Cytochrome P450 enzymes in oxygenation of prostaglandin endoperoxides and arachidonic acid: Cloning, expression and catalytic properties of CYP4F8 and CYP4F21

Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy 231

_____________________________ _____________________________

Cytochrome P450 Enzymes in Oxygenation of

Prostaglandin Endoperoxides and Arachidonic Acid

Cloning, Expression and Catalytic Properties of CYP4F8 and CYP4F21

BY

JOHAN BYLUND

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

ABSTRACT

Bylund, J. 2000. Cytochrome 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.

Cytochrome P450 (P450 or CYP) is an enzyme system involved in the oxygenation of a wide range of endogenous compounds as well as foreign chemicals and drugs. This thesis describes investigations of P450-catalyzed oxygenation of prostaglandins, linoleic and arachidonic acids.

The formation of bisallylic hydroxy metabolites of linoleic and arachidonic acids was studied with human recombinant P450s and with human liver 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 fatty acid 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 genes, 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 yeast system. The human enzyme was designated CYP4F8 and the ovine enzyme was designated CYP4F21. Comparison of their deduced protein sequences showed that they had 74 % amino acid identity. Recombinant CYP4F8 oxygenated two prostaglandin endoperoxides (PGH

and PGH

) and three stable PGH

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-hydroxylation of PGH

and PGH

, 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

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 Rat 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 MS

. Anal. Biochem. 265, 55-68.

III. Bylund, J., Finnström, N. and Oliw, E. H. (1999) Gene 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.

TABLE OF CONTENTS

INTRODUCTION ………. 7

Cytochrome P450 ...………. 7

Reactions ………8

Expression and substrate specificity ...……….. 9

Eicosanoid biosynthesis ...……… 10

Prostaglandin H synthase pathway ..……….. 11

Lipoxygenase 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 metabolism ...………. 17

Seminal prostaglandins ..………. 18

AIMS ………. 21

COMMENTS ON METHODOLOGY ……….. 22

Analysis of metabolites ...………... 22

Degenerate primers……….. 22

Recombinant cytochrome P450 ...………... 23

PGH

and stable PGH

analogues ……….…. 24

RESULTS ………. 25

Bisallylic hydroxylation of fatty acids…...………. 25

Identification of fatty acid metabolites with LC-MS ………..………... 26

Gene expression 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

ABBREVIATIONS

APCI atmospheric pressure chemical ionization

CYP cytochrome P450

EETepoxyeicosatrienoic acid ER endoplasmic reticulum 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 PGI

prostacyclin

RP-HPLC reverse phase-high performance liquid chromatography RT-PCR reverse transcriptase-polymerase chain reaction

SIM selective ion monitoring TXA

thromboxane A

12-HHT 12-hydroxyheptadecatrienoic acid

INTRODUCTION

Arachidonic acid is a polyunsaturated fatty acid, which is present in most human and animal cells. Arachidonic acid can be converted into oxygenated metabolites, the so-called eicosanoids. The eicosanoids are involved in many physiological and pathophysiological functions such as blood pressure regulation, blood platelet aggregation, inflammation, reproduction and cancer. 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, asthma 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 heme-thiolate enzymes whose Fe

-carbon monoxide 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 bacteria, fungi, yeast, plants, insects, 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-activation and catabolism of endogenous compounds, such as fatty acids, steroid hormones,

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

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 vitamin D. 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 oxidases or monooxygenases, because they incorporate one atom of molecular oxygen 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.

The catalytic cycle begins with the substrate binding to the ferric form of the enzyme. The enzyme-substrate complex is then reduced by an electron transferred from NADPH via NADPH- cytochrome P450 reductase. Molecular oxygen binds to the reduced enzyme-substrate complex to form an enzyme-O

-substrate complex followed by the introduction of a second electron transferred from NADPH via NADPH-cytochrome P450 reductase or from cytochrome b

. 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 electrons 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 animals 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 human genome (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 drug-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 polymorphism of the corresponding P450

genes, as well as physiological, environmental, and pathological factors. Mutations 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

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 active site of human P450s.

