No abstract

Among numerous groups of naturally occurring compounds examined so far, volatile monoterpenes have long been used as fragrances and flavouring agents. (ϩ)-Fenchone, a bicyclic monoterpene, is widely distributed in plants and found in essential oils from Foeniculum vulgare.1) This component has camphoraceous fragrance. (ϩ)-Fenchone is used as a food flavour and in perfumes. Although, there are report that (ϩ)-fenchone have toxic.2) P450 enzymes are shown to detoxify and/or toxify these compounds to metabolites more polar and sometimes more reactive metabolites. 3-7)There is no literature on the absorption, excretion of the (ϩ)-fenchone in human body. Therefore, it is interesting to examine how (ϩ)-fenchone is metabolized by P450 in human liver microsomes.There are several reports on the biotransformation of (ϩ)-fenchone by microorganisms, insect and mammals, e.g. Baeyer-Villiger type oxidation by Corynebacterium sp. 5-exo and 6-exo hydroxylation by Absidia orchidis, 8) 5-exo, 6-exo and H-7 glucosylation by cultured plant cells of Eucalyptus perriniana, 9) 5-endo, 6-endo hydroxylation by Aspergillus niger, 10) 5-exo, 6-exo, 6-endo, 10-hydroxylation by Spodoptera litura 11) and hydroxylation of methyl groups by rabbits. 12) However, there is no report on the biotransformation of (ϩ)-fenchone by the human body.Our previous studies have demonstrated that several terpenes, 1,4-cineole, 1,8-cineole, (ϩ)-and (Ϫ)-limonenes and (Ϫ)-verbenone are catalyzed by cytochrome P450 enzymes to their respective oxidation products in human liver microsomes. [13][14][15][16] The metabolite from monoterpene esthers have determined to be oxidized at the 2-position of the molecules (1,4-cineole and 1,8-cineole) by human CYP3A4 enzyme. 13,14) Limonene enatiomers are metabolized to their respective carveols and perillyl alcohols in human liver. 13,14) (Ϫ)-Verbenone has found to be oxidized at the 10-position of the molecules by human CYP2A6 and 2B6. 16) In this study, we examined oxidations of (ϩ)-fenchone by P450 enzymes in liver microsomes prepared from different human samples. The metabolites thus formed were analyzed on GC-MS. To determine which P450s are the major enzymes in the oxidations of (ϩ)-fenchone, we used specific P450 inhibitors and antibodies raised against purified human liver P450 enzymes. Catalytic rates with eleven forms of human P450 enzymes expressed in Trichoplusia ni cells in the oxidations of (ϩ)-fenchone are also reported. MATERIALS AND METHODSChemicals (ϩ)-Menthofuran, thioTEPA, NADP, glucose 6-phosphate, and glucose 6-phosphate dehydrogenase were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). (ϩ)-Fenchone (purity, Ͼ99%) was purchased from Fluka. 6-exo-Hydroxyfenchone, 6-endo-hydroxyfenchone and 10-hydroxyfenchone (purity, Ͼ99%) were isolated with Spodoptera litura.11) Other reagents and chemicals used in this study were obtained from sources as described previously or were of the highest quality commercially available. (3H, s, H-9), 1.16 (3H, s, H-10), 1.62 (1H, dd, Jϭ1.7, 11.0 Hz, H B -7; nearly C...

The in vitro metabolism of (-)-fenchone was examined in human liver microsomes and recombinant enzymes. The biotransformation of (-)-fenchone was investigated by gas chromatography-mass spectrometry. (-)-Fenchone was found to be oxidized to 6-exo-hydroxyfenchone, 6-endo-hydroxyfenchone and 10-hydroxyfenchone by human liver microsomal P450 enzymes. The formation of metabolites was determined by the relative abundance of mass fragments and retention times on gas chromatography (GC). CYP2A6 and CYP2B6 were major enzymes involved in the hydroxylation of (-)-fenchone by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 and CYP2B6 catalysed the oxidation of (-)-fenchone. Second, oxidation of (-)-fenchone was inhibited by thioTEPA and (+)-menthofuran. Finally, there was a good correlation between CYP2A6, CYP2B6 contents and (-)-fenchone hydroxylation activities in liver microsomes of 11 human samples. CYP2A6 may be more important than CYP2B6 in human liver microsomes. Kinetic analysis showed that the Vmax/Km values for (-)-fenchone 6-endo-, 6-exo- and 10-hydroxylation catalysed by liver microsomes of human sample HG-03 were 24.3, 44.0 and 1.3nM(-1)min(-1) , respectively. Human recombinant CYP2A6 and CYP2B6 catalysed (-)-fenchone 6-exo-hydroxylation with Vmax values of 2.7 and 12.9 nmol min(-1) nmol(-1) P450 and apparent Km values of 0.18 and 0.15 mM and (-)-fenchone 6-endo-hydroxylation with Vmax values of 1.26 and 5.33nmolmin(-l) nmol(-1) P450 with apparent Km values of 0.29 and 0.26mM. (-)-Fenchone 10-hydroxylation was catalysed by CYP2B6 with Km and Vmax values of 0.2 mM and 10.66 nmol min(-1) nmol(-1) P450, respectively.

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.