Arachidonic and eicosapentaenoic acid metabolism by human CYP1A1: highly stereoselective formation of 17(R),18(S)-epoxyeicosatetraenoic acid Dieter Schwarz a,* , Pyotr Kisselev b , Spencer S. Ericksen c , Grazyna D. Szklarz c , Alexey Chernogolov a,1 , Horst Honeck d , Wolf-Hagen Schunck d , Ivar Roots a a Institute of Clinical Pharmacology, University Medical Center Charite ´, Humboldt University of Berlin, Schumannstrasse 20-21, Berlin 10098, Germany b Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Minsk 220141, Belarus c Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26505, USA d Max Delbrueck Center for Molecular Medicine, Berlin 13125, Germany Received 27 August 2003; accepted 1 December 2003 Abstract Human cytochrome P450 1A1 (CYP1A1) and human NADPH-cytochrome P450 reductase were expressed and purified from Spodoptera frugiperda insect cells. A reconstituted enzymatically active system metabolized polyunsaturated fatty acids such as arachidonic (AA) and eicosapentaenoic acid (EPA). CYP1A1 was an AA hydroxylase which oxidizes this substrate at a rate of 650  10 pmol/min/nmol CYP1A1, with over 90% of metabolites accounted for by hydroxylation products and with 19-OH-AA as major product. Epoxyeicosatrienoic acid (EET), mainly 14,15-EET, accounted for about 7% of total metabolites. Unlike rat CYP1A1, the human enzyme exhibited no 20-OH-AA as product. In contrast, with EPA as substrate CYP1A1 was mainly an epoxygenase, oxidizing with over 68% of total metabolites EPA to 17(R),18(S)-epoxyeicosatetraenoic acid (17(R),18(S)-EETeTr). 19-OH-EPA accounted for about 31% of total metabolites. Significantly, the 17,18-olefinic bond of EPA was epoxidized to 17(R),18(S)-EETeTr with nearly absolute regio- and stereoselectivity. Molecular modeling analyses provided rationale for high efficiency of AA hydroxylation at C 19 and its gradual decrease down to C 14 , as well as for the limited EPA 17(S),18(R) epoxidation due to unfavorable enzyme–substrate interactions. The absence of o- hydroxylation for both substrates is not due to steric factors, but probably a consequence of different reactivities of o and (o  1) carbons for hydrogen abstraction. It is suggested that the capacity of human CYP1A1 to metabolize AA and EPA and its inducibility by polycyclic aromatic hydrocarbons may affect the production of physiologically active metabolites, in particular, in the cardiovascular system and other extrahepatic tissues including lung. # 2004 Elsevier Inc. All rights reserved. Keywords: Human cytochrome P450 1A1; CYP1A1; Polyunsaturated fatty acid; Arachidonic acid; Eicosapentaenoic acid; Vasoactive metabolite 1. Introduction AA can be metabolized by CYP enzymes to several classes of oxygenated metabolites. The product profile depends on the CYP isoform involved and may consist of a series of regio- and stereoisomeric EETs and hydro- xyeicosatetraenoic acids (midchain HETEs and 16- through 20-OH-AA) [1]. Several of these CYP-derived eicosanoids have potent biological activities and are increasingly recog- nized to play important roles in the regulation of vascular tone and of renal, pulmonary, and cardiac function (reviewed in [2–6]). Human CYP isoforms acting primarily Biochemical Pharmacology 67 (2004) 1445–1457 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2003.12.023 * Corresponding author. Tel.: þ49-30-450525004; fax: þ49-30-450525933. E-mail address: dieter.schwarz@charite.de (D. Schwarz). 1 On leave from the Research Institute for Physical and Chemical Problems, Belarussian State University, Minsk, Belarus. Present address: Research Institute for Molecular Pharmacology, 13125 Berlin-Buch, Germany. Abbreviations: CYP, cytochrome P450; CYP1A1, human cytochrome P450 1A1; P450 reductase, human NADPH cytochrome P450 reductase; Sf9, Spodoptera frugiperda insect cells; AA, arachidonic acid (eicosate- traenoic acid); EPA, eicosapentaenoic acid; EET, epoxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; EETeTr, epoxyeicosatetraenoic acid; RP-HPLC, reversed-phase HPLC; NP-HPLC, normal-phase HPLC.