Cytochrome
P450
JORGE
H.
and the arachidonate
CAPDEVILA,’ J. R. FALCL,1 AND RONALD
cascade1
W. ESTABROOK
of aMlice and Biochemistr)#{231} Vanderbilt Universit Nashville, Tennessee 37232, USA; and of tMolecular Genetics and 1Biochemistry and the *Cecil H. and Ida Green Center for Reproductive Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
Departments
Departments Biology
Arachidonic acid and many products of the arachidonate cascade serve as substrates for cytochrome P450-mediated metabolism via allylic oxidation, omega hydroxylation, and epoxygenation as well as peroxide rearrangement. Defining the physiological importance of these metabolites is an area of intense research interest. Cytochrome P450-catalyzed reactions play prominent roles in multiplying the structural and functional diversity of the arachidonate metabolic cascade. Capdevila, H.; Falck, J. R.; Estabrook, R. W. Cytochrome P450 and the arachidonate cascade. FASEBJ. 6: 731-736; 1992.
ABSTRACT
J.
Key Words.prostanoids
P450 . arachidonate eicosanoids . epoxygenase hydroxyeicosatetraenoic acids omega oxidation
LIPID-DERIVED
of cell and significance noids and
MEDIATORS
PLAY
role
A CENTRAL
in the
control
cells have developed several oxygenases that incorporate regioand stereo-specific chemical information into fatty acids. Cells use such recognition signals to convey physiologically
relevant
for
the
intraand/or of arachidonic part, common
intercellular messages. Although acid oxidative metabolism is, to all fatty acids possessing the r-C = C -CH2-C = C -1 H H H H functionality,
most
I
requisite pentadienyl arachidonic acid is uAique terified
to
hormonally
transmembrane lipases,
the
in that
sensitive
signaling release
results
acid,
to a variety
to
and
for oxidative Cyclooxygenase
es-
subsequent
of physiologically metabolism generates
peroxide from which prostaglandins, prostacyclin are derived; the various tially
in vivo,
glycerolipid pools. Thus in the activation of specific
of arachidonic
idative metabolism metabolites (1). The pathways acid are multiple.
it is prese11t,
ox-
important
of arachidonic a labile endo-
thromboxanes, and lipoxygenases lead ini-
hydroperoxyeicosatetraenoic
acids,
which
in
turn
can be converted to leukotrienes, lipoxins, and hepoxilins; in addition, cytochrome P450 can serve as the catalyst for the biotransformation of arachidonic acid to a variety of oxygenated metabolites, including epoxides and a series of fatty acid
alcohols.
The
cade, highlighting of cytochrome
P450,
HISTORICAL In spite
in the years
main the
of the
arachidonate
or proposed
are outlined
in Fig.
cas-
of a few early the
initial
0892-h61R/q2/fl00E.fl7l
reports, description
1/401
cft
(
acid
P450’s
was
of its role
evidence was prethat cytochrome P450 is an active catalyst for the metabolism of arachidonic acid. Capdevila et a!. (5) demonstrated that purified preparations of cytochrome P450, sented
showing
with
purified
NADPH-P450
reductase
and
cytochrome b5, metabolized arachidonic acid to a series of more polar metabolites that were chromatographically distinct from prostaglandins. Oliw and Oates (6), Capdevila et a!. (7), and Morrison and Pascoe (8) reported almost simultaneously the oxidation of arachidonic acid to metabolites by an NADPH-dependent microsomal reaction. These studies heralded the introduction of the third pathway of arachidonic acid metabolism and introduced a number of abbreviations to the lexicon of arachidonic acid metabolites (9, 10). Until the middle 1970s, studies of the metabolism of arachidonic acid were dominated by those reactions catalyzed by cyclooxygenase. In 1979, and with the discovery of leukotrienes, emphasis shifted toward the study of mammalian lipoxygenases (1). In both cases, the biological (functional) signficance of the main metabolites was established before the biochemical characterization of the products and/or enzyme (or enzymes) involved. The physiological significance of these metabolites has been one of the driving forces responsible for the intensive research in this area. In contrast, studies of the functional relevance of the cytochrome P450-system to the metabolism of arachidonic acid started with a well-characterized enzyme system capable of actively metabolizing the fatty acid to several products of unknown physiological significance. The cytochrome P450 system was extensively studied after the demonstration in the early 1960s of its role in the in the metabolism of drugs
biosynthesis
of steroids
(11) and
and xenobiotics (12). Considerable research during the last few years has resulted in the isolation and structural characterization of many cytochrome P450 isozymes (16), and in the demonstration that distinct
macromolecules
provide,
in
many
in-
stances, the molecular basis for their catalytic heterogeneity. Current interest in defining a functional role for cytochrome P450 as a catalyst for the monooxygenation of arachidonic acid emanates from the pivotal physiological role played by
1.
