ACCELERATED PAPER: Mutational spectra of the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) at the Chinese hamster hprt locus

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Abstract
The mutagenic ‘fingerprint’ of the cooked food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) was determined in a Chinese hamster cell line genetically engineered to express human CYP1A2 (XEMh1A2-MZ). The parental Chinese hamster V79 and XEMh1A2-MZ cells were exposed to PhIP at various concentrations for 24 h. There was a dose-dependent increase in frequency of mutations at the hypoxanthine-guanine phosphoribosyl-transferase (hprt)locus only in the metabolically competent XEMh1A2-MZ cells. The mutant frequency ranged from 25 to 90×106 with final concentrations of 2.5 to 100 μM PhIP compared to 8×106 in the solvent controls and the V79MZ cells. The molecular nature of PhlP-induced mutations in XEMh1AZ-MZ cells was determined by examining DNA sequence modifications at the hprt locus in forty five 6-thioguanine resistant (6-TG1) mutant clones. Single base substitutions, predominantly GC→TA transversions, were the major class of PhIP-induced mutation. However, a-1 frameshift ‘hotspot’ in a 5'-GGGA sequence was also observed, With the exception ofa compound modification, all of the PhIP-induced mutations involved G.C base pairs. This is consistent with the previouslyobserved PhIP-induced mutations in cultured mammalian cells and 32P-postlabelling experiments that show PhIP adducts to the guanine base and that the major adduct is at the C8 position. Furthermore, nearly all of these mutations involved guanine bases on the non-transcribed strand which is possibly indicative of preferential repair of PhIP adducts from the transcribed strand. Nearest neighbour analysis of induced base substitutions indicates a preference for 5' guanine and3' adenine. These data effectively define a mutation &fingerprint’ for PhIP, which may provide the basis fordefinitive studies on the role of PhIP in diet associated cancers such as tumours of colon. It is, therefore, intriguing that in their recent report of mutation in tumours of the colon induced by PhIP in male rats Kakiuchi et al.(Proc. Natl Acad. Sci. USA, 92, 910–914) report that four out of eight tumours had an identical mutation of the tumour suppressor gene apc which comprised of a -1 G grameshift in a 5'-GGGA sequence.

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