Cytochrome P450 and the Metabolism and Bioactivation of Arachidonic Acid and Eicosanoids

11 Cytochrome P450 and the Metabolism and Bioactivation of Arachidonic Acid and Eicosanoids Jorge H. Capdevila, Vijaykumar R. Holla, and John R. Faick 1. Introduction content stored in the AA metabolites, is limited compared to that contained in complex informa The convergence of important advances in the tional molecules such as proteins or nucleic acids, identification of several lipid-derived mediators as low energy cost, versatility, and rapid turnover, inter- and intracellular signaling molecules, and in makes them efficient on/off molecular switches the biochemistry of oxidative lipid metabolism, for rapid and efficient intra- or intercellular sig has focused interest in the functional roles of path naling. Metabolism by prostaglandin H2 synthase ways responsible for their formation, and the generates a cyclic endoperoxide, prostaglandin physiological significance of their products. H2 (PGH2) that serves as the precursor for the Among these, the studies of the enzymes of the formation of prostaglandins, prostacyclin, and arachidonic acid (AA) cascade, consisting of thromboxanes^' ^. Metabolism by lipoxygenases prostaglandin H2 synthase^'^, lipoxygenases^, and leads to the formation of several regioisomeric cytochrome P450'^' ^, constitute a premier example hydroperoxides, the precursors of leukotrienes, of the biological importance of these reactions and regioisomeric cis/trans conjugated hydroxye- of their products (eicosanoids). Studies of the last icosatetraenoic acids (HETEs), lipoxins, and hep- two decades, have implicated the enzymes of the oxilins^. Metabolism by microsomal cytochrome AA cascade in the pathophysiology of diseases P450s (P450s) generates several hydroxy- and such as hypertension, diabetes, and cancer, and epoxy-AA derivatives'^' ^. The reactions catalyzed some of these enzymes serve as molecular targets by prostaglandin H2 synthase and lipoxygenases for drugs of extensive use in clinical medicine, are mechanistically similar to those of the free- including many nonsteroidal anti-inflanmiatory, radical-mediated autooxidation of polyunsaturated antipyretic, and anti-asthmatic drugs^~^. The bio fatty acids in that they are initiated by hydrogen logical and signaling properties of eicosanoids are atom abstraction from a bis-allylic methylene derived from the enzymatic, regio-, and stereo carbon, followed by coupling of the resulting car selective oxygenation of AA, a rather simple bon radical to groimd state molecular oxygen. The molecular template. While the informational kinetics, regiochemistry, and chirality of these Jorge H. Capdevila • Departments of Medicine and Biochemistry, Vanderbilt University Medical School, Nashville, TN. Vijaykumar R. Holla • Department of Medicine, Vanderbilt University Medical School, Nashville, TN. John R. FaIck • Department of Biochemistry, Southwestern Medical Center, Dallas, TX. Cytochrome P450: Structure, Mechanism, and Biochemistry, 3e, edited by Paul R. Ortiz de Montellano Kluwer Academic / Plenum Publishers, New York, 2005. 531 532 Jorge H. Capdevila et al. reactions are under strict enzymatic control. In pathophysiology of genetically controlled experi contradistinction to the c)^ochrome P450-catalyzed, mental hypertension^' ^. These earlier studies estab redox coupled, activation of molecular oxygen and lished P450 AA monooxygenation as a formal delivery to ground state carbon, prostaglandin H2 metabolic pathway, P450 as an endogenous mem synthase and the lipoxygenases are typical dioxyge- ber of the AA metabolic cascade, and more impor nases that catalyze substrate carbon activation tantly, suggested functional roles for this enzyme in instead of oxygen activation. the bioactivation of the fatty acid and thus, in cell It is apparent from the literature that the P450 and organ physiology. Many of the biological activ gene superfamily of hemoproteins is, as a group, ities attributed to the P450-derived eicosanoids, one of the most intensively studied enzyme sys as well as the potential physiological importance of tems and yet our knowledge of their endogenous these reactions, have been reviewed^"^^. metabolic or physiological roles remains limited. Prior to the demonstration of AA metabolism This is partly due to the complexity of mammalian by P450, several groups demonstrated the role of P450 isoforms, and the wide structural diversity microsomal P450s in the (o/w-l hydroxylation of of substrates known to be metabolized by these prostanoids^^"^^ and, more recently, leukotrienes^^. proteins. This catalytic versatility pointed to, and Most of these reactions are considered to be served as the basis for, many of the documented involved in eicosanoid catabolism and excretion, roles for P450 in the metabolism of foreign