title: Metabolism of Delta(3)-carene by human cytochrome p450 enzymes: identification and characterization of two new metabolites.
abstract: The metabolism of the bicyclic monoterpene Delta(3)-carene was investigated in vitro using human liver microsomes as well as human smoker/non-smoker lung microsomes and 12 different recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. We detected two metabolites using GC-MS analysis. The mass fragmentation indicated for one metabolite hydroxylation in the allyl position and for the other metabolite epoxidation at the double bond. For clear identification the suggested metabolites were synthesized in a four-step reaction. Comparison of GC retention times and mass spectra lead to the identification of the metabolites as Delta(3)-carene-10-ol ((1S, 6R)-7,7-Dimethylbicyclo[4.1.0]hept-3-en-3-yl-methanol) and Delta(3)-carene-epoxide ((1S, 3S, 5R, 7R)-3,8,8-Trimethyl-4-oxa-tricyclo[5.1.0.0(3,5)]octane). Delta(3)-carene-10-ol was formed by human liver microsomes and recombinant human CYP2B6, CYP2C19 and CYP2D6. Delta(3)-Carene-epoxide was obviously catalyzed only by CYP1A2. In both cases there was a clear correlation between the metabolite formation, incubation time and enzyme concentration, respectively. Further kinetic analysis revealed that CYP2B6 exhibited the highest activity for Delta(3)-carene 10-hydroxylation. Michaelis-Menten K(m) and V(max) for oxidation of Delta(3)-carene were 0.6 mM and 28.4 nmol/min/nmol P450 using human CYP2B6. For the formation of Delta(3)-carene-epoxide 98.2 mM and 3.9 nmol/min/nmol P450 were determined as K(m) and V(max) by using human CYP1A2. To our knowledge, this is the first time that Delta(3)-carene-10-ol and Delta(3)-carene-epoxide are described as human metabolites of Delta(3)-carene.
authors.affiliation: Institute of Hygiene and Environmental Medicine, RWTH Aachen, Aachen, Germany.
authors.forename: Mike
authors.initials: M
authors.lastname: Duisken
authors.forename: Dorina
authors.initials: D
authors.lastname: Benz
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Peiffer
authors.forename: Brunhilde
authors.initials: B
authors.lastname: Blömeke
authors.forename: Juliane
authors.initials: J
authors.lastname: Hollender
mesh_terms: D000081008:Bicyclic Monoterpenes; D001643:Bridged Bicyclo Compounds / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006900:Hydroxylation; D008168:Lung / Q000378:metabolism; D000432:Methanol / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D039821:Monoterpenes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000378:metabolism; D012907:Smoking
chemical_list: D000081008:Bicyclic Monoterpenes; D001643:Bridged Bicyclo Compounds; D004852:Epoxy Compounds; D039821:Monoterpenes; D011994:Recombinant Proteins; C507856:delta(3)-carene-10-ol; C507857:delta(3)-carene-epoxide; D003577:Cytochrome P-450 Enzyme System; C030218:3-carene; D000432:Methanol