Eicosanoid biosynthesis

The eicosanoids are a family of structurally related lipid mediators with a wide range of biological functions in man and animals (16). They are derived from cis-polyunsaturated C

fatty acids, primarily arachidonic acid (20:4n-6), but also from eicosapentaenoic acid (20:5n-3), dihomo- γ-linolenic acid (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 phospholipids in the membranes. Upon cell stimulation, they are liberated from the membrane phospholipids via activation of lipid-cleaving enzymes, such as phospholipase A

(17). The free fatty acid can either be re-incorporated into the cellular membrane or converted into bioactive lipids 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 (PGI

) (collectively called prostanoids) and thromboxanes (TX).

2. The lipoxygenase pathway, leading to the formation of hydroperoxy and hydroxy fatty acids, and leukotrienes (LT).

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

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

Prostaglandin H synthase pathway

PGH synthases (sometimes also referred to as cyclooxygenases) 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 aspirin 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 G

(PGG

), which is subsequently reduced to the hydroxy prostaglandin endoperoxide H

(PGH

) (Fig. 3).

The conversion of arachidonic acid to PGG

by PGH synthases can also lead to the formation of the side products 11R-HETE and 15-HETE (20). The biological active compounds PGE

, PGF

, PGD

, PGI

and TXA

are formed from PGH

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

Membrane phospholipids

Arachidonic acid

Prostaglandins Thromboxanes Prostacyclin

EETs HETEs HPETEs

HETEs Leukotrienes

Phospholipases

Cytochrome P450s H synthases

Lipoxygenases

Prostaglandin

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 TXA

and PGI

are two P450 enzymes. CYP5A (TXA

synthase) of platelets and CYP8A (PGI

synthase) of the vascular endothelium rearrange PGH

into TXA

and PGI

, respectively. These two P450 enzymes catalyze isomerization reactions and do not require NADPH (22). CYP5A also converts PGH

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 PGH

(23).

Lipoxygenase pathway

Lipoxygenases constitute a family of dioxygenases, 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 mouse skin (26).

COOH

COOH O

COOH O

O O

OH

O

OOH

HO COOH

HO HO

O HO

Arachidonic acid

HO COOH

COOH

COOH

COOH OH O

O

OH OH

OH O

OH PGG2

PGH2 PGI₂

TXA2

PGE₂

PGD2 PGF2α

PGHS-1 PGHS-2

CYP5A CYP8A

PGDS PGFS

PGES

5-Lipoxygenases oxygenate arachidonic acid to 5S-hydroperoxyeicosatetraenoic acid (5S- HPETE). 5S-HPETE can either be reduced to 5S-HETE by peroxidases or converted to LTA

by 5-lipoxygenases. LTA

can be further metabolized to LTB

, LTC

, LTD

and LTE

, 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, lipoxins, hepoxilins, 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.

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 hormone 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-PGH

and 5,6-epoxy-PGH

, 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 linoleic acid (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)-hydroxylations 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

tissues including the kidney and liver (30, 31, 41). However, the CYP4A enzymes usually metabolize saturated fatty acids such as lauric acid 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 phospholipid 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-epoxides

(EETs). These EETs can further be hydrated by epoxide hydrolases 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).

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 epoxygenases are members of the CYP2 family. CYP2C8 has been identified as a prominent epoxygenase 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-toxins, 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 rats 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). Dexamethasone, 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.

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 phenobarbital-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 psoriasis lesions and might have important inflammatory effects (74). Studies have demonstrated that 12R-HETE is an agonist for the LTB

-receptor 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 ∆

double bond. The formed compounds 15-

keto-13,14-dihydroprostaglandins as well as the prostaglandins can also be metabolized by one or

two steps of ß-oxidation and ω-hydroxylation. Subsequent oxidation of ω-hydroxyprostaglandins may lead to the formation of aldehyde 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). LTB

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 K

values (86). CYP4A4 is highly induced during pregnancy or treatment with progesterone (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 LTB

with a very low K

value (0.7 µM) (89). The CYP4F3-catalyzed ω-hydroxylation of LTB

in polymononuclear leukocytes is considered to be a major catabolic pathway of LTB

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 LTB

, but with a much higher K

value (45 µM). CYP4F2 is considered to be involved in the inactivation of LTB

in the liver. In rat polymononuclear leukocytes, LTB

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-PGE

and 19R-hydroxy- PGE

, 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 PGE

, PGE

, PGE

, PGF

,

PGF

, 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

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 (EP

) (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).

