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Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 231

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Cytochrome P450 in Oxygenation of Endoperoxides and

Cloning, Expression and Catalytic Properties of CYP4F8 and CYP4F21

BY

JOHAN BYLUND

ACTA UNIVERSITATIS UPSALIENSIS UPPSALA 2000 Dissertation for the Degree of Doctor of Philosophy (Faculty of Pharmacy) in Pharmaceutical Pharmacology presented at Uppsala University in 2000

ABSTRACT

Bylund, J. 2000. P450 Enzymes in Oxygenation of Prostaglandin Endoperoxides and Arachidonic Acid: Cloning, Expression and Catalytic Properties of CYP4F8 and CYP4F21. Acta Universitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations from Faculty of Pharmacy 231 50 pp. Uppsala. ISBN 91-554-4784-8. (P450 or CYP) is an system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and . This thesis describes investigations of P450-catalyzed oxygenation of , linoleic and arachidonic acids. The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human microsomes. Several P450 enzymes catalyzed the formation of bisallylic hydroxy metabolites. Inhibition studies and stereochemical analysis of metabolites suggest that the enzyme CYP1A2 may contribute to the biosynthesis of bisallylic hydroxy metabolites in adult human liver microsomes. 19R-Hydroxy-PGE and 20-hydroxy-PGE are major components of human and ovine semen, respectively. They are formed in the seminal vesicles, but the mechanism of their biosynthesis is unknown. Reverse transcription-polymerase chain reaction using degenerate primers for mammalian CYP4 family , revealed expression of two novel P450 genes in human and ovine seminal vesicles. The full coding regions of the genes were cloned and the enzymes were expressed in a system. The human enzyme was designated CYP4F8 and the ovine enzyme was designated CYP4F21. Comparison of their deduced sequences showed that they had 74 % identity. Recombinant CYP4F8 oxygenated two prostaglandin endoperoxides (PGH1 and PGH2) and three stable PGH2 analogues into 19-hydroxy metabolites. Oxygenation of these substrates was mirrored when incubated with microsomes isolated from human seminal vesicles. These results suggest that CYP4F8 is present in human seminal vesicles and that 19R-hydroxy-PGE is formed by CYP4F8-catalyzed 19R- of PGH1 and PGH2, followed by PGE synthase-catalyzed isomerization. Studies of catalytic properties of recombinant CYP4F21 suggest that 20-hydroxy- PGE may be formed by similar mechanisms in ovine seminal vesicles. CYP4F8 is the first enzyme shown to hydroxylate prostaglandin endoperoxides. Johan Bylund, Division of Biochemical Pharmacology, Department of Pharmaceutical Biosciences, Biomedical Centre, Box-591, SE-751 24 Uppsala, Sweden

© Johan Bylund 2000

ISSN 0282-7484 ISBN 91-554-4784-8

Printed in Sweden by Universitetstryckeriet, Ekonomikum, Uppsala, 2000

2 3 List of original papers

This thesis is based on the following papers, which will be referred to by their Roman numerals in the text.

I. Bylund, J., Kunz, T., Valmsen, K., and Oliw, E. H. (1998) Cytochrome P450 with Bisallylic Hydroxylation Activity on Arachidonic and Linoleic Acids Studied with Human Recombinant Enzymes and with Human and Liver Microsomes. J. Pharmacol. Exp. Ther. 284, 51-60.

II. Bylund, J., Ericsson, J. and Oliw, E. H. (1998) Analysis of Cytochrome P450 Metabolites of Arachidonic and Linoleic Acids by Liquid Chromatography-Mass Spectrometry with Ion Trap MS2. Anal. Biochem. 265, 55-68.

III. Bylund, J., Finnström, N. and Oliw, E. H. (1999) Expression of a Novel Cytochrome P450 of the CYP4F Subfamily in Human Seminal Vesicles. Biochem. Biophys. Res. Commun. 261, 169-174.

IV. Bylund, J., Hidestrand, M., Ingelman-Sundberg, M. and Oliw, E. H. (2000) Identification of CYP4F8 in Human Seminal Vesicles as a Prominent 19-Hydroxylase of Prostaglandin Endo- peroxides. J. Biol. Chem. 275, 21844-21849.

V. Bylund, J. and Oliw, E. H. (2000) Characterization of a Prostaglandin ω-Hydroxylase of Ram Seminal Vesicles: cDNA Cloning and Expression of CYP4F21. Manuscript

The articles are reprinted with permission from the copyright holders.

4 TABLE OF CONTENTS

INTRODUCTION ……………………………………………………………………. 7 Cytochrome P450 ...………………………………………………………………. 7 Reactions ………………………………………………………………………8 Expression and specificity ...……………………………………….. 9 biosynthesis ...……………………………………………………… 10 Prostaglandin H synthase pathway ..……………………………………….. 11 pathway ..……………………………………………………. 12 Cytochrome P450 pathway ..………………………………………………. 13 Hydroxylation of the ω-side chain ..…………………………………… 14 Epoxidation …………………………………………………………….15 Bisallylic hydroxylation ……………………………………………….. 16 Hydroxylation with double bond migration ..…………………………. 17 Eicosanoid ...………………………………………………………. 17 Seminal prostaglandins ..……………………………………………………. 18

AIMS …………………………………………………………………………………. 21

COMMENTS ON METHODOLOGY …………………………………………….. 22 Analysis of metabolites ...………………………………………………………... 22 Degenerate primers……………………………………………………………….. 22 Recombinant cytochrome P450 ...………………………………………………... 23

PGH2 and stable PGH2 analogues …………………………………………….…. 24

RESULTS ……………………………………………………………………………. 25 Bisallylic hydroxylation of fatty acids…...………………………………………. 25 Identification of fatty acid metabolites with LC-MS …………………..………... 26 of P450s in human seminal vesicles …………………………….27 Catalytic properties of CYP4F8 ……………………………………………….....27 CYP4F21 in ovine seminal vesicles ……………………………………………… 29

DISCUSSION ………………………………………………………………………. 30 Bisallylic hydroxylation of fatty acids (papers I-II) …………………………..… 30 Biosynthesis and metabolism of seminal prostaglandins (papers III-V) ……….... 32

CONCLUSIONS…………………………………………………………………..… 38

ACKNOWLEDGEMENTS…………………………………………..……………… 39

REFERENCES ………………………………………………………………………..40

5 ABBREVIATIONS

APCI atmospheric pressure chemical ionization CYP cytochrome P450 EETepoxyeicosatrienoic acid ER ESI electrospray ionization DHET dihydroxyeicosatrienoic acid GC-MS gas chromatography-mass spectrometry HETE hydroxyeicosatetraenoic acid HODE hydroxyoctadecadienoic acid HPETE hydroperoxyeicosatetraenoic acid HPLC high performance liquid chromatography LC-MS liquid chromatography-mass spectrometry LTleukotriene P450 cytochrome P450 PG prostaglandin PGH prostaglandin H PGG prostaglandin G

PGI2 RP-HPLC reverse phase-high performance liquid chromatography RT-PCR reverse transcriptase-polymerase chain reaction SIM selective ion monitoring

TXA2 A2 12-HHT 12-hydroxyheptadecatrienoic acid

6 INTRODUCTION

Arachidonic acid is a polyunsaturated fatty acid, which is present in most human and cells. Arachidonic acid can be converted into oxygenated metabolites, the so-called . The eicosanoids are involved in many physiological and pathophysiological functions such as blood pressure regulation, blood platelet aggregation, , reproduction and . There are three major pathways involved in the formation of eicosanoids, the prostaglandin H synthase, the lipoxygenase, and the cytochrome P450 pathways. The understanding of the physiological functions of the eicosanoids and their mechanisms of formation has generated many new drugs and treatments of common disorders e.g. inflammation, pain, and cardiovascular diseases. This study focuses on the involvement of cytochrome P450 enzymes in the biosynthesis of two groups of eicosanoids, hydroxyeicosatetraenoic acids and ω/(ω-1)-hydroxyprostaglandins. An increased knowledge of the mechanism of biosynthesis and metabolism of these eicosanoids may contribute to a better understanding of their physiological functions.

Cytochrome P450

Cytochrome P450 (P450) was discovered in the late 1950’s (1, 2). P450 refers to a superfamily of -thiolate enzymes whose Fe2+- complex shows an absorption spectrum with a maximum at 450 nm. The diversity of P450s has been found to be enormous. The P450 enzymes can be found in virtually all organisms including , fungi, yeast, , , fish and mammals. All P450 genes have probably evolved from one single ancestral gene, which existed before the time of prokaryote/eukaryote divergence (3). Due to the large number of P450s, a standardized nomenclature system has been developed. The enzymes have been organized on the basis of identities in protein sequence. The enzymes are named CYP, representing cytochrome P450, followed by a number denoting the family, a letter designating the subfamily and a second numeral representing the individual enzyme. The corresponding gene name is written in italics. Enzymes that have >40 % amino acid sequence identity are considered to belong to the same family, whereas enzymes with >55 % sequence identity are in the same subfamily (3). The P450 enzymes have two major functions (3, 4). They are involved in the biosynthesis, bio- and catabolism of endogenous compounds, such as fatty acids, , vitamins, bile acids, and eicosanoids. They are also involved in the metabolism of foreign chemicals and drugs. Drugs and other chemicals are often converted by P450 to more polar compounds that can be directly excreted or further conjugated by other enzymes. The conjugate makes the modified compound more water-soluble so it can be excreted into the urine. Some of

7 these P450 reactions can lead to bio-activation of drugs or to the formation of more toxic compounds with cancerogenic properties (4, 5). Within the cell the P450s are mainly located to the endoplasmic reticulum (ER) and to the inner membrane of the mitochondria (4). The mitochondria house several P450 enzymes involved in biosynthesis and metabolism of steroid hormones, bile acids and . This study focuses exclusively on P450 enzymes located in the ER.