cytochrome
of arachidonic
on the metabolism of several drugs. was not until 1981 that conclusive
structurally
contributions
PERSPECTIVE
metabolism after
features
established
acid It
reconstituted
organ physiology. Among these, the functional of arachidonic acid as the precursor of prostaleukotrienes is well established (1). Mammalian
the enzymology
Studies characterizing the spectral change occurring when arachidonic acid interacts with liver microsomal cytochrome P450 were reported in 1967 (2); little further work was done until 1976 when Cinti and Feinstein (3) demonstrated that microsomes prepared from human platelets contained cytochrome P450 and that the arachidonic acid-induced aggregation of platelets was inhibited by metyrapone and carbon monoxide, two recognized inhibitors of P450; also, about this time Pessayre et a!. (4) documented the effect of arachidonic
involvement
recognized
as an
oxygenase.
20
tDedicated
to MinorJ.
scientist, a warm
person,
Coon-a and
respected
a good
colleague,
a splendid
friend.
FAcIR
www.fasebj.org by UNIVERSITY OF TOLEDO LIBRARIES (18.218.56.169) on September 27, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
Cytochrome
P450 and
16-.17-,&18-OH
,.
01’IEGA-
-.::
i9-keto
HYDROXYLATION
--“
Cascade
Arachidonate
the
,
20-carboxyl
19-&20-OH
-
19- & 20-OH POs
glulathionc
Epoxy genase
-
adducts
5,6-oxygenated P05 vic-DHET5
LETs
oxid.
w/w-I
-
EET-Phosphoiipids diepoxides / epoxy-alcohols
/
Cyto. P450 AIIyI!c
/
Oxid. dihydro-HETEs HETEs
rARAcHuoNic (ACIo
-
wiw-i oxid.
lipoxins DIHETES
Lipoxygenase
HPEFES
--.::
-
glutathioneadduct
hepoxilin thoxilins Ailene oxide dihydro-LTB LTA
-
__I
Solid lines =
Figure
the substrate metabolites,
and as well
of the
significance 10 years
plosion of interest in the cytochrome olites of arachidonic acid. Their
This
article
will
roles
discuss of the
recent different
has
advances types
that
cytochrome
of its pharma-
of the cytochrome have witnessed an ex-
P450-generated role in many
reactions of biological significance area of intense, but often controversial, characterization
multiple
the potent biological activities as the established biochemical, last
metabimportant
developed research. in the
into
an
biochemical
of cytochrome
P450-
directed monooxygenase systems involved in the metabolism of arachidonic acid. Many of these reactions are summarized in Fig. 1, which also outlines additional cytochrome P450catalyzed reactions for the metabolism of eicosanoids and the rearrangment of (hydro)peroxides. Because some excellent reviews have summarized the potential physiological significance of these metabolites (1, 13, 14), we refer only briefly to their biological activities and physiological implications.
PROPERTIES ARACHIDONIC
OF
THE ACID
v,-.I c.
1QQ
-
P01
-
CY1DCHROME MONOOXYGENASE
P450
oxid.
-
w/w-l
-
w/w-I oxid. w/w-i oxid. w/w-i oxid.
LTC LTD
-
LTE
-
w/w-i/w-2 oxid. w/w-I oxid. P01
5,6-epoxy
HHT = hydroxyhepiadecalrienoic PG =prostagiandin P01 = prostacyciin
acid
TX = thromboxane
plays
in eicosanoid
metabolism.
liver microsomes are known to contain multiple cytochrome P450 isoforms and because many different metabolites were formed, the hypothesis was proposed that metabolite diversity resulted from catalysis by mutiple cytochrome P450s (15). Reconstitution studies, utilizing solubilized and purified components of the microsomal electron transport system, demonstrated conclusively that the metabolism of arachidonic acid was cytochrome P450 dependent and that metabolites of different polarity were formed, depending on the type of cytochrome P450 used (5, 13). The detailed structural characterization of the reaction products proved that during microsomal arachidonate metabolism cytochrome P450 can catalyze three reaction types. Allylic
oxidation
Allylic
oxidation
six regioisomeric acids (HETE5).2
is a lipoxygenase-like
reaction
that
affords
cis, trans-conjugated hydroxyeicosatetraenoic Stereochemical analysis demonstrated that
liver microsomal fractions catalyzed the formation of 5-, 8-, 9-, 11-, and 15-HETEs as nearly racemic mixtures whereas 12-HETE was formed enantioselectively as the R isomer (17). Although the contribution of cytochrome P450 to the
P450
Initial studies of arachidonate metabolism by microsomal fractions, isolated from the livers of phenobarbital treated rats, showed the rapid conversion of the fatty acid to many products (7). These studies demonstrated that the reaction was enzymatic, had an absolute requirement for NADPH, and was inhibited by typical cytochrome P450 inhibitors. As
7s)
TX5 HHT
=
description
cological, and toxicological P450 enzyme system. The
-
LET epoxyeicosatnenoic acid DHET = dihydroxyeicosatrienoic acid HYETE/HETE hydroperoxy/hydroxyeicosatelraenoi.acid LT = leukolrlene
cyto. P450or related enzymes other enzymes =
1. A comprehensive
P00 P0111
j
Cyclooxygenase
Dashed lines
LTB
Th
FAfFR
2Abbreviations: HETE, hydroxyeicosatetraenoic acid; EET, cisepoxyeicosatrienoic acid; /-NF’, fl-naphthoflavone; PB, phenobarbital. The nomenclature and designation of various cytochrome P450s follow the recommendations described by nebert et a!. (16).
