Mutagenesis of the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine DNA adduct in mammalian cells. Sequence context effects.
Site-specifically modified oligodeoxynucleotides were used to investigate the mutagenic properties of a major cooked food mutagen-derived DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (dG-C8-PhIP). dG-C8-PhIP-modified oligodeoxynucleotides were prepared by reacting an oligodeoxynucleotide containing a single dG (5'-TCCTCCTXGCCTCTC, where X = C, A, G, or T) with N-acetoxy-PhIP. The unmodified and dG-C8-PhIP-modified oligomers were inserted into single-stranded phagemid vectors. These single-stranded vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. When dC was at the 5'-flanking position to dG-C8-PhIP, preferential incorporation of dCMP, the correct base, was observed opposite the dG-C8-PhIP. Targeted G --> T transversions were detected, along with lesser amounts of G --> A transitions and G --> C transversions. No mutations were detected for the unmodified vector. The influence of sequence context on the dG-C8-PhIP mutation frequency and spectrum was also explored. When the dC 5'-flanking base was replaced by dT, dA, or dG, the mutational spectra were similar to that observed with dC-flanking base. Higher mutational frequencies (28-30%) were observed when dC or dG was 5' to dG-C8-PhIP. A lower mutational frequency (13%) was observed when dA was at the 5' to the lesion. Single-base deletions were detected only when dG or dT flanked the adduct. We conclude that dG-C8-PhIP is mutagenic, generating primarily G --> T transversions in mammalian cells. The mutational frequency and specificity of dG-C8-PhIP vary depending on the neighboring sequence context.
Shibutani
S
S
Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794-8651, USA. shinya@pharm.som.sunysb.edu
Fernandes
A
A
Suzuki
N
N
Zhou
L
L
Johnson
F
F
Grollman
A P
AP
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D004247:DNA / Q000235:genetics* / Q000378:metabolism*; D018736:DNA Adducts; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007093:Imidazoles / Q000633:toxicity*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D014162:Transfection
D018736:DNA Adducts; D007093:Imidazoles; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; C078255:N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D004247:DNA; D003849:Deoxyguanosine