chem but their potential relevance in eicosanoid bioacti icals, and has contributed to establish its toxico- vation or inactivation, and/or in the control of logical and pharmacological importance. In the organ/cell eicosanoid levels has only begun to be last few years, there has been an increasing inter explored. We will first discuss the role of P450 est in the understanding of the physiological sig in the metabolism of eicosanoids, and then con nificance of the P450 enzyme system, and its centrate on the studies of its role in AA metabolism role(s) in the metabolism of endogenous sub and bioactivation. strates. In this regard, the studies of the P450 branch of the AA cascade have provided a new focus to these efforts, and far-reaching results 2. Metabolism of Eicosanoids from several laboratories are generating new par adigms in fatty acid metabolism, as well as in cell During the metabolism of eicosanoids, and organ physiology^^^. depending on the nature of the oxygenated sub The studies of the role of P450 in the metabo strate, P450 catalyzes both NADPH-dependent lism and bioactivation of AA were initiated in 1981 and -independent reactions. This differential with the demonstration that liver and kidney requirement for NADPH-mediated changes in the microsomal fractions, as well as purified P450 redox state of the heme-iron illustrates the marked isoforms^ ^"^^ actively catalyzed the NADPH- differences in oxygen chemistries for these reac dependent, oxidative metabolism of AA to prod tions, that is, the isomerization of AA peroxides, ucts that were different from prostanoids and vs the more demanding activation and delivery of leukotrienes"'^. The widely documented physio a reactive form of atomic oxygen to ground state logical importance of AA suggested that these carbon-hydrogen bonds. observations were unique and likely to be func tionally significant, and led to the rapid structural characterization of most P450-eicosanoids, their 2.1. NADPH-lndependent chemical synthesis, and subsequent biological Reactions evaluation"^'^. Interest in these novel P450 reac tions was stimulated by: (a) the initial demonstra P450 catalyzes the isomerization of a variety tion that some of the products displayed potent of fatty acid hydroperoxides, including 15-hydro- biological activities, including the inhibition of peroxyeicosatetraenoic acid (15-HPETE)^^' ^^, Na^ reabsorption in the distal nephron^^, (b) the and of the prostaglandin H2 (PGH2) endoperox- documentation of P450 participation in the in vivo ide^^. A distinctive feature of some P450 fatty acid metabolism of endogenous AA pools^^, and (c) the peroxide isomerases is their inability to accept proposal of a role for these enzymes in the electrons from NADPH and to activate molecular Cytochrome P450 and the Metabolism of AA and Eicosanoids 533 oxygen^ ^"^^. Moreover, while all these enzymes terminal (C20 or co-carbon) or penultimate carbon possess a heme-thiolate prosthetic group, their atoms (C J9 or (0-1 carbon). However, the epoxida- overall homology to other members of the P450 tion of infused PGI2 by a perfused kidney prepa gene superfamily is limited and suggests an early ration'^, and the metabolism of 5,6- and evolutionary functional specialization^^"^^. The 8,9-EET by prostaglandin H2 synthase, were mechanism by which the hemoprotein cleaves the described several years ago'^' ^^. The former leads peroxide oxygen-oxygen bond, that is, homolytic to a variety of 5,6-oxygenated prostanoids'^. or heterolytic scission, plays a decisive role in Oxidation of the latter was stereodependent, determining the catalytic outcome of these reac that is, 8(5),9(i?)-EET formed ll(i?)-hydroxy- tions and is highly dependent on the nature of 8(-S),9(i?)-epoxyeicosatrienoic acid exclusively, the P450 isoform, the chemical properties of the whereas the 8(/?),9(*S)-enantiomer formed both organic peroxide, and the nature of the oxygen Cjj and Cj5 hydroxylated metabolites^^. A acceptor^^"^^' ^^. A homolytic pathway was proposed detailed study of the secondary metabolism of for the formation of 11- and 13-hydroxy-14, 12(i?)-HETE and 14,15-EET by P450 has been 15-epoxyeicosatrienoic acids (EETs) from 15- reported^ ^' ^'. The efficient w/w-l oxidation of HPETE by rat liver microsomes'^. Prostacyclin EETs by rat CYP4A isoforms to the correspon and thromboxane synthases are P450-like proteins ding regioisomeric epoxy-alcohols at rates com containing a heme-thiolate prosthetic group'^~'^. parable to those observed with lauric acid, a The heterolytic cleavage of PGH2 and an oxygen prototype substrate for these enzymes, was pub atom transfer or oxenoid mechanism has been lished recently^^. One of these metabolites, the proposed to account for the P450-catalyzed forma o)-alcohol of 14,15-EET,

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