Identification of aprt gene mutations induced in repair-deficient and P450-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 UV5P3 cells [a Chinese hamster ovary (CHO) cell line]. Sequence analysis of the PhIP-induced mutations in the adenine phosphoribosyltransferase (aprt) gene, which is heterozygous in the UV5P3 cells, can provide insight into the mutagenic mechanism in these repair-deficient cells expressing P4501A2. Two allele-specific 20 mer oligonucleotide primer pairs were used in the polymerase chain reaction and the allele of interest was amplified. Single-base transversions occurred in 31/32 PhIP-induced 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. On mutant had a change from C.G-->G.C at the 3' splice site of intron 4, thereby creating a new AG splice acceptor site. Another mutant had an insertion of T within a run of repeated sequences and resulted in a frameshift mutation. There were three 'hot-spots', two at the 3' end of exon 2 and one at the beginning of exon 3; 6 (19%) mutants showed a change 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%) mutants from C.G-->A.T (exon 3, amino acid residue 66). Consequently, 75% of the mutations were observed at these three sites. In contrast, none of the 20 spontaneous mutants had alterations at these hotspot sites. 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 more susceptible to mutation from these food mutagens than genes not containing these sequences.
Wu
R W
RW
Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, CA 94551-0808, USA.
Wu
E M
EM
Thompson
L H
LH
Felton
J S
JS
D000228:Adenine Phosphoribosyltransferase / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D003062:Codon; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000378:metabolism; D017931:DNA Primers; D005091:Exons; D005502:Food; D016368:Frameshift Mutation; D006579:Heterozygote; D006720:Homozygote; D007093:Imidazoles / Q000494:pharmacology*; D051379:Mice; D008969:Molecular Sequence Data; D009153:Mutagens / Q000494:pharmacology*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection
D003062:Codon; D017931:DNA Primers; D007093:Imidazoles; D009153:Mutagens; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D000228:Adenine Phosphoribosyltransferase