lotirnal
CAPriC/ll
A
CT
Al
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pool of HETEs found in vivo remains uncertain, interest in this reaction has been encouraged by reports of: 1) the presence of 12(R)-HETE in human skin and its increased formation during psoriatic inflammation (18); 2) the potent activity of 12(R)-HETE as an inhibitor of Na/KATPase (13, 14); and 3) the formation of 12(R)-HETE after incubation of microsomes from cornea! epithelium with arachidonic acid and NADPH (13, 14). The mechanism of HETE formation by cytochrome P450 remains undefined. Although the products of this reaction are structurally similar to the HETEs generated by several lipoxygenases, attempts to demonstrate the intermediacy of a hydroperoxide, similar to that formed in lipoxygenase reactions, were unsuccessful (7, 15). Hydrogen peroxide formation is stimulated during the metabolism of arachidonic acid by liver microsomal cytochrome P450 (7, 15). Differences in the rate of formation of HETEs by liver microsomes can be modulated after treatment of animals with agents that induce different P450s. However, no studies have yet been reported using purified P450s that link a specific cytochrome P450 form (or forms) with HETE formation.
Omega-hydroxylation
reactions
omega-oxidation of fatty acids has been known for more than 50 years (19). During this time it has served as an interesting biochemical curiosity contributing to the catabolism of medium chain-length fatty acids. The role of cytochrome P450 in these reactions was established by studies of stearic and lauric acid hydroxylation by liver microsomes (20). The classic paper by Lu and Coon (21) appeared about this time, showing the ability to solubilize and resolve rabbit liver cytochrome P450 from other components of the microsomal electron transport system. Lu and Coon (21) selected the omega-oxidation of laurie acid as their assay system to demonstrate the reconstitution of cytochrome P450 activity in the presence of cytochrome P450 reductase and a heatstable factor (phospholipid). The 1970s saw many studies extending these observations. Of particular interest were studies that showed 1) the formation of both omegaand omega-i hydroxylation products of lauric acid, 2) the influence of fatty acid chain length on the rate of the reaction, 3) the incorporation of 180 from molecular oxygen into the product, and 4) the presence of an isotope effect and stereoselectivity for the omega-i reaction. The omegahydroxylation of prostaglandins and their further metabolism to dicarboxylic acids was shown by Granstrom (22), and the hypothesis developed that omega-oxidation may be the first step of biological inactivation of prostaglandins, and therefore, may be important in controlling organ concentrations and/or the potency of these bioactive molecules. What delayed the study of arachidonic acid as a possible substrate for this cytochrome P450-directed reaction? Perhaps earlier studies (23) that showed a marked influence of chain length on the ability of cytochrome P450 to catalyze the omega-hydroxylation of fatty acids discouraged investigators from testing arachidonic acid as a substrate. For arachidonic acid, this reaction entails the delivery of a hemoprotein-bound active oxygen to the terminal and adjacent sp3 carbon atoms of the arachidonate molecule, i.e. C-19 and C-20. Recently, the cytochrome P450-dependent oxidation of arachidonic acid to 16-, 17-, and 18-hydroxyeicosatetraenoic acids by microsomal fractions has been reported (13). Hydroxylation at thermodynamically less reactive C16 through C20 and not at the chemically comparable C2 through C4 suggests a rigid and highly strucThe
P450 AND
ARACTHIDONATF
CASCADF
tured hemoprotein substrate binding site. Thus, this binding site must be capable of positioning the acceptor carbon in optimal proximity to the heme-bound active oxygen and with almost complete exclusion of the reactive olefins and the bis-allylic methylene carbons. Several different cytochrome P450s with marked specificity toward the omega oxidation of saturated fatty acids, arachidonic acid, and arachidonic acid metabolites have been purified and characterized (24-27). As of this writing 12 different P450s active in fatty acid omega and/or omega-i oxidation (gene 4 family) have been cloned and sequenced. Many more P450s with omega-oxidation activities exist in plants, insects, and other mammalian species, and many of these remain to be characterized. Fatty acid omega-oxidation is regulated by factors such as animal age, diet, starvation, and the administration of fatty acids, hypolipidemic drugs, steroids such as dehydroepiandrosterone, or aspirin (24-27). Interest in the role of this cytochrome P450-directed reaction has been stimulated by its contributions to the metabolism of cyclooxygenase, lipoxygenase, and epoxygenase products of the arachidonic acid cascade (see Fig. 1). Although fatty acid hydroxylation at the omega and omega-I positions has long been considered a catabolic process, recent reports of potent biological activities associated with 19- and 20-hydroxyeicosatetraenoic acids (13, 14) suggest that this view warrants further investigation. Olefin
epoxidation
In mammals, the fatty acid epoxygenase activity is unique to cytochrome P450, and in contradistinction to fatty acid omega oxidation, more or less selective for arachidonic acid (24). Epoxidation involves the reductive cleavage of molecular oxygen with the formation of water and an activated form of atomic oxygen, presumably an oxene. This intermediate is regio- and stereoselectively delivered to a ground state olefin. On the other hand, lipoxygenases and cyclooxygenase, rather than catalyzing oxygen activation instead activate their substrates by abstracting a hydrogen atom from a bis-allylic methylene carbon. Oxygenation is then accomplished by coupling the activated substrate and ground state molecular oxygen. To date, the catalysis of EET formation by purified cytochrome P450s, microsomal fractions, or isolated cell preparations has been demonstrated in numerous tissues, inter alia, kidney, liver, brain, pituitary, adrenal, and endothelium (13, 14).