Reactions

The P450s catalyze many types of reactions including oxygenations, dehalogenations, dealkylations, deaminations, dehydrogenations and isomerizations (4). The quantitatively most important reaction is the oxygenation. The P450 enzymes are often called mixed-function or , because they incorporate one atom of molecular into the substrate and one atom into water. The catalytic mechanism of the microsomal P450 mediated mono-oxygenation is shown in Fig. 1.

Fig. 1. Catalytic mechanism of microsmal P450 mediated monooxygenation (6, 7). RH, substrate.

8 The catalytic cycle begins with the substrate binding to the ferric form of the enzyme. The enzyme-substrate complex is then reduced by an transferred from NADPH via NADPH- cytochrome P450 . Molecular oxygen binds to the reduced enzyme-substrate complex to form an enzyme-O2-substrate complex followed by the introduction of a second electron transferred from NADPH via NADPH-cytochrome P450 reductase or from cytochrome b5. The next step involves addition of a proton leading to the formation of an enzyme-OOH-substrate complex. The addition of a second proton results in homolytic cleavage of the oxygen-oxygen bond with one atom of oxygen being released as water. The retained oxygen atom is then inserted into the substrate, the oxidized product is released, and the ferric form of the enzyme is regenerated (6, 7). In mitochondria, the are transferred from NADPH to the P450s via an alternative pathway (4).

Expression and substrate specificity

P450 is a ubiquitously expressed enzyme system, which has been identified in all mammalian cell types investigated thus far (3). The main site of P450 expression in is the liver, although tissues such as the kidney, intestine and lung contain rather high concentrations of mammalian P450. There are considered to be about 55 different P450 genes that code for functional enzymes in the (8). These enzymes are divided into 17 different families. Enzymes belonging to the CYP1-3 families are considered to be mostly involved in metabolism of drugs and other exogenous compounds (9). These P450s are mainly expressed in the liver, although they are found in numerous extrahepatic tissues. They often exhibit a broad, and in many cases, overlapping substrate specificity. The enzymes belonging to the other 14 families are involved in metabolism of endogenous compounds (9). These physiologically important enzymes often show a strict substrate specificity and tissue distribution in contrast to the versatile -metabolizing enzymes. However, the possibility cannot be excluded that some of the enzymes in the CYP1-3 families can catalyze physiological important reactions of endogenous compounds that have not yet been discovered. There are large species differences within the mammalian CYP2-4 families (9). The number of enzymes in their subfamilies as well as their substrate specificity and expression distribution differs. This makes it difficult to extrapolate results from animal metabolism studies to man. There is also a large variability in expression and metabolic capacity of individual P450s within the same species (10, 11). This is due to genetic of the corresponding P450 genes, as well as physiological, environmental, and pathological factors. in a P450 gene can result in protein variants with altered substrate specificity and catalytic activity. They can also lead to formation of an inactive protein or to changes in the expression of the gene. Several mutations of P450s are known to cause diseases (9). The levels of P450 enzymes may change as a

9 consequence of induction or inhibition because of concomitant drug treatment or exposure to other environmental factors. Also physiological factors such as hormones, cytokines and growth factors, as well as diseases, contribute to the variability between individuals. Alterations in expression levels of P450s may arise as a consequence of changes in synthesis, degradation or a combination of these processes (4, 12). In the absence of a crystal structure of a eukaryotic, membrane-bound P450 enzyme, inferences about the structure of mammalian P450s active sites has been drawn from the crystal structures of several soluble bacterial enzymes (13). However, recently the first structure of a mammalian P450 was published (14). The rabbit CYP2C5 protein was crystallized after modifications to the membrane-binding portion of the enzyme (14, 15). The availability of a crystal structure of a mammalian P450 will undoubtedly increase the knowledge of how substrates bind and interact with the of human P450s.

Eicosanoid biosynthesis

The eicosanoids are a family of structurally related mediators with a wide range of biological functions in man and animals (16). They are derived from cis-polyunsaturated C20 fatty acids, primarily arachidonic acid (20:4n-6), but also from (20:5n-3), dihomo-γ- (20:3n-6), eicosatrienoic acid “mead” (20:3n-9) and eicosatetraenoic acid (20:4n-3). These fatty acids are obtained from food intake or from biosynthesis of their precursors, linoleic, α-linolenic and oleic acids. Arachidonic acid and the other eisosanoid precursors are components of cellular membranes. They are esterified preferentially at the sn-2 position of various in the membranes. Upon cell stimulation, they are liberated from the membrane phospholipids via activation of lipid-cleaving enzymes, such as (17). The free fatty acid can either be re-incorporated into the cellular membrane or converted into bioactive by the eicosanoid metabolizing enzymes (16, 18). There are at least three major pathways involved in the formation of eicosanoids (Fig. 2).

1. The prostaglandin H synthase pathway, leading to the formation of prostaglandins (PG) and

prostacyclin (PGI2) (collectively called prostanoids) and (TX). 2. The lipoxygenase pathway, leading to the formation of hydroperoxy and hydroxy fatty acids, and (LT).

3. The cytochrome P450 pathway, leading to the formation of hydroxy and epoxy fatty acids.

10 Membrane phospholipids

Phospholipases

Arachidonic acid Cytochrome P450s

Prostaglandin H synthases

HPETEs EETs HETEs HETEs Leukotrienes Prostaglandins Thromboxanes Prostacyclin

Fig. 2. Metabolism of arachidonic acid via the three major pathways. HETE, hydroxyeicosatetra- enoic acid, and EET, .

Prostaglandin H synthase pathway

PGH synthases (sometimes also referred to as ) are bifunctional heme- containing enzymes, which catalyze the initial two steps in the biosynthesis of prostaglandins and thromboxanes (19). There exist two different isoforms of PGH synthase. PGH synthase-1 is constitutively expressed in many mammalian cells and tissues and is considered to be responsible for the formation of prostaglandins and thromboxanes involved in the regulation of physiological functions. PGH synthase-2 is considered to be mainly an inducible enzyme that is expressed in low levels under basal conditions, but is strongly induced in response to inflammatory stimuli. The PGH synthases are important pharmacological targets for and other non-steroidal anti-inflammatory drugs (19). Prostanoids of the first, second and third series are formed by PGH synthase from dihomo- γ-linolenic, arachidonic and eicosapentaenoic acids, respectively (16). Arachidonic acid is converted by PGH synthases to the hydroxyperoxy prostaglandin endoperoxide G2 (PGG2), which is subsequently reduced to the hydroxy prostaglandin endoperoxide H2 (PGH2) (Fig. 3). The conversion of arachidonic acid to PGG2 by PGH synthases can also lead to the formation of the side products 11R-HETE and 15-HETE (20). The biological active compounds PGE2, PGF2α, PGD2, PGI2 and TXA2 are formed from PGH2 by isomerization catalyzed by various tissue- specific synthases (Fig. 3). The prostaglandins and thromboxanes are involved in many physiological and pathophysiological functions such as, blood pressure regulation, blood platelet

11 COOH

Arachidonic acid

O COOH PGHS-1 O PGHS-2 OOH PGG2

O O COOH COOH COOH CYP8A CYP5A O O O HO OH OH TXA OH PGH2 2

PGI2 PGDS PGFS PGES O HO HO COOH COOH COOH HO HO O OH OH OH PGE 2 PGF α PGD2 2

Fig. 3. The PGH synthase pathway. PGHS, PGH synthase; PGES, PGE synthase; PGFS, PGF synthase; PGDS, PGD synthase. aggregation, inflammation, reproduction and cancer. The prostaglandins and thromboxanes mainly mediate their effects by the stimulation of G-coupled prostanoid receptors (21).

The enzymes catalyzing the formation of TXA2 and PGI2 are two P450 enzymes. CYP5A (TXA2 synthase) of platelets and CYP8A (PGI2 synthase) of the vascular endothelium rearrange PGH2 into TXA2 and PGI2, respectively. These two P450 enzymes catalyze isomerization reactions and do not require NADPH (22). CYP5A also converts PGH2 to 12-hydroxyhepta- decatrienoic acid (12-HHT) and malondialdehyde. Recently, it was shown that human drug- metabolizing P450 and liver microsomes also could catalyze the formation of 12-HHT and malondialdehyde from PGH2 (23).

Lipoxygenase pathway

Lipoxygenases constitute a family of , which catalyze stereospecific insertion of molecular oxygen into polyunsaturated fatty acids (24, 25). In mammals, the lipoxygenases are divided into three major groups, 5-, 12-, and 15-lipoxygenases, depending on their positional specificity of arachidonic acid oxygenation. In addition, an 8-lipoxygenase has been identified in skin (26).

12 5-Lipoxygenases oxygenate arachidonic acid to 5S-hydroperoxyeicosatetraenoic acid (5S-

HPETE). 5S-HPETE can either be reduced to 5S-HETE by or converted to LTA4 by 5-lipoxygenases. LTA4 can be further metabolized to LTB4, LTC4, LTD4 and LTE4, which are potent mediators in allergy and inflammation (25, 27). 12-Lipoxygenases mainly metabolize arachidonic acid, depending on the isoform, to 12S-HPETE or 12R-HPETE, while 15- lipoxygenases form 15S-HPETE (25, 28, 29). These HPETEs can further be reduced to their corresponding hydroxy compounds (HETEs). The formation of HPETEs and HETEs by lipoxygenases occurs in many different kind of cells, but the biological roles of these products generated by 8-, 12-, and 15-lipoxygenases are largely unknown. However, numerous biological activities have been implicated for individual HPETEs and HETEs (25). The lipoxygenases can further convert HPETEs into biologically active products, such as, , , 15-epi- lipoxins, and di- and tri- H(P)ETEs (25). HETEs can also be formed by the cytochrome P450 pathway (30, 31).

