Roles of cytochrome P450 3A enzymes in the 2-hydroxylation of 1,4-cineole, a monoterpene cyclic ether, by rat and human liver microsomes.
1. Oxidation of 1,4-cineole, a monoterpene cyclic ether, was studied in rat and human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes expressed in insect cells in which human P450 and NADPH-P450 reductase cDNAs have been introduced. On analysis with gas chromatography/mass spectrometry, 2-exo-hydroxy-1,4-cineole was identified as a principal oxidation product of 1,4-cineole catalysed by rat and human P450 enzymes. 2. CYP3A4 was a major enzyme involved in the 2-hydroxylation of 1,4-cineole by human liver microsomes, based on the following lines of evidence. First, 1,4-cineole 2-hydroxylation activities catalysed by human liver microsomes were inhibited by ketoconazole, a potent inhibitor of CYP3A activities, and an anti-CYP3A4 antibody. Second, there was a good correlation beteeen CYP3A4 contents and 1,4-cineole 2-hydroxylation activities in liver microsomes of eighteen human samples examined. Finally, of 10 recombinant human P450 enzymes examined, CYP3A4 had the highest activity for 1,4-cineole 2-hydroxylation. 3. Liver microsomal 1,4-cineole 2-hydroxylation activities were induced in rat by pregnenolone 16alpha-carbonitrile and dexamethasone and extensively inhibited by ketoconazole, indicative of the possible roles of CYP3A enzymes in this reaction. 4. Kinetic analysis showed that Vmax/Km for 1,4-cineole 2-hydroxylation catalysed by liver microsomes was higher in a human sample HL-104 (4.6 microM(-1) min(-1)) than those of rat treated with pregnenolone 16alpha-carbonitrile (0.49 microM(-1) min(-1)) and dexamethasone (0.36 microM(-1) min(-1)). 5. 1,8-Cineole, a structurally related monoterpene previously shown to be catalysed by CYP3A enzymes, inhibited 1,4-cineole 2-hydroxylation catalysed by human liver microsomes, whereas 1,4-cineole did not inhibit 1,8-cineole 2-hydroxylation activities. Both compounds caused inhibition of testosterone 6beta-hydroxylation by human liver microsomes, the former compound being more inhibitory than the latter. 6. These results suggest that 1,4-cineole and 1,8-cineole, two plant essential oils present in Citrus medica L. var. acida and Eucalyptus polybractea, respectively, are converted to 2-hydroxylated products by CYP3A enzymes in rat and human liver microsomes. It is unknown at present whether the 2-hydroxylation products of these compounds are more active biologically than the parent compound.
Miyazawa
M
M
Department of Applied Chemistry, Faculty of Science and Engineering, Kinki University, Higashiosaka, Osaka, Japan. miyazawa@apch.kindai.ac.jp
Shindo
M
M
Shimada
T
T
D000328:Adult; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002478:Cells, Cultured; D000081005:Cyclohexane Monoterpenes; D003511:Cyclohexanols / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D000077591:Eucalyptol; D005260:Female; D005421:Flavoring Agents / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006900:Hydroxylation; D066298:In Vitro Techniques; D007313:Insecta; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008875:Middle Aged; D039821:Monoterpenes; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013729:Terpenes / Q000378:metabolism*; D013739:Testosterone / Q000378:metabolism
D000081005:Cyclohexane Monoterpenes; D003511:Cyclohexanols; D005421:Flavoring Agents; D039821:Monoterpenes; D013729:Terpenes; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; C058951:1,4-cineole; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D000077591:Eucalyptol