous mutants had alterations at these hotspot si tes. The mutations induced by PHIP in these repair-deficient CHO cells were unique and specific, and suggest that these sequences, if found in important genes controlling cell replication and survival, may be m ore susceptible to mutation from these food mutagens than genes not co ntaining these sequences.