Human CYP2B6: expression, inducibility and catalytic activities.
Human cytochrome (CYP)2B6 cDNA was cloned and expressed in bacteria and in yeast. Its expression in Saccharomyces cerevisiae enabled us to obtain, at a high level, an active yeast-expressed CYP2B6 protein, so as to assess its role in the metabolism of ethoxyresorufin, pentoxyresorufin, benzyloxyresorufin, ethoxycoumarin, testosterone and cyclophosphamide. Kinetic analysis showed that human CYP2B6 preferentially metabolized benzyloxyresorufin and pentoxyresorufin, although other CYPs also metabolized these substrates in human liver microsomes. CYP2B6 also manifested a strong 4-hydroxycyclophosphamide activity. Its expression in Escherichia coli enabled us to produce a very specific anti-human CYP2B6 antibody. No cross reactivity of this antibody was observed with CYPs1A1, 1A2, 3A4, 3A5, 2C8, 2C9, 2C18, 2C19, 2D6 or 2E1. This antibody enabled us to study the hepatic and extrahepatic expression of CYP2B6 in man, as well as its expression and inducibility in primary cultured human hepatocytes and in different human cell lines. Immunoblot analysis revealed that the CYP2B6 protein was expressed in 43 of the 48 human liver samples tested, with levels ranging from 0.4 to 8 pmol/mg of microsomal protein with a mean of 1.7 pmol/mg protein. CYP2B was also expressed in human brain, intestine and kidney, and at a lower level in the lung. CYP2B mRNA was detected in human liver, kidney, lung, trachea and intestine. We also found that CYP2B6 is induced at protein and mRNA levels by phenobarbital (2 mM) and cyclophosphamide (1 mM), an anticancer drug known to be metabolized by CYP2B6. No expression or inducibility of CYP2B6 was observed in any of the human cell lines tested.
Gervot
L
L
INSERM U490, Université René Descartes, Paris, France.
Rochat
B
B
Gautier
J C
JC
Bohnenstengel
F
F
Kroemer
H
H
de Berardinis
V
V
Martin
H
H
Beaune
P
P
de Waziers
I
I
D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D003001:Cloning, Molecular; D003520:Cyclophosphamide / Q000494:pharmacology; D065702:Cytochrome P-450 CYP2B6; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004790:Enzyme Induction; D006801:Humans; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D017931:DNA Primers; D011994:Recombinant Proteins; D003520:Cyclophosphamide; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D010089:Oxidoreductases, N-Demethylating; D010634:Phenobarbital