Cytochrome P450 pathway

ω-Oxidation of fatty acids was first described in the 1930’s, many years before the cytochrome P450 system was discovered (32). Oxygenation of arachidonic acid by P450 was first reported in 1969. Sih et al. showed that arachidonic acid could be converted by a fungal root pathogen of wheat into the monohydroxy metabolites, 19-HETE and 18-HETE (33). But it was not until 1981 that the role of mammalian P450s in the oxidative metabolism of arachidonic acid was demonstrated (34-39). Since then detailed characterization of NADPH-dependent P450 metabolism of arachidonic acid has been carried out (30, 31, 40). P450 can oxygenate arachidonic acid by one or more of the reactions shown in Fig. 4.

Fig. 4. Reactions catalyzed by the cytochrome P450 pathway of arachidonic acid metabolites. Only the primary oxygenation products are shown.

13 The biological significance of the arachidonic acid metabolites formed by the P450 pathway remains to be fully understood (30, 31, 41). Many studies have suggested that the metabolites have a wide range of physiological implications. They appear to act primarily within the cells of origin and do not need to be extruded into the extracellular space to stimulate membrane receptors. Within the cell they have been proposed to be involved in the regulation of ion channels and transporters and to act as mitogens. In whole animal physiology, they have been implicated in the mediation of peptide release, regulation of vascular tone and regulation of volume homeostasis. Several studies have indicated that some of the biological effects mediated by P450 metabolites of arachidonic acid are PGH synthases-dependent (30, 31, 41). Some of these effects might be due to modulation of the biosynthesis of prostanoids. P450 metabolites of arachidonic acid can be substrates for PGH synthases. 20-HETE and

5,6-EET can be converted by PGH synthase to 20-hydroxy-PGH2 and 5,6-epoxy-PGH1, respectively (42-44). Both of these compounds can further be metabolized to bioactive prostanoids. 8,9-, 11,12-, and 14,15-EET lack the necessary double bonds to be converted to prostanoids by PGH synthases, but they might undergo abortive PGH synthase reactions or can inhibit the enzyme. It has been shown that 8,9-EET can be hydroxylated at C11 and C15 by PGH synthase (45). 18-HETE and 19-HETE can be converted into prostanoids by PGH synthase, but they are considered to be poor substrates (33). Whether endogenous pools of arachidonic acid metabolites generated by P450 are transformed by PGH synthase in vivo is not known. The individual P450 isoforms that oxygenate arachidonic acid have been studied in microsomes isolated from different human and animal tissues, and by recombinant or purified enzymes (30, 31, 40). In microsomes, correlation analysis and inhibition studies have been used to determine which P450 isoform is primarily responsible for the formation of a certain metabolite. Experiments involving comparisons of stereochemistry of endogenous metabolites with those formed by recombinant or purified enzymes have been used to determine which isoform catalyzes the reaction in vivo. In plants, fungi and algae, arachidonic acid occurs in small amounts and thus eisosanoids are usually not formed (16). Instead, polyunsaturated acids such as (18:2n-6) and α- linolenic acid (18:3n-3), which occur in large amounts, are converted into bioactive products by different oxidative pathways. Linoleic and α-linolenic acids can be oxidized by P450 to monohydroxy and epoxy products through mechanisms similar to those for arachidonic acid (40).

Hydroxylation of the ω-side chain

Arachidonic acid ω- and (ω-1)- have been demonstrated in microsomes isolated from many different tissues (30, 31). Many studies have shown that P450s belonging to the CYP4A subfamily are the predominant fatty acid ω/(ω-1)-hydroxylases in most mammalian

14 tissues including the kidney and liver (30, 31, 41). However, the CYP4A enzymes usually metabolize saturated fatty acids such as at much higher rates than arachidonic acid (46). Thus, the CYP4A enzymes probably play an important role in cellular fatty acid homeostasis. It is in the kidney that the formation of 20-HETE and 19-HETE is best characterized, as they are most prevalent there and have several postulated functional roles in renal physiology (31, 41). Microsomes from human kidney cortex convert arachidonic acid mainly to 20-HETE, which has been proposed to be involved in renal ion transport and vascular tone. 20-HETE and a glucuronide conjugate of 20-HETE have been identified in human urine (47). It has been reported that 16R-, 18R-, and 19-HETE exert vasodilator effects and that 16S-, and 17S-HETE inhibit renal proximal tubular Na+/K+ATPase activity (48). ω-Side chain HETEs can be stored in the pool in the rabbit kidney, and released in response to angitotesin II stimulation (48). Recently, it was shown that human PMNL contained endogenous pools of 16-HETE and 20-HETE (49). The P450 isoforms that are responsible for the in vivo formation of ω-side chain HETEs in man have not been identified. Both CYP4A11 and CYP4F2 have been implicated to form 20- HETE in vitro, but the participation of isoform in the 20-HETE formation is controversial (50- 52). This might be due to the usage of different recombinant expression systems, or the existence of polymorphic or closely related isoforms of CYP4A11 and CYP4F2. Several P450s belonging to the CYP1-3 families have been shown to oxidize arachidonic acid at the ω-side chain in vitro (53). However, most of these isoforms oxidize arachidonic acid with less regioselectivity than the enzymes belonging to the CYP4 family. In microsomes of monkey seminal vesicles, arachidonic acid is almost exclusively converted to 18R-HETE by an unidentified P450 (54).

Epoxidation

Arachidonic acid has four double bonds all of which P450 can oxygenate to cis- (EETs). These EETs can further be hydrated by to dihydroxyeicosatrienoc acids (DHETs) (30, 31). The EETs/DHETs have been detected in many tissues and body fluids, including human liver, kidney, heart, plasma and urine. In rat liver, a majority of EETs are esterified to cellular glycerophospho-lipids (55). Racemic EETs can be formed non- enzymatically, but strong evidence for enzymatic formation of endogenous EETs has been provided by chiral analysis of EETs in phospholipids. It has been proposed that EETs play a role in the structure and hence the function of cellular membranes (30, 31). Many studies have indicated that EETs are involved in regulation of vascular tone and might serve as an endothelium- derived hyperpolarization factor (31, 56-58). EETs might also to play a role in vascular inflammation (59) and be involved in transcriptional regulation of PGH synthase-2 and P450 (60- 62).

15 Studies with recombinant P450 enzymes have demonstrated that several P450s catalyze the formation of EETs with regio- and stereochemical selectivity (31). However, no mammalian P450 enzyme has so far been shown to only catalyze the formation of one single epoxide of arachidonic acid. Most of the arachidonic acid are members of the CYP2 family. CYP2C8 has been identified as a prominent in human liver, whereas CYP2J2 has been identified as an epoxygenase in different human extrahepatic tissues (63-66). The two epoxides of linoleic acid can be further converted to the pro-, leukotoxin and isoleukotoxin, in mammalian cells and have therefore attracted some biological attention (67).

Bisallylic hydroxylation

Arachidonic acid has four double bonds and three bisallylic carbons in positions 7, 10 and 13. These bisallylic carbons can be hydroxylated by P450 (40). The bisallylic hydroxy metabolites are chemically unstable at acidic pH, which is commonly used for extractive isolation of fatty acid metabolites. At acidic pH the bisallylic HETEs will undergo a non-enzymatic rearrangement to cis-trans conjugated HETEs (68, 69). The formation of bisallylic HETEs and 11-hydroxyoctadecadienoic acid (HODE) (the corresponding bisallylic product of linoleic acid) has been studied in liver microsomes of treated with different kinds of P450 inducers (68-71). Liver microsomes prepared from rats treated with phenobarbitol converted arachidonic acid to 7- HETE, 10-HETE and 13-HETE, and linoleic acid to 11-HODE (68, 69). Steric analysis revealed that these metabolites were almost racemic. Mechanistic studies have shown that 11-HODE is formed by hydrogen abstraction from C11 followed by oxygen insertion with retention of configuration (68). , which is an inducer of the CYP3A isoforms, efficiently and selectively increased the bisallylic hydroxylation activity in rat liver microsomes (71). Rats treated with inducers of other P450s isoforms, e.g. CYP1A also increased the bisallylic hydroxylation activity (71). This indicates that the formation of bisallylic hydroxy metabolites can be catalyzed by rat CYP3A isoforms, but that other P450 isoforms may also possess this activity. The formation of 13-HETE and 11-HODE has been detected in microsomes of human liver (70). The human P450 isoforms that are involved in the formation of bisallylic hydroxy fatty acids have not been studied. However, it has been reported that human CYP1A2, CYP2C8 and CYP2C9 formed large amounts of cis-trans conjugated HETEs following acidic isolation (53). In this study, it is likely that bisallylic HETEs decomposed to cis-trans conjugated HETEs during the acidic extractive isolation. Whether the bisallylic HETEs are formed in vivo and whether they exert biological effects is not known.

