Characterization of a human cell line expressing high levels of cDNA-derived CYP2D6.
We have developed a human B-lymphoblastoid cell, designated h2D6v2, which expresses high levels of CYP2D6 cDNA. Microsomal P450 contents of 160 pmol mg-1 protein were observed. NADPH-fortified microsomes exhibited a substantial capacity to hydroxylate the prototype CYP2D6 substrates bufuralol and debrisoquine. Kinetic parameters, apparent Km, turnover number, Ki for quinidine inhibition and stereospecificity of bufuralol hydroxylation, observed with the human lymphoblast expressed enzyme were similar to those observed in human liver microsomes or purified liver CYP2D6 proteins. Therefore, the human lymphoblast expressed material appears to faithfully reflect the authentic protein. Relative to control cells, h2D6v2 cells were more sensitive to the cytotoxicity and mutagenicity of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), supporting our previous observation with a cell line expressing lower levels of CYP2D6. h2D6v2 microsomes were capable of metabolizing NNK and NNK metabolism and mutagenicity were markedly inhibited by the addition of quinidine, a CYP2D6 inhibitor. h2D6v2 cells coupled with control cells, represent a useful in vitro system for studying xenobiotic metabolism by the clinically important, polymorphic CYP2D6. The human lymphoblast system offers the desirable ability to couple metabolic transformation studies with toxicological endpoints such as cytotoxicity and mutagenicity.
Penman
B W
BW
GENTEST Corporation, Woburn, MA 01801.
Reece
J
J
Smith
T
T
Yang
C S
CS
Gelboin
H V
HV
Gonzalez
F J
FJ
Crespi
C L
CL
D001402:B-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D002458:Cell Fractionation; D002460:Cell Line; D019389:Cytochrome P-450 CYP2D6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004247:DNA / Q000235:genetics*; D003647:Debrisoquin / Q000378:metabolism; D004983:Ethanolamines / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009153:Mutagens / Q000378:metabolism / Q000633:toxicity; D009602:Nitrosamines / Q000378:metabolism / Q000633:toxicity; D013379:Substrate Specificity
D004983:Ethanolamines; D009153:Mutagens; D009602:Nitrosamines; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; C010831:bufuralol; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D019389:Cytochrome P-450 CYP2D6; D003647:Debrisoquin