Human CYP2A6 activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) : mutational specificity in the gpt gene of AS52 cells

• Published in: Carcinogenesis (New York) 1994-12, Vol.15 (12), p.2859-2866
• Main Authors: TIANO, H. F, REN-LI WANG, HOSOKAWA, M, CRESPI, C, TINDALL, K. R, LANGENBACH, R
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Aryl Hydrocarbon Hydroxylases; Bacterial Proteins - genetics; Base Sequence; Biological and medical sciences; Biotransformation; Cell Line; CHO Cells; Cricetinae; Cytochrome P-450 CYP2A6; Cytochrome P-450 Enzyme System - genetics; Cytochrome P-450 Enzyme System - metabolism; Escherichia coli Proteins; Genes, ras; Genes, Reporter - drug effects; Hypoxanthine Phosphoribosyltransferase - genetics; Medical sciences; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Molecular Sequence Data; Mutagenicity Tests; Nitrosamines - metabolism; Nitrosamines - toxicity; Pentosyltransferases; Polymerase Chain Reaction; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Tetrahydrofolate Dehydrogenase - genetics; Tobacco, tobacco smoking; Toxicology
• ISSN: 0143-3334; 1460-2180
• DOI: 10.1093/carcin/15.12.2859

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK) is a potential human carcinogen that is known to be metabolized to DNA-reactive intermediates by the cytochromes P450. We have examined the nature of NNK's DNA damaging effects in a mammalian cell system expressing a specific human cytochrome P450 (2A6) and containing a target gene for mutagenesis. Human CYP2A6, which is known to activate NNK to a mutagen, was lipofected via a retroviral vector into the Chinese hamster ovary AS52 cell line, which contains the bacterial gpt gene and can be mutated to 6-thioguanine resistance. AS52 cells expressed negligible CYP2A6-specific coumarin 7-hydroxylase activity (0.7 pmol/mg protein/min), while a CYP2A6 transfected clone (AS52-E8) expressed 30 pmol/mg protein/min. Both cell lines were equally sensitive to the cytotoxic and mutagenic effects of the direct-acting mutagen ethylmethanesulfonate; however, only the AS52-E8 cells exhibited a dose-dependent increase in cytotoxicity and mutant frequency upon treatment with NNK. At the highest NNK dose (1200 micrograms/ml), the mutant frequency in AS52-E8 cells was 14-fold (339 x 10(-6)) greater than the spontaneous frequency of 24 x 10(-6). Ninty-eight mutant clones were isolated following NNK treatment. Based on PCR analysis, 21 clones contained deletions/rearrangements and 77 were putative point mutants. Sequencing potential point mutants showed that 81% contained G:C to A:T transitions. Four of six G:C to A:T hotspots were at the second G of the GGT motif, which is the motif and major mutation found in codon 12 of Ki-ras from NNK-induced lung tumors in strain A mice. Since NNK may be metabolized via different pathways to pyridyloxobutylate or methylate DNA, the data suggest that methylation damage causes the major mutagenic events in AS52-E8 cells when NNK is activated by human CYP2A6.