CYTOCHROME
P450
EPOXYGENASE:
A PRODIGAL
ARACHIDONIC
ACID
CHILD
Of the three reactions described previously, the one that has received the most attention is arachidonic acid epoxidation. This is based, in part, on the potent biological activity of the metabolites and the documentation of the EETs as products of the in vivo metabolism of arachidonic acid. Chiral analysis of the epoxygenase metabolites generated by microsomal incubates or by reconstituted systems containing different P450 isoforms showed that, except for P450 2C11 (28) (Table 1), oxidation proceeded with an unprecedented high degree of enantiofacial selectivity for such an unbiased, acyclic molecule. The key role that specific forms of cytochrome P450 play in the control of the stereoselectivity of the epoxygenase is illustrated using the 11,12olefin of arachidonic acid. As revealed in Table 1, treatment of animals with selective P450 inducers can change the inventory of microsomal cytochrome P450s. This results in a 733
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TABLE
1. Enantioselective
properties
of the cylochrome
P450
cascade. Endogenous EETs have been detected in rat, human, and rabbit kidney, human urine, and rat brain (13, 14, 29, 30). A unique feature of the hepatic endogenous pools of EET is their presence as esters of cellular glycerolipids (31). ApEnzyme source % of totalEETS Il(R)12(S).,% Il(S)12(R)-,% proximately 92% of the total liver EETs were found esterified to phospholipids. Of the remaining EETs, 4% was present in Control microsomes 42 81 19 diglycerides, 4% in neutral lipids, and a small amount as -NF microsomes 43 75 25 free acids (less than 1% of the total). Analysis of the phosPB microsomes 32 17 83 pho!ipid pools demonstrated that the EETs were esterified at 58 95 5 CYT P450 IA! the sn-2 position (31). Most phospholipid-associated EETs CYT P450 1A2 22 13 87 were present in phosphatidylcholine (55% of the total), with CYT P450 2Bl 29 14 86 the hepatic phosphatidylinositol pools containing the organs CYT P450 2B2 37 16 84 highest relative EET concentrations (Table 2) (31). Studies CYT P450 2Cli 26 54 46 of the mechanism of EET-phospholipid formation indicate a The conditions used during incubations, product extraction, multistep process initiated by the cytochrome P450-catalyzed purification,and enantiomer resolution are detailed in ref 28. Values shown enantioselective epoxidation of arachidonic acid, ATPare means calculated from at least three different experiments with SE < dependent activation to the corresponding EET-CoA deriva10% of the mean. tive, and EET enantiomer-selective transfer to a lyso-lipid acceptor (31). This in vivo esterification process for enprofound alteration in the profile of positional and stereodogenous EETs appears to be unique. Although the in vitro chemical characteristics of the metabolites, e.g., treatment esterification of exogenously added HETEs has been demonwith PB results in an inversion in the absolute configuration strated (32), most bioactive eicosanoids are either secreted, of the epoxides vs. control or 3-NF treated animals (Table I) excreted, or undergo oxidative metabolism and excretion. (28). The concept of P450-enzyme-specific control of regioThe above data describe a new and potentially important and stereoselectivity of epoxidation was unequivocally demfunctional role for cytochrome P450 in the biosynthesis of onstrated using purified enzyme forms (28). Although cytounique membrane lipid pools. The EET-phospholipids are chrome P450 lA2 showed a high enantiotopic selectivity for biosynthetized, in vivo, under physiological conditions and the re,si-face of the lI,12-olefin (19:1 ratio of antipodes), from endogenous precursors. These data also indicate that, cytochromes P450 iAi, 2B1, and 2B2 catalyzed highly asymin contrast to most eicosanoids, cells have the potential for metric epoxidation at the opposite enantiotopic face of the free-EET generation through hydrolysis of the EET from phos11,12-olefin (sz,re-face), and 2C11 yielded almost racemic mixpholipids, independent of oxidative arachidonate metabolism. tures of enantiomers (28) (Table 1). Thus, the