Human CYP2A6 activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): mutational specificity in the gpt gene of AS52 cells.
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.
Tiano
H F
HF
Laboratory of Environmental Carcinogenesis and Mutagenesis, NIEHS, RTP, NC 27709.
Wang
R L
RL
Hosokawa
M
M
Crespi
C
C
Tindall
K R
KR
Langenbach
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001711:Biotransformation; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D017930:Genes, Reporter / Q000187:drug effects; D011905:Genes, ras; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009152:Mutagenicity Tests; D009602:Nitrosamines / Q000378:metabolism* / Q000633:toxicity; D010430:Pentosyltransferases; D016133:Polymerase Chain Reaction; D011506:Proteins; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009602:Nitrosamines; D011506:Proteins; D011993:Recombinant Fusion Proteins; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D013762:Tetrahydrofolate Dehydrogenase; D010430:Pentosyltransferases; C071962:Gpt protein, E coli; D007041:Hypoxanthine Phosphoribosyltransferase