A tobacco smoke-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, is activated by multiple human cytochrome P450s including the polymorphic human cytochrome P4502D6.
We have developed a human B-lymphoblastoid cell line, designated 2D6/Hol, which stably expresses human cytochrome P450 CYP2D6 cDNA. This cell line exhibits bufuralol 1'-hydroxylase activity and immunologically detectable CYP2D6 protein. The specific activity of (+)-bufuralol 1'-hydroxylase in microsomes from 2D6/Hol cells was comparable to that observed in human liver microsomes. This cell line was used to examine the mutagenicity activation of three tobacco smoke-derived nitrosamines, N-nitrosonornicotine (NNN), 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal) (NNA) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2D6. Exposure of 2D6/Hol cells to NNK concentrations of 30-90 micrograms/ml induced a concentration-dependent decrease in relative survival and increase in mutant fraction at the hypoxanthine guanine phosphoribosyl transferase (hprt) locus. In contrast, NNK was non-mutagenic and non-cytotoxic to control cells at exposure concentrations up to 150 micrograms/ml. NNK mutagenicity in 2D6/Hol cells was compared to the responses observed in isogenic cell lines expressing human CYP1A2 (1A2/Hol), human CYP2A3 (2A3/Hol) and human CYP2E1 (2E1/Hol). These three additional human cytochrome P450-expressing cell lines were also found to be sensitive to NNK-induced mutagenicity and cytotoxicity. We found no evidence for CYP2D6-mediated activation of NNN or NNA. NNN was non-cytotoxic and non-mutagenic to both control and 2D6/Hol cells. NNA was equally cytotoxic and mutagenic to control cells and 2D6/Hol cells. The activation of NNA to a mutagen may have been carried out by P450 native to the AHH-1 TK +/- cell line. The 2D6/Hol cell line, in conjunction with the control cell line and other isogenic cell lines expressing other human cytochrome P450 cDNAs provides a useful system for the examination of the role of the polymorphic CYP2D6 in human procarcinogen activation and drug metabolism.
Crespi
C L
CL
GENTEST Corporation, Woburn, MA 01801.
Penman
B W
BW
Gelboin
H V
HV
Gonzalez
F J
FJ
D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism*; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000502:physiology*; D004128:Dimethylnitrosamine / Q000633:toxicity; D006801:Humans; D007527:Isoenzymes / Q000502:physiology*; D006899:Mixed Function Oxygenases / Q000032:analysis; D009153:Mutagens / Q000378:metabolism; D009602:Nitrosamines / Q000378:metabolism* / Q000633:toxicity; D014162:Transfection
D002273:Carcinogens; D007527:Isoenzymes; D009153:Mutagens; D009602:Nitrosamines; C047760:4-(methylnitrosamino)-4-(3-pyridyl)butanal; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C043872:bufuralol 1'-hydroxylase; D004128:Dimethylnitrosamine; C008655:N'-nitrosonornicotine