asymmetry of the epoxygenase reaction is under the control of a single protein catalyst. This suggests that the active site molecular PROSPECTS FOR THE FUTURE coordinates responsible for heme-fatty acid spatial orientation are remarkably rigid and structured, with cytochromes Membrane biology P450 1AI, 2B1, and 2B2 sharing a similar binding site geomThe formation and incorporation of epoxyarachidonoyl etry significantly different from that of cytochrome P450 1A2. lipids into membranes may underlie the mechanism responIn contrast to the lipoxygenase and cyclooxygenase memsible for some of the biological activities attributed to the bers of the arachidonic acid cascade, the stereoselectivity of EETs, and more important, indicate a functional role for the epoxygenase is under in vivo regulatory control. These cytochrome P450 in the control of membrane microenvironstudies also demonstrate the multienzyme nature of the ments and function (31). Evidence accumulated from studies epoxygenase reaction and they argue against the existence of of lipid peroxidation indicates that the presence of membrane a single protein responsible for catalysis of all epoxidations. phospholipids containing oxidized moieties has important consequences for cell membrane physicochemical properties, IN VIVO SIGNIFICANCE OF ARACHIDONIC ACID and consequently their structural and functional properties. These consequences include changes in membrane ion perEPOXIDES meability (33), alterations in the enzymatic activities of Even though in vitro studies are an important tool for the enmembrane enzymes (34), and membrane turnover (34) as zymatic characterization of metabolic pathways, they prowell as fluidity and fusogenic properties (35). Published revide only limite information concerning the in vivo significance of the metabolites and enzymes involved. An important step in defining a function for the epoxygenase reaction TABLE 2. Composition of the rat liver EET-containing was the demonstration that its products were endogenous 11, l2-epoxygenase”
constituents of several tissues (13, 14). Chiral analysis of the EET pools in rat liver and human kidney established their enantioselective formation, and unequivocally demonstrated their biosynthetic origin (29, 30). Animal treatment with phenobarbital resulted in a 3.7-fold increase in microsomal cytochrome P450 concentration and a concomitant 6.8- and 3.4-fold increase in the liver concentrations of 8(S),9(R)and i4(R),15(S)-EET, respectively. Both regioisomers were formed as nearly optically pure enantiomers (29). These results are important because they document a novel metabolic function for cytochrome P450, and also establish the arachidonate epoxygenase as a new member of the arachidonic acid metabolic
phospholipid
pools
Phospholipidpool EET-PC EET-PE EET-PI Concentrations
% Distribution
Concentration, smol/mol
55
70 85 106
32 12 are expressed as rmol
of either EET-PC,
EET-PE,
or EET-PI/mol of totalphosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylinositol (P1)in ratliver.The different lipidpoo1swere isolatedand characterizedas detailedin ref31. Values are means calculated from at leastthreedifferentexperiments with SF.
P450
JORGE
H.
and the arachidonate
CAPDEVILA,’ J. R. FALCL,1 AND RONALD
cascade1
W. ESTABROOK
of aMlice and Biochemistr)#{231} Vanderbilt Universit Nashville, Tennessee 37232, USA; and of tMolecular Genetics and 1Biochemistry and the *Cecil H. and Ida Green Center for Reproductive Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA
Departments
Departments Biology
Arachidonic acid and many products of the arachidonate cascade serve as substrates for cytochrome P450-mediated metabolism via allylic oxidation, omega hydroxylation, and epoxygenation as well as peroxide rearrangement. Defining the physiological importance of these metabolites is an area of intense research interest. Cytochrome P450-catalyzed reactions play prominent roles in multiplying the structural and functional diversity of the arachidonate metabolic cascade. Capdevila, H.; Falck, J. R.; Estabrook, R. W. Cytochrome P450 and the arachidonate cascade. FASEBJ. 6: 731-736; 1992.
ABSTRACT
J.
