Metabolism of Δ³-Carene by Human Cytochrome P450 Enzymes: Identification and Characterization of Two New Metabolites

Mike      Duisken; Dorina      Benz; Thomas   H.   Peiffer; Brunhilde      Blomeke; Juliane      Hollender

doi:10.2174/138920005774832614

Abstract

The metabolism of the bicyclic monoterpene Δ³-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 Δ³-carene-10-ol ((1S, 6R)- 7,7-Dimethylbicyclo[4.1.0]hept-3-en-3-yl-methanol) and Δ³-carene-epoxide ((1S, 3S, 5R, 7R)-3,8,8-Trimethyl-4-oxatricyclo[ 5.1.0.03,5]octane). Δ³-carene-10-ol was formed by human liver microsomes and recombinant human CYP2B6, CYP2C19 and CYP2D6. Δ³-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 Δ³-carene 10-hydroxylation. Michaelis-Menten Km and Vmax for oxidation of Δ³-carene were 0.6 mM and 28.4 nmol/min/nmol P450 using human CYP2B6. For the formation of Δ³-carene-epoxide 98.2 mM and 3.9 nmol/min/nmol P450 were determined as Km and Vmax by using human CYP1A2. To our knowledge, this is the first time that Δ³-carene-10-ol and Δ³-carene-epoxide are described as human metabolites of Δ³- carene.

Keywords: Metabolism, Δ³-Carene, Cytochrome P450, Δ³-Carene-10-ol, Δ³-Carene-epoxide

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