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 PGE

to 19-hydroxy-PGE

, 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 ∆

- and ∆

-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 PGE

into 20-hydroxy-PGE

slowly with a K

value of about 0.1 mM (96). Most other species studied

have relatively low concentrations of prostaglandins in their semen (111).

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)

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, [

C]-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 [

C]-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 MS

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 MS

spectra.

However, several ω- and (ω-1)-hydroxy metabolites of prostaglandins and PGH

analogues had both similar retention times and MS

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 cysteine

residue that forms a thiolate bond with the heme-group (13). Alignment analysis revealed two

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 CYP4A2rat --G --- --A --- --- --- -- CYP4A3rat --G --- --A --- --- --- -- CYP4A4rab --C --C --C --- --- --- -- CYP4A5rat --C --C --C --- --- --- -- CYP4A6rat --C --C --C --- --- --- -- CYP4A7rat --C --C --C --- --- --- -- CYP4A8rat --G --- --- --A --- --T -- CYP4A11hum --C --- --- --- --- --- -- CYP4B1hum --C --G --- --A --- --- -- CYP4B1rat --C --G --- --A --- --- -- CYP4B1rab --C --C --- --A --- --- -- CYP4F1rat A-C A-A --A --- -CC --- -- CYP4F2hum A-- A-A --A --A -CT --- -- CYP4F3hum A-- A-A --A --A -CT --- -- CYP4F4rat A-T A-A --- --- -CT --- -- CYP4F5rat A-C --G --A --- -CT --- -- CYP4F6rat A-C --A --A --- -CT --- -- 5' CTI MGI GCI GAR GYI GAY AC 3'

CYP4A1rat TTC CCA ATG CAG TTC CTC GCT CC CYP4A2rat --- --- --- --- --- --T --- -- CYP4A3rat --- --- --- --- --- --T --- -- CYP4A4rab --- --G --- --- --- -GT --- -- CYP4A5rab --- --- --- --- --- -GT --- -- CYP4A6rab --- --G --- --- --- -GT -G- -- CYP4A7rat --- --G --- --- --- --- -A- -- CYP4A8rat --- --- --- --- --- --T --- -- CYP4A11hum --- --- --- --- --- --T -A- -- CYP4B1hum -G- --- --- --- --- --G -GC -- CYP4B1rat -G- --G --- --A --- --G -GC -- CYP4B1rab -G- --G --- --- --- --G -GC -- CYP4F1rab -GA --T --- --- --- --G -GC -- CYP4F2hum -G- --- --- --- --- --G -GC -- CYP4F3hum -G- --- --- --- --- --G -GC -- CYP4F4rat -GT --T --- --- --- --G -GC -- CYP4F5rat -GT --T --- --- --- --G -GC -- CYP4F6rat -GT --T --- --- --- --G -G- -- 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, insect 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 promoter in the expression plasmid and in the yeast genome was

used to control the expression of the P450s and the NADPH-cytochrome P450 reductase.

PGH

and stable PGH

analogues

The prostaglandin endoperoxides are unstable in aqueous solutions. In buffer, PGH

and PGH

decompose with a half-life of ∼5 min at 37°C (132). PGH

will mainly decompose to PGD

, PGE

, PGF

and 12-HHT, and PGH

will decompose to the corresponding prostaglandins of the first series. Hydroxy metabolites of PGH

and PGH

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-PGH

, chemical reduction with SnCl

was utilized (132). SnCl

will reduce PGH

compounds to their corresponding PGF

compounds, whereas PGE

and PGD

compounds are unaffected. PGH

was incubated without any pre-incubation time. After 2 minutes incubation, one-half of the incubation was terminated with buffered SnCl

, and the other half was terminated with ethanol. The formation of hydroxy-PGE

and hydroxy-PGF

compounds was then analyzed by LC-MS using SIM of their carboxylated anions.

PGH

and PGH

were stored in acetone 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 N

prior to incubation start. This handling of PGH

in combination with the fact that microsomal fractions of seminal vesicles and yeast may contain compounds and enzymes that interfere with the stability of PGH

led to problems with estimations of kinetic parameters. Due to these analytical problems, three stable PGH

analogues were used as model substrates in many experiments. Fig. 7 shows the chemical structure of PGH

and the three stable PGH

analogues U44069, U-46619 and U-51605.