Key Words.prostanoids
P450 . arachidonate eicosanoids . epoxygenase hydroxyeicosatetraenoic acids omega oxidation
LIPID-DERIVED
of cell and significance noids and
MEDIATORS
PLAY
role
A CENTRAL
in the
control
cells have developed several oxygenases that incorporate regioand stereo-specific chemical information into fatty acids. Cells use such recognition signals to convey physiologically
relevant
for
the
intraand/or of arachidonic part, common
intercellular messages. Although acid oxidative metabolism is, to all fatty acids possessing the r-C = C -CH2-C = C -1 H H H H functionality,
most
I
requisite pentadienyl arachidonic acid is uAique terified
to
hormonally
transmembrane lipases,
the
in that
sensitive
signaling release
results
acid,
to a variety
to
and
for oxidative Cyclooxygenase
es-
subsequent
of physiologically metabolism generates
peroxide from which prostaglandins, prostacyclin are derived; the various tially
in vivo,
glycerolipid pools. Thus in the activation of specific
of arachidonic
idative metabolism metabolites (1). The pathways acid are multiple.
it is prese11t,
ox-
important
of arachidonic a labile endo-
thromboxanes, and lipoxygenases lead ini-
hydroperoxyeicosatetraenoic
acids,
which
in
turn
can be converted to leukotrienes, lipoxins, and hepoxilins; in addition, cytochrome P450 can serve as the catalyst for the biotransformation of arachidonic acid to a variety of oxygenated metabolites, including epoxides and a series of fatty acid
alcohols.
The
cade, highlighting of cytochrome
P450,
HISTORICAL In spite
in the years
main the
of the
arachidonate
or proposed
are outlined
in Fig.
cas-
of a few early the
initial
0892-h61R/q2/fl00E.fl7l
reports, description
1/401
cft
(
acid
P450’s
was
of its role
evidence was prethat cytochrome P450 is an active catalyst for the metabolism of arachidonic acid. Capdevila et a!. (5) demonstrated that purified preparations of cytochrome P450, sented
showing
with
purified
NADPH-P450
reductase
and
cytochrome b5, metabolized arachidonic acid to a series of more polar metabolites that were chromatographically distinct from prostaglandins. Oliw and Oates (6), Capdevila et a!. (7), and Morrison and Pascoe (8) reported almost simultaneously the oxidation of arachidonic acid to metabolites by an NADPH-dependent microsomal reaction. These studies heralded the introduction of the third pathway of arachidonic acid metabolism and introduced a number of abbreviations to the lexicon of arachidonic acid metabolites (9, 10). Until the middle 1970s, studies of the metabolism of arachidonic acid were dominated by those reactions catalyzed by cyclooxygenase. In 1979, and with the discovery of leukotrienes, emphasis shifted toward the study of mammalian lipoxygenases (1). In both cases, the biological (functional) signficance of the main metabolites was established before the biochemical characterization of the products and/or enzyme (or enzymes) involved. The physiological significance of these metabolites has been one of the driving forces responsible for the intensive research in this area. In contrast, studies of the functional relevance of the cytochrome P450-system to the metabolism of arachidonic acid started with a well-characterized enzyme system capable of actively metabolizing the fatty acid to several products of unknown physiological significance. The cytochrome P450 system was extensively studied after the demonstration in the early 1960s of its role in the in the metabolism of drugs
biosynthesis
of steroids
(11) and
and xenobiotics (12). Considerable research during the last few years has resulted in the isolation and structural characterization of many cytochrome P450 isozymes (16), and in the demonstration that distinct
macromolecules
provide,
in
many
in-
stances, the molecular basis for their catalytic heterogeneity. Current interest in defining a functional role for cytochrome P450 as a catalyst for the monooxygenation of arachidonic acid emanates from the pivotal physiological role played by
1.
cytochrome
of arachidonic
on the metabolism of several drugs. was not until 1981 that conclusive
structurally
contributions
PERSPECTIVE
metabolism after
features
established
acid It
reconstituted
organ physiology. Among these, the functional of arachidonic acid as the precursor of prostaleukotrienes is well established (1). Mammalian
the enzymology
Studies characterizing the spectral change occurring when arachidonic acid interacts with liver microsomal cytochrome P450 were reported in 1967 (2); little further work was done until 1976 when Cinti and Feinstein (3) demonstrated that microsomes prepared from human platelets contained cytochrome P450 and that the arachidonic acid-induced aggregation of platelets was inhibited by metyrapone and carbon monoxide, two recognized inhibitors of P450; also, about this time Pessayre et a!. (4) documented the effect of arachidonic
involvement
recognized
as an
oxygenase.
20
tDedicated
to MinorJ.
scientist, a warm
person,
Coon-a and
respected
a good
colleague,
a splendid
friend.
FAcIR
www.fasebj.org by UNIVERSITY OF TOLEDO LIBRARIES (18.218.56.169) on September 27, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
Cytochrome
P450 and
16-.17-,&18-OH
,.
