Roles of human CYP2A6 and rat CYP2B1 in the oxidation of (+)-fenchol by liver microsomes.
The metabolism of (+)-fenchol was investigated in vitro using liver microsomes of rats and humans and recombinant cytochrome P450 (P450 or CYP) enzymes in insect cells in which human/rat P450 and NADPH-P450 reductase cDNAs had been introduced. The biotransformation of (+)-fenchol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Fenchol was oxidized to fenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on GC. Several lines of evidence suggested that CYP2A6 is a major enzyme involved in the oxidation of (+)-fenchol by human liver microsomes. (+)-Fenchol oxidation activities by liver microsomes were very significantly inhibited by (+)-menthofuran, a CYP2A6 inhibitor, and anti-CYP2A6. There was a good correlation between CYP2A6 contents and (+)-fenchol oxidation activities in liver microsomes of ten human samples. Kinetic analysis showed that the Vmax/Km values for (+)-fenchol catalysed by liver microsomes of human sample HG03 were 7.25 nM-1 min-1. Human recombinant CYP2A6-catalyzed (+)-fenchol oxidation with a Vmax value of 6.96 nmol min-1 nmol-1 P450 and apparent Km value of 0.09 mM. In contrast, rat CYP2A1 did not catalyse (+)-fenchol oxidation. In the rat (+)-fenchol was oxidized to fenchone, 6-exo-hydroxyfenchol and 10-hydroxyfenchol by liver microsomes of phenobarbital-treated rats. Recombinant rat CYP2B1 catalysed (+)-fenchol oxidation. Kinetic analysis showed that the Km values for the formation of fenchone, 6-exo- hydroxyfenchol and 10-hydroxyfenchol in rats treated with phenobarbital were 0.06, 0.03 and 0.03 mM, and Vmax values were 2.94, 6.1 and 13.8 nmol min-1 nmol-1 P450, respectively. Taken collectively, the results suggest that human CYP2A6 and rat CYP2B1 are the major enzymes involved in the metabolism of (+)-fenchol by liver microsomes and that there are species-related differences in the human and rat CYP2A enzymes.
Miyazawa
M
M
Faculty of Science and Engineering, Department of Applied Chemistry, Kinki University, Osaka, Japan. miyazawa@apch.kindai.ac.jp
Gyoubu
K
K
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D001892:Camphanes; D065637:Cytochrome P-450 CYP2A6; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009636:Norbornanes / Q000737:chemistry / Q000493:pharmacokinetics*; D010084:Oxidation-Reduction; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D015262:Xenobiotics / Q000737:chemistry / Q000493:pharmacokinetics
D001892:Camphanes; D009636:Norbornanes; D011994:Recombinant Proteins; D015262:Xenobiotics; C027328:fenchol; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D019362:Cytochrome P-450 CYP2B1