Fig. 7. Chemical structure of PGH

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

COOH

H

C

H

C

COOH O

OH

O COOH O

OH

U-44069

O

U-46619

OH

COOH

PGH ₂

N

U-51605

N

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

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.

Enzyme / Microsomes 13-HETE S R

CYP1A2 75 25

CYP2C9 90 10

CYP3A4 50 50

Adult HL 81

19

Adult HL + αNF (100 µM) 60 40

Fetal HL 22

78

Dex RL 50 50

HL; human liver, αNF; 7,8-Benzoflavone,

DexRL; liver from rats treated with dexamethasone

a

Average data from three different subjects

b

Average data from two different subjects.

Dexamethasone and erythromycin 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 MS

analysis (135-141). Similar MS

-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.

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 chromosome 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 PGD

, PGE

, PGE

, PGF

and LTB

appeared to be poor substrates. Three stable PGH

analogues (U-44069, U- 46619 and U-51605) were rapidly metabolized by ( ω-1)- and (ω-2)-hydroxylations. U-44069 was oxygenated with a V

of ∼260 pmol min

pmol P450

and a K

of ∼7 µM.

PGH

is an unstable compound that decomposes in buffer to PGD

, PGE

, PGF

and 12-

HHT. Hydroxy metabolites of PGH

will decompose to hydroxy metabolites of prostaglandins in

the same way. Recombinant CYP4F8 was incubated with PGH

and the formation of

prostaglandins and hydroxylated prostaglandins was analyzed with LC-MS (Fig. 8). The first

eluting product was identified as 19-hydroxy-PGF

, whereas the second peak mainly contained

19-hydroxy-PGE

, but trace amounts of 19-hydroxy-PGD

were detected on the right shoulder of

this peak. 18-Hydroxy-PGE

was identified in the third eluting peak. PGF

, PGE

and PGD

eluted after 18, 24 and 29 min, respectively. The formation of 18-hydroxyprostaglandins was

about 17% of total hydroxyprostaglandins.

Fig. 8. LC-MS chromatogram of hydroxyprostaglandins and prostaglandins formed during an incubation of CYP4F8 with PGH

. The chromatogram shows SIM of the intervals m/z 367- 369 and m/z 351-353.

Additional experiments with chemical reduction of hydroxy-PGH

metabolites with SnCl

confirmed that PGH

was metabolized to 19-hydroxy-PGH

and 18-hydroxy-PGH

(Fig. 9). LC- MS analysis showed that a sample reduced with SnCl

mainly formed two peaks containing 19- hydroxy-PGF

and 19-hydroxy-PGE

, while the control sample, terminated with ethanol, mainly formed one peak containing 19-hydroxy-PGE

. Small amounts of 18-hydroxy-PGE

were also present in both chromatograms and small amounts of 19-hydroxy-PGF

were identified in the sample reduced with SnCl

. PGH

was similarly metabolized by CYP4F8 to 19-hydroxy-PGH

and 18-hydroxy-PGH

.

Fig. 9. LC-MS chromato- gram of hydroxyprosta- glandins formed during an incubation of CYP4F8 with PGH

when terminated with SnCl

. Left, sample reduced with SnCl

. 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 PGH

was mirrored

when incubated with microsomes isolated from human seminal vesicles. The relative amounts of

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 PGH

analogues (U-44069, U-46619 and U- 51605) to their 20-hydroxy metabolites. PGE

was a relatively poor substrate, but detectable amounts of a 20-hydroxy-PGE

metabolite were formed. PGE

and PGF

were only converted into trace amounts of hydroxyprostaglandins.

CYP4F21 was also assayed for activity towards PGH

and PGH

. Very short incubation times in combination with SnCl

reduction were utilized in order to distinguish between hydroxylation of PGH

/PGH

and PGE

/PGE

. Attempts to demonstrate hydroxylation of PGH

and PGH

were not successful. However, LC-MS analysis revealed that PGH

was transformed into four products. Analysis of their MS

spectra and comparison with authentic standards revealed that the four products were likely to be stereoisomers of 5-hydroxy-PGI

.

Microsomes of ram seminal vesicles and NADPH metabolized all three PGH

analogues

and PGE

in the same way as CYP4F21. It is therefore likely that CYP4F21 is expressed in the

ovine seminal vesicle tissue.