16 Hydroxylation with double bond migration

Hydroxylation with double bond migration leads to enzymatic biosynthesis of cis-trans conjugated hydroxy fatty acids (30, 31). Arachidonic acid can be converted by P450 to six cis- trans conjugated HETEs (5-, 8-, 9-, 11-, 12-, and 15-HETE). Cis-trans conjugated HETEs can also be formed non-enzymatically by acid-catalyzed rearrangement of bisallylic HETEs (69), by the lipoxygenase pathway (25) or by PGH synthase (20). The mechanism of formation has been investigated in microsomes of -treated rats with linoleic acid stereo-specifically deuterated at C11 as substrate (68). Linoleic acid was converted to the cis-trans conjugated hydroxy metabolites 9-HODE (80% R) and 13-HODE (85% R) by initial hydrogen abstraction at C11 with subsequent double bond migration and oxidation at C9 or C13. The human P450 isoforms, which catalyzing for the formation of these metabolites have not been well characterized. Previous studies on the formation of HETEs by P450, which have used acidic extractions, may have partly overlooked the stereoselectivity and the quantitative importance of the cis-trans conjugated HETEs. Cis-trans conjugated hydroxy fatty acids have been implicated as signal molecules involved in the regulations of genes (72, 73). 12R-HETE is formed in human lesions and might have important inflammatory effects (74). Studies have demonstrated that 12R-HETE is an + + agonist for the LTB4- and might exert inhibitory effects on Na /K ATPase activity (75, 76). P450 enzymes of human liver microsomes form 12-HETE with stereospecificity (>90% 12R-HETE) (70). Recombinant CYP2C9 has been implicated to form 12-HETE, but the stereo- specificity of this product was not been determined (53). The recent finding of a human lipoxygenase that oxygenate arachidonic acid at C12 with selectivity for the R enatiomer (28, 29) has called into question the role that P450 may play in the in vivo generation of 12R-HETE. The formation of 12R-HETE in psoriasis lesions is probably catalyzed by a 12R-lipoxygenase (28, 29). Whether P450 enzymes catalyze the formation of cis-trans conjugated HETEs in vivo from endogenous pools of arachidonic acid is not known. However, intact human lung vasculature has been shown to contain 5-, 8-, 9-, 11-, and 12-HETE (77). Whether any of these products are formed by P450 is not known.

Eicosanoid metabolism

Studies identifying the urinary metabolites of eicosanoids have shown that eicosanoids can undergo several catabolic pathways in various combinations in vivo (78-82). In general, the metabolism of eicosanoids leads to the formation of an inactive compound or a compound with reduced biological activity. Prostaglandins often undergo dehydrogenation of their 15-hydroxyl group, which can be followed by reduction of the ∆13 double bond. The formed compounds 15- keto-13,14-dihydroprostaglandins as well as the prostaglandins can also be metabolized by one or

17 two steps of ß-oxidation and ω-hydroxylation. Subsequent oxidation of ω-hydroxyprostaglandins may lead to the formation of compounds and finally to ω-carboxylic metabolites. After this step has taken place, one or two steps of ß-oxidation can also occur at this end of the prostaglandin compound (81, 82). LTB4 and stable thromboxanes can also be metabolized by ß- oxidation and ω-oxidation. In addition, many eicosanoids have been shown to undergo (ω-1)- hydroxylation. Several rabbit P450 enzymes belonging to the families 1-4 have been shown to catalyze ω- side chain hydroxylations of eicosanoids in vitro (83-88). The NADPH-dependent P450 reactions are much slower compared to the reactions catalyzed by other enzymes involved in the biosynthesis of eicosanoids, and therefore the ω/(ω-1)-hydroxylation of eicosanoids are generally considered to be mainly a catabolic pathway. However, the possibility cannot be excluded that some ω/(ω-1)-hydroxylated eicosanoids can have specific biological effects. Several enzymes belonging to the subfamilies CYP4A and CYP4F have been shown to catalyze ω/(ω-1)-hydroxylations of eicosanoids (83, 86, 89-92). CYP4A4 of rabbit lung catalyzes

ω-hydroxylation of several prostaglandins with low Km values (86). CYP4A4 is highly induced during pregnancy or treatment with (88). Hydroxylations of prostaglandins also occur in the seminal vesicles of man, primate and ovine and may be of physiological relevance (93, 94). The P450 enzymes catalyzing these reactions in the seminal vesicles are probably under the regulation of sex hormones (95, 96). These prostaglandin hydroxylases have not yet been identified.

The human enzyme CYP4F3 catalyses ω-hydroxylation of LTB4 with a very low Km value (0.7 µM) (89). The CYP4F3-catalyzed ω-hydroxylation of LTB4 in polymononuclear leukocytes is considered to be a major catabolic pathway of LTB4 in these cells (97). The other human CYP4F enzyme that has been characterized is CYP4F2 (90). CYP4F2, which is expressed in human liver and kidney, also catalyzes the ω-hydroxylation of LTB4, but with a much higher Km value (45 µM). CYP4F2 is considered to be involved in the inactivation of LTB4 in the liver. In rat polymononuclear leukocytes, LTB4 undergo (ω-1)- and (ω-2)-hydroxylations catalyzed by an unidentified P450 enzyme (98).

Seminal prostaglandins

Prostaglandins were discovered in human semen in the 1930’s by Goldblatt and von Euler (99-103). In human seminal vesicles von Euler found a related substance, which he designated

“vesiglandin” (102, 103). Vesiglandin likely consisted of 19R-hydroxy-PGE1 and 19R-hydroxy- PGE2, which later were found to be the major prostaglandins of human seminal fluids (104-106).

Other prostaglandins that have been identified in human semen are PGE1, PGE2, PGE3, PGF1α, 18 19 PGF2α, as well as 18-hydroxy-PGE, 19-hydroxy-PGF, ∆ -PGE, and ∆ -PGE of the first and second series (107-112). The dominant prostaglandins are 19R-hydroxy-PGE and PGE of the

18 first and second series. The total concentration of prostaglandins in human seminal plasma approaches millimolar levels, giving a concentration some 10,000 times higher than that found at the site of inflammation. Men have an average ratio of 19R-hydroxy-PGE to PGE of about 4:1 (105, 113). However, this ratio varies considerably among men. Ratios of 19R-hydroxy-PGE to PGE between 0.4-30 have been observed in different human semen samples (111-115). A majority of men can be defined as “rapid” hydroxylators with 19R-hydroxy-PGE to PGE ratios above 2.5 (114). Fig. 5 shows a mass-chromatogram of the 19R-hydroxy-PGEs (~73%) and PGEs (~27%) in a sample from human semen.

Fig. 5. LC-MS chromato- gram of seminal PGE com- pounds in human semen. The PGE compounds were detected as their correspon- ding PGB compounds. The chromatogram shows selec- tive ion monitoring (SIM) of the intervals m/z 349-351 and m/z 333-335.

Exposure of sperm to prostaglandins is not required for in vitro fertilization (116). It therefore seems likely that seminal prostaglandins contribute to fertility by ensuring maximum efficiency in vivo, but the mechanism is largely unknown. There have been implications of a correlation of the composition of PGE compounds in semen and fertility (113, 117). However, due to the wide range of prostaglandins in fertile men, any significant correlation between fertility and the composition of PGE compounds in semen has not been determined (115). Seminal PGE compounds may have immunosuppressive actions in the female genital tract, induce tolerance to sperm antigens, promote sperm survival and contribute to the acrosome reaction (118-120). 19R- hydroxy-PGE is pharmacologically active and has been shown to act as a selective agonist of one of the PGE receptors (EP2) (119, 121). However, any distinct effects of 19R-hydroxy-PGE, which differ significantly from PGE’s own contribution to fertility have so far not been convincingly determined. Targeted disruption of PGH synthase and prostanoid receptor genes has shown that prostanoids are of physiological importance in rodent reproduction (21, 122, 123).

19 The seminal prostaglandins are formed by PGH synthase of seminal vesicles (124), but the mechanism of biosynthesis of 19R-hydroxy-PGE has not been resolved. It seems likely that the 19R-hydroxylation of prostaglandins is catalyzed by a P450 in the seminal vesicles, but the enzyme has not been characterized and cloned. Microsomes of human seminal vesicles only slowly metabolize PGE2 to 19-hydroxy-PGE2, which differs from the rapid biosynthesis of 19- hydroxy-PGE in vivo (93, 105). Many properties of the prostaglandin 19-hydroxylase can be deduced from the analysis of PGE compounds in semen. The prostaglandin 19-hydroxylase would be expected to oxygenate at C19 and to some extent at C18, whereas the ∆18- and ∆19-PGE compounds might be formed by P450 catalyzed desaturations (108, 109). Semen analysis also suggests that the enzyme catalyze the formation 19-hydroxyprostaglandins of the first and second series equally well. In addition, the activity of prostaglandin 19-hydroxylase must be closely linked to that of PGH synthase, since the ratio of 19R-hydroxy-PGE to PGE changes little in ejaculates obtained at long or short time intervals (93). Semen from monkeys also contains large amounts of PGE compounds. The ratio of 19- hydroxy-PGE to PGE in semen from monkeys is about 100:1 (125). In ovine seminal vesicles, a presumably related hydroxylation of prostaglandins occurs (94, 96). Ovine semen contains large amounts of PGE and 20-hydroxy-PGE. Microsomes of ovine seminal vesicles metabolize PGE2 into 20-hydroxy-PGE2 slowly with a Km value of about 0.1 mM (96). Most other species studied have relatively low concentrations of prostaglandins in their semen (111).

20 AIMS

The cytochrome P450 enzyme system is involved in the oxygenation of a wide range of physiologically important fatty acids. The general objectives of this thesis were to investigate the cytochrome P450-catalyzed formation of bisallylic hydroxy metabolites of arachidonic and linoleic acids, and to study the mechanism of biosynthesis of hydroxyprostaglandins in human and ovine semen.

Specific aims of the investigations were to:

1. Study the bisallylic hydroxylation of arachidonic and linoleic acids with human cytochrome P450 enzymes. (papers I and II)

2. Evaluate LC-MS as a method for identifying P450 metabolites of arachidonic and linoleic acid. (paper II)

3. Identify gene expression of a putative prostaglandin 19-hydroxylase (CYP4F8) in human seminal vesicles. (paper III)

4. Characterize the catalytic properties of CYP4F8 and to compare them with microsomes isolated from human seminal vesicles. (paper IV)

5. Clone and characterize a novel enzyme of the CYP4F subfamily in ovine seminal vesicles. (paper V)

21 COMMENTS ON METHODOLOGY

Analysis of metabolites

Reversed phase-high performance liquid chromatography (RP-HPLC) was used for separation of fatty acid metabolites. In paper I, [14C]-labeled arachidonic and linoleic acids were used and the formation of metabolites was monitored by an on-line radioactivity detector in combination with an on-line UV-detector. The identification of [14C]-labeled metabolites was based on the retention times of authentic standards. The monohydroxy metabolites of arachidonic acid that did not resolve well on RP-HPLC were further analyzed by straight phase-HPLC. Stereochemical analysis of HETEs was performed by different chiral HPLC methods. UV- absorption was mainly used for monitoring internal standards, which were used for estimation of recovery during extractions. In papers II, IV and V, liquid chromatography-mass spectrometry (LC-MS) was mainly used for identification and quantification of metabolites. The metabolites were separated on RP-HPLC and the effluent first passed by an UV-detector and then subjected to negative ion electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) (126, 127) in an ion trap mass spectrometer (LCQ, TermoQuest) with MSn capacity. The fragmentation of the metabolites in the mass spectrometer is influenced by the position of the oxygen substituents and the metabolites were identified based on their specific MS2 spectra.