01’IEGA-
-.::
i9-keto
HYDROXYLATION
--“
Cascade
Arachidonate
the
,
20-carboxyl
19-&20-OH
-
19- & 20-OH POs
glulathionc
Epoxy genase
-
adducts
5,6-oxygenated P05 vic-DHET5
LETs
oxid.
w/w-I
-
EET-Phosphoiipids diepoxides / epoxy-alcohols
/
Cyto. P450 AIIyI!c
/
Oxid. dihydro-HETEs HETEs
rARAcHuoNic (ACIo
-
wiw-i oxid.
lipoxins DIHETES
Lipoxygenase
HPEFES
--.::
-
glutathioneadduct
hepoxilin thoxilins Ailene oxide dihydro-LTB LTA
-
__I
Solid lines =
Figure
the substrate metabolites,
and as well
of the
significance 10 years
plosion of interest in the cytochrome olites of arachidonic acid. Their
This
article
will
roles
discuss of the
recent different
has
advances types
that
cytochrome
of its pharma-
of the cytochrome have witnessed an ex-
P450-generated role in many
reactions of biological significance area of intense, but often controversial, characterization
multiple
the potent biological activities as the established biochemical, last
metabimportant
developed research. in the
into
an
biochemical
of cytochrome
P450-
directed monooxygenase systems involved in the metabolism of arachidonic acid. Many of these reactions are summarized in Fig. 1, which also outlines additional cytochrome P450catalyzed reactions for the metabolism of eicosanoids and the rearrangment of (hydro)peroxides. Because some excellent reviews have summarized the potential physiological significance of these metabolites (1, 13, 14), we refer only briefly to their biological activities and physiological implications.
PROPERTIES ARACHIDONIC
OF
THE ACID
v,-.I c.
1QQ
-
P01
-
CY1DCHROME MONOOXYGENASE
P450
oxid.
-
w/w-l
-
w/w-I oxid. w/w-i oxid. w/w-i oxid.
LTC LTD
-
LTE
-
w/w-i/w-2 oxid. w/w-I oxid. P01
5,6-epoxy
HHT = hydroxyhepiadecalrienoic PG =prostagiandin P01 = prostacyciin
acid
TX = thromboxane
plays
in eicosanoid
metabolism.
liver microsomes are known to contain multiple cytochrome P450 isoforms and because many different metabolites were formed, the hypothesis was proposed that metabolite diversity resulted from catalysis by mutiple cytochrome P450s (15). Reconstitution studies, utilizing solubilized and purified components of the microsomal electron transport system, demonstrated conclusively that the metabolism of arachidonic acid was cytochrome P450 dependent and that metabolites of different polarity were formed, depending on the type of cytochrome P450 used (5, 13). The detailed structural characterization of the reaction products proved that during microsomal arachidonate metabolism cytochrome P450 can catalyze three reaction types. Allylic
oxidation
Allylic
oxidation
six regioisomeric acids (HETE5).2
is a lipoxygenase-like
reaction
that
affords
cis, trans-conjugated hydroxyeicosatetraenoic Stereochemical analysis demonstrated that
liver microsomal fractions catalyzed the formation of 5-, 8-, 9-, 11-, and 15-HETEs as nearly racemic mixtures whereas 12-HETE was formed enantioselectively as the R isomer (17). Although the contribution of cytochrome P450 to the
P450
Initial studies of arachidonate metabolism by microsomal fractions, isolated from the livers of phenobarbital treated rats, showed the rapid conversion of the fatty acid to many products (7). These studies demonstrated that the reaction was enzymatic, had an absolute requirement for NADPH, and was inhibited by typical cytochrome P450 inhibitors. As
7s)
TX5 HHT
=
description
cological, and toxicological P450 enzyme system. The
-
LET epoxyeicosatnenoic acid DHET = dihydroxyeicosatrienoic acid HYETE/HETE hydroperoxy/hydroxyeicosatelraenoi.acid LT = leukolrlene
cyto. P450or related enzymes other enzymes =
1. A comprehensive
P00 P0111
j
Cyclooxygenase
Dashed lines
LTB
Th
FAfFR
2Abbreviations: HETE, hydroxyeicosatetraenoic acid; EET, cisepoxyeicosatrienoic acid; /-NF’, fl-naphthoflavone; PB, phenobarbital. The nomenclature and designation of various cytochrome P450s follow the recommendations described by nebert et a!. (16).
lotirnal
CAPriC/ll
A
CT
Al
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pool of HETEs found in vivo remains uncertain, interest in this reaction has been encouraged by reports of: 1) the presence of 12(R)-HETE in human skin and its increased formation during psoriatic inflammation (18); 2) the potent activity of 12(R)-HETE as an inhibitor of Na/KATPase (13, 14); and 3) the formation of 12(R)-HETE after incubation of microsomes from cornea! epithelium with arachidonic acid and NADPH (13, 14). The mechanism of HETE formation by cytochrome P450 remains undefined. Although the products of this reaction are structurally similar to the HETEs generated by several lipoxygenases, attempts to demonstrate the intermediacy of a hydroperoxide, similar to that formed in lipoxygenase reactions, were unsuccessful (7, 15). Hydrogen peroxide formation is stimulated during the metabolism of arachidonic acid by liver microsomal cytochrome P450 (7, 15). Differences in the rate of formation of HETEs by liver microsomes can be modulated after treatment of animals with agents that induce different P450s. However, no studies have yet been reported using purified P450s that link a specific cytochrome P450 form (or forms) with HETE formation.