However, several ω- and (ω-1)-hydroxy metabolites of prostaglandins and PGH2 analogues had both similar retention times and MS2 spectra. In those cases, the structural identification was elucidated with gas chromatography-mass spectrometry (GC-MS) (ITS40 ion trap mass spectrometer, Finnigan, MAT). The metabolites were methylated and converted to trimethylsilyl ethers and were then identified with GC-MS based on their C-values (number of apparent carbons) and their specific MS spectra (128). The C-values were determined from the retention times of fatty acid methyl esters. Metabolite formation was estimated by LC-MS, using standard curves of authentic standards or parent compounds, or by percent conversion of substrate. The UV-detector was used for monitoring internal standards.

Degenerate primers

Degenerate primers used for identification of CYP4F8 and CYP4F21 were designed based on conserved regions of 18 human, rat and rabbit cDNA sequences of the CYP4A, CYP4B and CYP4F subfamilies (Fig. 6). Multiple alignments using the GCG program (Seq Web version 1.0, Wisconsin package) revealed three conserved regions in the CYP4 family genes. One conserved region was found to be located between nucleotides ~1290-1425 (from the starting codon). This region is highly conserved in all P450 enzymes. It codes for the amino acids around the residue that forms a thiolate bond with the heme-group (13). Alignment analysis revealed two

22 additional conserved regions, which were located between nucleotides ~930-1020 and ~390-480. The degenerate primers 5’-CTIMGIGCIGARGYIGAYAC-3’, 5’-CCRCTRGYYGTGGTGTC RTG-3’, 5’-CCAYTWYRACATYCTGAARYC-3’ and 5’-TKICCIATGCARTTCCKIGVYCC- 3’ were designed on these three conserved regions.

CYP4A1rat CTA CGT GCT GAG GTG GAC AC CYP4A1rat TTC CCA ATG CAG TTC CTC GCT CC CYP4A2rat --G --- --A ------CYP4A2rat ------T --- -- CYP4A3rat --G --- --A ------CYP4A3rat ------T --- -- CYP4A4rab --C --C --C ------CYP4A4rab --- --G ------GT --- -- CYP4A5rat --C --C --C ------CYP4A5rab ------GT --- -- CYP4A6rat --C --C --C ------CYP4A6rab --- --G ------GT -G- -- CYP4A7rat --C --C --C ------CYP4A7rat --- --G ------A- -- CYP4A8rat --G ------A --- --T -- CYP4A8rat ------T --- -- CYP4A11hum --C ------CYP4A11hum ------T -A- -- CYP4B1hum --C --G --- --A ------CYP4B1hum -G------G -GC -- CYP4B1rat --C --G --- --A ------CYP4B1rat -G- --G --- --A --- --G -GC -- CYP4B1rab --C --C --- --A ------CYP4B1rab -G- --G ------G -GC -- CYP4F1rat A-C A-A --A --- -CC --- -- CYP4F1rab -GA --T ------G -GC -- CYP4F2hum A-- A-A --A --A -CT --- -- CYP4F2hum -G------G -GC -- CYP4F3hum A-- A-A --A --A -CT --- -- CYP4F3hum -G------G -GC -- CYP4F4rat A-T A-A ------CT --- -- CYP4F4rat -GT --T ------G -GC -- CYP4F5rat A-C --G --A --- -CT --- -- CYP4F5rat -GT --T ------G -GC -- CYP4F6rat A-C --A --A --- -CT --- -- CYP4F6rat -GT --T ------G -G- -- 5' CTI MGI GCI GAR GYI GAY AC 3' 5' TKI CCI ATG CAR TTC CKI GVY CC

Fig. 6. Design of two degenerate primers used for amplification of CYP4F8 and CYP4F21. Abbreviations: hum, human; rab, rabbit. Letters used: I= inosine; M= A or C; R= A or G; Y= C or T; K= G or T; V= A, C or T.

Recombinant cytochrome P450

P450s have been expressed in many different systems, such as mammalian cells, bacteria, cells and yeast (129). CYP4F8 and CYP4F21 were expressed in a yeast system, whereas the other recombinant P450 enzymes used in the study were purchased from Gentest Corp. (Woburn, MA). CYP1A2, CYP2A6, CYP2B6, CYP2D6 and CYP2E1 were expressed in a human lymphoblastic cell line, whereas CYP2C8, CYP2C9, CYP2C19, and CYP3A4 were co-expressed with NADPH-cytochrome P450 reductase in insect cells. The expression levels, as well as the activity of the recombinant enzymes differ depending on isoform and cell system. This makes it difficult to draw conclusions from the rate of formation of metabolites formed by different isoforms. Additional experimentation with the determination of kinetic parameters and comparisons with biosynthesis in microsomes might be needed to determine which P450 isoform is primarily responsible for the formation of a certain metabolite. Saccharomyces cerevisiae has been used for expression of many P450 isoforms. It is in many ways a rather suitable system because it is easy to manipulate and expresses low background levels of P450. It is a eukaryotic cell and heterologous P450s are incorporated into the ER (130). The S. cerevisiae strain W(R), which has been genetically modified to overexpress the yeast NADPH-cytochrome P450 reductase (131) was used for expression of CYP4F8 and CYP4F21. A galactose inducible in the expression plasmid and in the yeast genome was used to control the expression of the P450s and the NADPH-cytochrome P450 reductase.

23 PGH2 and stable PGH2 analogues

The prostaglandin endoperoxides are unstable in aqueous solutions. In buffer, PGH1 and PGH2 decompose with a half-life of ∼5 min at 37°C (132). PGH2 will mainly decompose to

PGD2, PGE2, PGF2α and 12-HHT, and PGH1 will decompose to the corresponding prostaglandins of the first series. Hydroxy metabolites of PGH1 and PGH2 will decompose to hydroxyprostaglandins in the same way. To confirm that the hydroxyprostaglandins, formed during incubations with CYP4F8 and microsomes from human seminal vesicles, originated from hydroxy-PGH2, chemical reduction with SnCl2 was utilized (132). SnCl2 will reduce PGH2 compounds to their corresponding PGF2α compounds, whereas PGE2 and PGD2 compounds are unaffected. PGH2 was incubated without any pre-incubation time. After 2 minutes incubation, one-half of the incubation was terminated with buffered SnCl2, and the other half was terminated with . The formation of hydroxy-PGE2 and hydroxy-PGF2α compounds was then analyzed by LC-MS using SIM of their carboxylated anions.

PGH1 and PGH2 were stored in at –80°C. Acetone was found to inhibit the activity of CYP4F8, and therefore the volume of acetone was reduced by evaporation under a flow of N2 prior to incubation start. This handling of PGH2 in combination with the fact that microsomal fractions of seminal vesicles and yeast may contain compounds and enzymes that interfere with the stability of PGH2 led to problems with estimations of kinetic parameters. Due to these analytical problems, three stable PGH2 analogues were used as model substrates in many experiments. Fig. 7 shows the chemical structure of PGH2 and the three stable PGH2 analogues U44069, U-46619 and U-51605.

O H2C COOH COOH

O O OH PGH2 OH U-46619

O N COOH COOH

H2C N OH U-44069 U-51605

α α Fig. 7. Chemical structure of PGH2, (15S)-Hydroxy-[9 ,11 -epoxymethano]prosta-5,13(Z,E)- dienoic acid (U-44069), (15S)-hydroxy-[11α,9α-epoxymethano]prosta-5,13(Z,E)-dienoic acid (U- 46619), [9α,11α-diazo]prosta-5,13(Z,E)-dienoic acid (U-51605).

24 RESULTS

Bisallylic hydroxylation of fatty acids (papers I and II)

The formation of monohydroxylated products from arachidonic and linoleic acid was studied with human and rat liver microsomes, and with human recombinant CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. To avoid non- enzymatic decomposition of bisallylic hydroxy compounds, the metabolites were extracted at neutral pH. CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 were found to convert linoleic acid to the bisallylic hydroxy metabolite 11-HODE and to the cis-trans conjugated hydroxy metabolites 9-HODE and 13-HODE as major monohydroxy metabolites. In addition, CYP1A2 and CYP2C19 converted linoleic acid to large amounts of 17-HODE. The formation of 11-HODE was studied in human liver microsomes with specific chemical inhibitors towards CYP1A2, CYP3A4/7, and CYP2C8/9. CYP1A2 inhibitors reduced the bisallylic hydroxylation activity by about 50%, whereas inhibitors of CYP3A4/7 and CYP2C8/9 were without effect on this activation. CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 were also found to convert arachidonic acid into bisallylic hydroxy metabolites. CYP1A2 and CYP3A4 formed 7-HETE, 10- HETE and 13-HETE as major monohydroxy compounds. CYP1A2 also formed large amounts of 19-HETE and 18-HETE, while CYP3A4 only formed trace amounts of ω-side chain hydroxy metabolites. CYP2C8 and CYP2C9 formed 13-HETE and 10-HETE together with cis-trans conjugated HETEs. CYP2C19 formed 19-HETE as the main metabolite with relatively little biosynthesis of other HETEs. CYP2A6, CYP2B6, CYP2D6 and CYP2E1 did not form any bisallylic hydroxy metabolites of arachidonic and linoleic acids. The formation of HETEs by CYP2C9 was studied in detail. CYP2C9 metabolized arachidonic acid to 19-HETE, 15R-HETE (72% R enatiomer), 13S-HETE (90%), 12R-HETE (>95%), 11R-HETE (57%) and 10-HETE. To examine the mechanism of HETE formation, CYP2C9 was incubated with arachidonic acid under oxygen-18 gas. LC-MS analysis showed that all HETEs contained oxygen from air in the same amount. This indicates that 15-HETE, 12- HETE and 11-HETE are formed enzymatically and not from decomposition of 13-HETE and 10- HETE. Human liver microsomes converted arachidonic acid to 13-HETE as the major bisallylic hydroxy metabolite. Steric analysis of 13-HETE formed by human adult liver microsomes and recombinant P450 showed that CYP1A2 and CYP2C9 formed 13-HETE with similar stereochemical selectivity as adult human liver microsomes (Table 1). High concentrations of 7,8- benzoflavone have been shown to augment the enzyme activity of CYP3A4 (133). Incubation with adult human liver microsomes in the presence of 7,8-benzoflavone (100 µM) stimulated the bisallylic hydroxylation activity. Human fetal liver microsomes possessed a relatively prominent bisallylic hydroxylation activity. Interestingly, adult and fetal human liver microsomes appeared

25 to form 13-HETE with different chirality (Table 1). Studies with inhibitors towards CYP1A2, and CYP3A4/7 did not interfere with the bisallylic hydroxylation activity in microsomes isolated from fetal liver.

Table 1. Steric analysis of 13-HETE. 13-HETE Enzyme / Microsomes S R CYP1A2 75 25 CYP2C9 90 10 CYP3A4 50 50 Adult HL 81a 19a Adult HL + αNF (100 µM) 60 40 Fetal HL 22b 78b Dex RL 50 50 HL; human liver, αNF; 7,8-Benzoflavone, DexRL; liver from rats treated with dexamethasone aAverage data from three different subjects bAverage data from two different subjects.

Dexamethasone and are known to induce enzymes belonging to the CYP3A subfamily in rat liver (134). Liver microsomes isolated from rats treated with these drugs showed an increased bisallylic hydroxylation activity compared to liver microsomes isolated from control rats. Microsomes from rats treated with dexamethasone formed 13-HETE with racemic stereochemistry. Inhibitors of CYP3A efficiently decreased the bisallylic hydroxylation activity in these microsomes.

Identification of fatty acid metabolites with LC-MS (paper II)

The fragmentation of monohydroxy, dihydroxy and epoxy fatty acids in the mass spectrometer is influenced by the position of the oxygen substituents. Previous work has shown that cis-trans conjugated HETEs, EETs and cis-trans conjugated HODEs will form characteristic fragments during MS2 analysis (135-141). Similar MS2-spectra of these compounds were observed in this study. In addition, metabolites of arachidonic and linoleic acids with hydroxyl groups in the ω-side chain and at bisallylic carbons, as well as epoxy and dihydroxy metabolites of linoleic acid and DHETs were analyzed. The dihydroxy, epoxy, and the bisallylic monohydroxy metabolites showed characteristic fragments of high intensity, whereas the ω-side chain metabolites showed rather uncharacteristic fragments. Nevertheless, monohydroxy metabolites with their hydroxyl group within the ω-side chain showed at least one characteristic signal in the upper mass range that could be used for identification. The ionization methods, APCI and ESI, yielded similar results.

26 Gene expression of P450s in human seminal vesicles (paper III)

In pursuit of identifying the prostaglandin 19-hydroxylase, gene expression of P450s in human seminal vesicles (n=4) was studied with RT-PCR. mRNA of CYP1B1, CYP2E1, CYP2J2, CYP3A5, and CYP4B1 were detected in all subjects. The gene expression of CYP4B1 was analyzed in detail. Sequencing analysis revealed two different forms of the gene, which were expressed in all four subjects. Both the previously published form of CYP4B1 (142, 143) and a novel form with an insertion of three nucleotides coding for an extra serine in position 207, were detected. RT-PCR using degenerate primers for the CYP4 family yielded a novel cDNA sequence, which was derived from a previously reported genomic sequence on 19 (Lamerdin et al., unpublished data, accession number AD000685). cDNA cloning using RT-PCR showed that the deduced amino acid sequence consisted of 520 amino acids. The deduced amino acid sequence was similar to the human sequences of CYP4F2 (90) and CYP4F3 (89) with 81% and 77% amino acid identities, respectively. This novel P450 was designated CYP4F8 by the P450 nomenclature committee. The gene was detected by RT-PCR in all subjects investigated. RT-PCR analysis also indicated that the gene was highly expressed in the seminal vesicle tissue. Gene expression of CYP4F8 was also detected in human prostate glands and human liver. However, a higher number of cycles were needed to generate positive detection of CYP4F8 in these tissues. Northern blot analysis suggested that the gene had two transcripts. A 3’-rapid amplification of cDNA ends method showed that this was probably due to usage of two different polyadenylation sites.

Catalytic properties of CYP4F8 (paper IV)

CYP4F8 was expressed in a yeast system and its catalytic properties were investigated.

Recombinant CYP4F8 oxygenated arachidonic acid to 18R-HETE, whereas PGD2, PGE1, PGE2,

PGF2α and LTB4 appeared to be poor substrates. Three stable PGH2 analogues (U-44069, U- 46619 and U-51605) were rapidly metabolized by (ω-1)- and (ω-2)-hydroxylations. U-44069 -1 -1 was oxygenated with a Vmax of ∼260 pmol min pmol P450 and a Km of ∼7 µM.

PGH2 is an unstable compound that decomposes in buffer to PGD2, PGE2, PGF2α and 12- HHT. Hydroxy metabolites of PGH2 will decompose to hydroxy metabolites of prostaglandins in the same way. Recombinant CYP4F8 was incubated with PGH2 and the formation of prostaglandins and hydroxylated prostaglandins was analyzed with LC-MS (Fig. 8). The first eluting product was identified as 19-hydroxy-PGF2α, whereas the second peak mainly contained 19-hydroxy-PGE2, but trace amounts of 19-hydroxy-PGD2 were detected on the right shoulder of this peak. 18-Hydroxy-PGE2 was identified in the third eluting peak. PGF2α, PGE2 and PGD2 eluted after 18, 24 and 29 min, respectively. The formation of 18-hydroxyprostaglandins was about 17% of total hydroxyprostaglandins.

27 Fig. 8. LC-MS chromatogram of hydroxyprostaglandins and prostaglandins formed during an incubation of CYP4F8 with PGH2. The chromatogram shows SIM of the intervals m/z 367- 369 and m/z 351-353.

Additional experiments with chemical reduction of hydroxy-PGH2 metabolites with SnCl2 confirmed that PGH2 was metabolized to 19-hydroxy-PGH2 and 18-hydroxy-PGH2 (Fig. 9). LC- MS analysis showed that a sample reduced with SnCl2 mainly formed two peaks containing 19- hydroxy-PGF2α and 19-hydroxy-PGE2, while the control sample, terminated with ethanol, mainly formed one peak containing 19-hydroxy-PGE2. Small amounts of 18-hydroxy-PGE2 were also present in both chromatograms and small amounts of 19-hydroxy-PGF2α were identified in the sample reduced with SnCl2. PGH1 was similarly metabolized by CYP4F8 to 19-hydroxy-PGH1 and 18-hydroxy-PGH1.

Fig. 9. LC-MS chromato- gram of hydroxyprosta- glandins formed during an incubation of CYP4F8 with PGH2 when terminated with SnCl2. Left, sample reduced with SnCl2. Right, control sample terminated with ethanol. The chro- matogram shows SIM of the interval m/z 367-369.

Oxygenation of arachidonic acid, U-44069, U-46619, U-51605 and PGH2 was mirrored when incubated with microsomes isolated from human seminal vesicles. The relative amounts of

28 18-hydroxy-PGEs and 19-hydroxy PGEs were analyzed in human seminal fluids. 18-Hydroxy- PGE compounds were found to be present to about 8-10 % of total hydroxy-PGE. In addition, 18-HETE could not be identified in human seminal fluids with LC-MS analysis.

CYP4F21 in ovine seminal vesicles (paper V)

RT-PCR using degenerate primers for the CYP4 family revealed expression of a novel CYP4F gene in ovine seminal vesicles. The full coding region of the gene was cloned using RT- PCR. The deduced amino acid sequence consisted of 528 amino acids. The deduced amino acid sequence was similar to the CYP4F8 sequence with 74 % identity. The P450 nomenclature committee named this novel P450 enzyme CYP4F21. CYP4F21 was expressed in yeast and its catalytic properties were examined. The expression of CYP4F21 in yeast microsomes was too low for chromophore detection at 450 nm with spectrophotometry. Nevertheless, yeast microsomes with recombinant expressed CYP4F21 possessed NADPH-dependent activities that were not present in the control microsomes. CYP4F21 metabolized three stable PGH2 analogues (U-44069, U-46619 and U- 51605) to their 20-hydroxy metabolites. PGE2 was a relatively poor substrate, but detectable amounts of a 20-hydroxy-PGE2 metabolite were formed. PGE1 and PGF2α were only converted into trace amounts of hydroxyprostaglandins.

CYP4F21 was also assayed for activity towards PGH1 and PGH2. Very short incubation times in combination with SnCl2 reduction were utilized in order to distinguish between hydroxylation of PGH1/PGH2 and PGE1/PGE2. Attempts to demonstrate hydroxylation of PGH1 and PGH2 were not successful. However, LC-MS analysis revealed that PGH2 was transformed into four products. Analysis of their MS2 spectra and comparison with authentic standards revealed that the four products were likely to be stereoisomers of 5-hydroxy-PGI1. Microsomes of ram seminal vesicles and NADPH metabolized all three PGH2 analogues and PGE2 in the same way as CYP4F21. It is therefore likely that CYP4F21 is expressed in the ovine seminal vesicle tissue.

29 DISCUSSION

Bisallylic hydroxylation of fatty acids (Papers I-II)

Bisallylic hydroxylation requires the bisallylic carbons to be positioned near the active site of the enzyme. It follows that the two adjacent double bonds will also be in the vicinity of the heme-bound reactive oxygen. During the hydroxylation reaction, one of the bisallylic hydrogens is abstracted from the bisallylic carbon and oxygen is inserted with retention of configuration (68). Hydrogen abstraction from a bisallylic carbon requires less energy than abstraction of other hydrogens from other carbons of the fatty acid (144). Alternatively, the reactive oxygen may react with one of the double bonds or with the allylic carbon after migration of the double bond to cis-trans configuration. Linoleic acid is structurally similar to arachidonic acid and was therefore mainly used as a model substrate. CYP1A2, CYP2C8, CYP2C9, CYP2C19 and CYP3A4 were found to metabolize linoleic and arachidonic acids into bisallylic hydroxy metabolites. Effects of inhibitors on the formation of 11-HODE suggest that CYP1A2 contributes to the formation of the bisallylic hydroxy metabolite of linoleic acid in adult liver microsomes. Steric analysis of 13-HETE, the major bisallylic hydroxy HETE formed from arachidonic acid, revealed that CYP1A2 formed this metabolite with similar stereospecificity as microsomes from adult liver microsomes. It seems possible that CYP1A2 also contributes to the formation of 13-HETE in adult liver microsomes. However, other enzymes such as CYP2C9 and CYP2C8 may also be involved in the formation of 13-HETE, whereas CYP3A4 probably is of minor importance. Human fetal liver microsomes possessed a relatively prominent bisallylic hydroxylation activity. Inhibitors of CYP1A2 and CYP3A4/7 had no effect on the formation of 11-HODE from linoleic acid in fetal liver microsomes. Steric analysis of 13-HETE revealed that adult and fetal liver microsomes formed this compound with different chirality. These data suggest that the bisallylic hydroxylation activity in fetal liver microsomes may be catalyzed by one or several P450 enzymes that have not been included in this study. The finding that the CYP3A enzymes seemed not to contribute to the bisallylic hydroxylation under normal conditions was unexpected, since CYP3A4 and CYP3A7 are major P450 enzymes in the adult and fetal liver, respectively. CYP2C9 was found to convert arachidonic acid to 12R-HETE by hydroxylation with double bond migration (Fig. 10). Biosynthesis of 13R-HODE from stereospecifically deuterated linoleic acid occurs by abstraction of the pro-S hydrogen at C11 followed by suprafacial oxygen insertion at C13 (68). It is likely that CYP2C9 forms 12R-HETE by a similar mechanism, by abstraction of the pro-S hydrogen at C10, followed by double bond migration and suprafacial oxygen insertion at C12. CYP2C9 was the first enzyme reported to form 12R-HETE. The formation of 12R-HETE has attracted some interest due to its proposed biological effects (30, 31). It was thought that 12R-HETE was exclusively produced by P450, since all l2-lipoxygenases known at that time were selective for 12S-HETE. However, since then 12R-lipoxygenases as well

30 as other P450 enzymes, have been found to catalyze the formation of this compound (28, 29, 145). The contribution of P450s and 12-lipoxygenases to the in vivo biosynthesis of 12R-HETE in different tissue merits further studies.

HH

R α R ω 10 Arachidonic acid

8 9 11 12

CYP2C9 H α ω R • R

10

8 9 11 12

α H ω α H ω R R R R CYP2C9 10 10 • 8 9 11 12 8 9 11 12 O H 12R -HETE

Fig. 10. In vitro biosynthesis of 12R-HETE generated by CYP2C9.

Paper II

GC-MS has been widely used for structural analysis and quantification of eicosanoids. GC- MS analysis often requires methylation and derivatization prior to analysis. This is time consuming and not suitable for many unstable compounds. Recently, LC-MS has been developed into a powerful technique due to new ionization methods, such as ESI and APCI. While many mass spectra of eicosanoids obtained by GC-MS are available (146), mass spectra of eicosanoids obtained by LC-MS analysis are so far inadequate. LC-MS was used to register MS2 spectra of carboxylate anions of roughly all P450 monohydroxy-, epoxy-, and dihydroxy- metabolites of arachidonic and linoleic acids. All metabolites yielded characteristic MS2 spectra that could be used for identification. LC-MS was also used to identify fatty acid metabolites in complex mixtures formed by recombinant P450. LC-MS with MS2 was found to be very useful for rapid identification of fatty acid metabolites in complex mixtures formed by cytochrome P450. LC-MS may also be used for quantitative purposes. However, in experiments with radioactivity monitoring of metabolites and with LC-MS analysis we noted somewhat different apparent amounts of the metabolites. This finding was not unexpected, since MacPherson et al. (138) have shown that carboxylate anions of cis-trans conjugated HETEs have different MS response factors. For quantitative analysis of metabolites with LC-MS, standard curves for each compound may therefore be needed. Quantification of isobaric metabolites that do not resolve on RP-HPLC can be difficult, but these metabolites can be accomplished by separation by straight phase HPLC and radioactivity monitoring. Since the elution order of most P450 metabolites on HPLC is

31 known (69), LC-MS may be used to for identification of metabolites of interest and then complemented with individual analysis of the metabolites in a suitable chromatographic system.

Biosynthesis and metabolism of seminal prostaglandins (papers III-V)

Paper III

RT-PCR using degenerate primers based on conserved regions of mammalian CYP4 family genes, revealed expression of a novel P450 gene in the seminal vesicles of man. cDNA cloning of the gene, which was derived from a previously reported genomic sequence on , showed that the deduced amino acid sequence was similar to other enzymes in the CYP4F subfamily. The gene was named CYP4F8. RT-PCR analysis suggested that CYP4F8 was highly expressed in the seminal vesicle tissue. The CYP4F subfamily was discovered in rat hepatic tumors by Chen and Hardwick in 1993 (147). Additional enzymes were soon found in rat and man (89, 90, 148). There are at least five CYP4F genes that code for functionally enzymes in the human genome. In addition to CYP4F2, CYP4F3 and CYP4F8, two human CYP4F cDNA sequences designated CYP4F11 (Strobel and Cui, unpublished data) and CYP4F12 (Bylund, J., Bylund, M. and Oliw, E. H, manuscript in preparation), have been reported (8). The catalytic properties of CYP4F11 and CYP4F12 have not yet been published. In addition, CYP4F22, which seems to code for a functional P450 protein, and eight CYP4F pseudo genes have been identified in the genomic DNA databases (8). An alignment of human CYP4F enzymes is shown in Fig. 11 and their deduced amino acid sequence identity is shown in Table 2.

Table.2. Amino acid identity of human CYP4F enzymes. 4F2 4F3 4F8 4F11 4F12 4F22a 4F2 100 87 81 86 82 64 4F3 100 77 82 78 63 4F8 100 79 78 59 4F11 100 82 61 4F12 100 61 4F22a 100

aThe amino acid sequence of CYP4F22 has been deduced from a draft sequence of the genomic clone AC011492.

Alignment of the human CYP4F enzymes shows that the enzymes share a very high . However, there are large differences in the residue region 67-114, which represents exon III (149, 150). Detailed studies of the CYP4F2 and CYP4F3 genes have shown that a region

32 with high homology to exon III of CYP4F2 exists in intron II of the CYP4F3 gene and vice versa (149-151). Recently, Christmas et al. showed liver-specific expression of a functionally distinct CYP4F3 isoform (151), which consisted of an alternative exon III, coding for the residues 67-114. Alternative splice forms of other CYP4F enzymes have not been observed.

Fig. 11. Alignments of the deduced amino acid sequences of human CYP4F2, CYP4F3, CYP4F8, CYP4F11 and CYP4F12. Residues conserved in all sequences are shaded.

33 RT-PCR analysis of P450 expression in the seminal vesicles revealed a novel variant of the human CYP4B1. This gene contained an insert of three nucleotides (AGC), which coded for an extra serine at position 207 (CYP4B1Ser207). cDNA cloning of the full coding region revealed no other differences compared to the previously reported sequences of CYP4B1 (142, 143). Strangely, all CYP4B1 amplicons, generated from four different subjects, contained a mixture of CYP4B1 and CYP4B1Ser207. The region coding for residue 207 is not associated with any splicing site (143) and it is therefore not likely that it originates from . Whether CYP4B1Ser207 is a polymorphic form of CYP4B1 or if it is generated from a distinct gene is not known. CYP4B1 is mainly expressed in extrahepatic tissue (152). The CYP4B1 genes are highly conserved among different species. Recombinant rabbit CYP4B1 has been shown to catalyze ω- hydroxylation of several short and medium fatty acids as well as hydrocarbons (153). However, the catalytic activity of human CYP4B1 has not been successfully demonstrated. This is probably due to an amino acid change, relative to other mammalian CYP4B1 enzymes, located in the so-called meander region (152). Whether the human CYP4B1Ser207 codes for a functional P450 protein is not known.

Paper IV

The catalytic properties of recombinant CYP4F8 and microsomes of human seminal vesicles were investigated. Recombinant CYP4F8 was found to efficiently metabolize PGH1 and PGH2 to 19-hydroxy-PGH1 and 19-hydroxy-PGH2, respectively. Metabolic studies of different fatty acids and prostaglandins formed by CYP4F8 and microsomes of seminal vesicles suggest that CYP4F8 is expressed in the seminal vesicle tissue and that PGH1 and PGH2 are likely to be its endogenous substrates. CYP4F8 can thus be designated PGH 19-hydroxylase. CYP4F8 is the first enzyme shown to hydroxylate prostaglandin endoperoxides.

Due to the instability of PGH1 and PGH2, three stable PGH2 analogues were used as model -1 substrates. The PGH2 analogue U-44069 was oxygenated with a Vmax of ∼260 pmol min pmol P450-1. This is one of the fastest mammalian P450-catalyzed hydroxylation reactions described, especially considering that microsomal fractions of CYP4F8 were used and not a purified enzyme in a reconstituted system. U-44069 was oxygenated with a Km of ∼7 µM. For comparison, PGH synthases oxygenate arachidonic acid with a similar Km value (~5 µM). Human semen contains the 19R-stereoisomers of 19-hydroxy-PGEs. It remains to be determined whether CYP4F8 oxidizes PGH1 and PGH2 to their 19R-stereoisomers. However, in collaboration with Dr. Hamberg at the Karolinska Institute, we have found that the PGH2 analogue U-51605 was hydroxylated by CYP4F8 at C19 to the 19R-stereoisomer at about 90% (unpublished data). Analysis of prostaglandins in human semen suggests that the 19R-hydroxy-

PGE and PGE are formed enzymatically, as no non-enzymatic breakdown products of PGH2 and PGH1, e.g. PGD compounds, have been detected. The rapid biosynthesis of 19R-hydroxy-PGE1

34 and 19R-hydroxy-PGE2 in vivo suggests that the activities of PGH synthase, PGH 19- hydroxylase and PGE synthase are closely linked. It is generally assumed that PGH synthase-1 is the enzyme involved in the production of

PGH1 and PGH2 in human seminal vesicles, since PGH synthase-1 is expressed in high levels in ovine seminal vesicles (154). However, constitutive expression of PGH synthase-2 has been shown to occur in the male reproductive system of rat. (155). PGH synthases are integral membrane in the ER and nuclear membranes (156). In the ER, the active sites of PGH synthases have been located to the lumen side (157-159). The substrate channel of PGH synthase-1 faces the ER. Its entrance is surrounded by the membrane-binding surface, which does not extend beyond one leaflet of the biolayer. Mammalian P450 enzymes are also integral membrane proteins, but their catalytic domains are located on the cytosolic side of the ER (160).

PGH2 must cross the lipid bilayer to gain access to CYP4F8 (Fig. 12). Mammalian P450s are anchored to the ER by their NH2-terminal side and hydrophobic compounds can probably also enter and exit the active site of P450 directly from the biolayer (14, 160).

Fig. 12. Proposed metabolism of arachidonic acid in the ER of human seminal vesicles. PGHS; PGH synthase.

PGE synthase is probably also associated with the same membrane system (161, 162). Immunoprecipitation studies have shown that there are at least two different enzymes in ovine seminal vesicles that are capable of catalyzing PGE isomerization reactions (161). Recently, two human -dependent PGE synthases were identified and characterized. A membrane bound PGE synthase, which is induced by inflammatory stimuli, has been proposed to be functionally coupled to PGH synthase-2 (163, 164). The second human PGE synthase, which is a cytosolic enzyme, is constitutively expressed and may be functionally coupled to PGH synthase-1 (165). Interestingly, a functional coupling between PGH synthase-1 and the

35 membrane bound PGE synthase seems to occur when a high concentration of arachidonic acid is available (164). Studies of the human membrane bound PGE synthase, overexpressed in a cell line, showed that it co-localized with perinuclear PGH synthases (164). Whether any of these two human PGE synthases are expressed in the human seminal vesicles is not known. However, a specific antibody for the membrane bound PGE synthase has been shown to cross-react with partially purified PGE synthase from ovine seminal vesicles (163). The coupling between the activities of PGH synthase, PGH 19-hydroxylase and PGE synthase in human seminal vesicles merits further investigation.

A proposed mechanism of the in vivo biosynthesis of 19R-hydroxy-PGE1 and 19R- hydroxy-PGE2 in human seminal vesicles is shown in Fig. 13. Alternative biosynthesis pathways of 19R-hydroxy-PGE, i.e.19R-hydroxylation of PGE or PGH synthase conversion of 19R-HETE and 19R-hydroxyeicostrienoic acid, are very unlikely.

Fig. 13. Proposed mechanism of in vivo biosynthesis of 19R-hydroxy-PGE1 and 19R-hydroxy-PGE2 in human seminal vesicles.

There is marked inter-individual variation of 19R-hydroxy-PGE and PGE compounds in normal human seminal fluid (110, 111). This may be due to variable expression of CYP4F8 or due to isoforms with reduced enzyme activity. Whether polymorphic forms of CYP4F8 exist is not known. Molecular characterization of the mechanism leading to inter-individual variation of 19R- hydroxyprostaglandins in semen may be important in future investigation of infertility.

36 NADPH-dependent P450 enzymes have, in general, low turnover numbers and are therefore considered to be not particularly well suited for oxidation of unstable substrates.

Unstable eicosanoids like PGH1 and PGH2 have therefore not been fully investigated as substrates for these P450s. The fact that the hydroxylated prostaglandin endoperoxides are further metabolized to bioactive prostaglandins is an interesting observation. It shows that the biosynthesis of prostaglandins is linked to the P450 system. This mechanism may not be unique to CYP4F8, as other P450 enzymes may also have the capacity to hydroxylate PGH1 and PGH2, which subsequently could undergo isomerization by tissue-specific eicosanoid synthases and form hydroxylated prostaglandins with specific biological effects. The expression of CYP4F8 in tissues other than the seminal vesicles should be determined, as well as the hydroxylation of prostaglandin endoperoxides by other P450s.

Paper V

The mechanism of biosynthesis of 20-hydroxy-PGE1 and 20-hydroxy-PGE2 in the ovine seminal vesicles is unknown. A novel gene, CYP4F21, was cloned from sheep seminal vesicles. Its deduced amino acid sequence showed 74% identity with that of CYP4F8. CYP4F21 was expressed in yeast, but the yield of recombinant enzyme was very low. However, by using a high concentration of microsomal protein and long incubation times, recombinant CYP4F21 was found to metabolize three stable PGH2 analogues to their ω-hydroxy metabolites. PGE2 appeared to be a relatively poor substrate, but detectable amounts of 20-hydroxy-PGE2 were formed. Attempts to demonstrate hydroxylation of PGH1 and PGH2 were not successful. This might be due to the low expression of CYP4F21 in combination with the instability of PGH1 and PGH2. During incubations of PGH2 with CYP4F21, four stereoisomers of 5-hydroxy-PGI1 were formed. The mechanism of biosynthesis of these 5-hydroxy-PGI1 compounds was not further investigated. It seems unlikely that CYP4F21 would catalyze 20-hydroxylation of three stable analogues of PGH2 and not PGH2 itself. PGH2 is an unstable compound, and in combination with a high concentration of microsomal protein, our in vitro conditions may not mimic in vivo synthesis. Further studies will be needed to clarify this issue. Ovine seminal vesicles contain large amounts of PGH synthase-1 and PGE synthase (154, 161, 163). Catalytic studies of microsomes of ram seminal vesicles suggest that CYP4F21 also is present in the seminal vesicle tissue. It is possible that 20-hydroxy-PGE1 and 20-hydroxy-PGE2 are formed in the ovine seminal vesicles by similar mechanisms to those that have been proposed for the 19-hydroxyprostaglandins in human seminal vesicles.

37 CONCLUSIONS

1. Several human liver P450 enzymes possess bisallylic hydroxylation activity towards arachidonic and linoleic acids. Inhibition studies and stereochemical analysis of bisallylic hydroxy metabolites suggest that CYP1A2 contributes to the formation of the bisallylic hydroxy metabolites in adult human liver microsomes.

2. Liquid chromatography-mass spectrometry with MS2 was found to be a powerful method for rapid identification of fatty acid metabolites in complex mixtures formed by cytochrome P450.

3. A novel cytochrome P450 gene was found to be highly expressed in human seminal vesicles. The full coding region of the gene was cloned, sequenced and designated CYP4F8.

4. Recombinant CYP4F8, expressed in a yeast system, was found to catalyze (ω-1)- hydroxylation of prostaglandin endoperoxides. Catalytic studies carried out in microsomes isolated from human seminal vesicles suggest that CYP4F8 is expressed in the human seminal

vesicles tissue. 19R-hydroxy-PGE1 and 19R-hydroxy-PGE2, the major prostaglandins in human semen, are likely to be formed in the seminal vesicles by CYP4F8-catalyzed 19R-

hydroxylation of PGH1 and PGH2 followed by PGE synthase-catalyzed isomerization.

5. A novel cytochrome P450 was cloned from ovine seminal vesicles, sequenced and designated

CYP4F21. Recombinant CYP4F21 catalyzed ω-hydroxylation of PGH2 analogues, suggesting

that 20-hydroxy-PGE1 and 20-hydroxy-PGE2 are formed in ovine seminal vesicles by similar mechanisms to those that have been proposed for the 19-hydroxyprostaglandins in human seminal vesicles.

38 ACKNOWLEDGEMENTS

This work was carried out at the Department of Pharmaceutical Biosciences, Division of Pharmacology, Faculty of Pharmacy, Uppsala University, Sweden. I would like to express my sincere gratitude to:

Prof. Ernst Oliw for being a great supervisor. My co-authors for their contributions to my thesis. Chao, Erica, Lena, Maria and Tina for making my time in Oliw’s lab enjoyable. All present and previous people at Division of Pharmacology as well as other friends at BMC for making my time at BMC enjoyable. Philip and Alex for proof-reading my thesis. The Swedish Pharmaceutical Society and IF’s stiftelse for giving me the opportunity to attend international conferences. The Swedish Society of Medical Research and Gunnar Hylténs minnesfond for financial support.

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