Omega-hydroxylation
reactions
omega-oxidation of fatty acids has been known for more than 50 years (19). During this time it has served as an interesting biochemical curiosity contributing to the catabolism of medium chain-length fatty acids. The role of cytochrome P450 in these reactions was established by studies of stearic and lauric acid hydroxylation by liver microsomes (20). The classic paper by Lu and Coon (21) appeared about this time, showing the ability to solubilize and resolve rabbit liver cytochrome P450 from other components of the microsomal electron transport system. Lu and Coon (21) selected the omega-oxidation of laurie acid as their assay system to demonstrate the reconstitution of cytochrome P450 activity in the presence of cytochrome P450 reductase and a heatstable factor (phospholipid). The 1970s saw many studies extending these observations. Of particular interest were studies that showed 1) the formation of both omegaand omega-i hydroxylation products of lauric acid, 2) the influence of fatty acid chain length on the rate of the reaction, 3) the incorporation of 180 from molecular oxygen into the product, and 4) the presence of an isotope effect and stereoselectivity for the omega-i reaction. The omegahydroxylation of prostaglandins and their further metabolism to dicarboxylic acids was shown by Granstrom (22), and the hypothesis developed that omega-oxidation may be the first step of biological inactivation of prostaglandins, and therefore, may be important in controlling organ concentrations and/or the potency of these bioactive molecules. What delayed the study of arachidonic acid as a possible substrate for this cytochrome P450-directed reaction? Perhaps earlier studies (23) that showed a marked influence of chain length on the ability of cytochrome P450 to catalyze the omega-hydroxylation of fatty acids discouraged investigators from testing arachidonic acid as a substrate. For arachidonic acid, this reaction entails the delivery of a hemoprotein-bound active oxygen to the terminal and adjacent sp3 carbon atoms of the arachidonate molecule, i.e. C-19 and C-20. Recently, the cytochrome P450-dependent oxidation of arachidonic acid to 16-, 17-, and 18-hydroxyeicosatetraenoic acids by microsomal fractions has been reported (13). Hydroxylation at thermodynamically less reactive C16 through C20 and not at the chemically comparable C2 through C4 suggests a rigid and highly strucThe
P450 AND
ARACTHIDONATF
CASCADF
tured hemoprotein substrate binding site. Thus, this binding site must be capable of positioning the acceptor carbon in optimal proximity to the heme-bound active oxygen and with almost complete exclusion of the reactive olefins and the bis-allylic methylene carbons. Several different cytochrome P450s with marked specificity toward the omega oxidation of saturated fatty acids, arachidonic acid, and arachidonic acid metabolites have been purified and characterized (24-27). As of this writing 12 different P450s active in fatty acid omega and/or omega-i oxidation (gene 4 family) have been cloned and sequenced. Many more P450s with omega-oxidation activities exist in plants, insects, and other mammalian species, and many of these remain to be characterized. Fatty acid omega-oxidation is regulated by factors such as animal age, diet, starvation, and the administration of fatty acids, hypolipidemic drugs, steroids such as dehydroepiandrosterone, or aspirin (24-27). Interest in the role of this cytochrome P450-directed reaction has been stimulated by its contributions to the metabolism of cyclooxygenase, lipoxygenase, and epoxygenase products of the arachidonic acid cascade (see Fig. 1). Although fatty acid hydroxylation at the omega and omega-I positions has long been considered a catabolic process, recent reports of potent biological activities associated with 19- and 20-hydroxyeicosatetraenoic acids (13, 14) suggest that this view warrants further investigation. Olefin
epoxidation
In mammals, the fatty acid epoxygenase activity is unique to cytochrome P450, and in contradistinction to fatty acid omega oxidation, more or less selective for arachidonic acid (24). Epoxidation involves the reductive cleavage of molecular oxygen with the formation of water and an activated form of atomic oxygen, presumably an oxene. This intermediate is regio- and stereoselectively delivered to a ground state olefin. On the other hand, lipoxygenases and cyclooxygenase, rather than catalyzing oxygen activation instead activate their substrates by abstracting a hydrogen atom from a bis-allylic methylene carbon. Oxygenation is then accomplished by coupling the activated substrate and ground state molecular oxygen. To date, the catalysis of EET formation by purified cytochrome P450s, microsomal fractions, or isolated cell preparations has been demonstrated in numerous tissues, inter alia, kidney, liver, brain, pituitary, adrenal, and endothelium (13, 14).
CYTOCHROME
P450
EPOXYGENASE:
A PRODIGAL
ARACHIDONIC
ACID
CHILD
Of the three reactions described previously, the one that has received the most attention is arachidonic acid epoxidation. This is based, in part, on the potent biological activity of the metabolites and the documentation of the EETs as products of the in vivo metabolism of arachidonic acid. Chiral analysis of the epoxygenase metabolites generated by microsomal incubates or by reconstituted systems containing different P450 isoforms showed that, except for P450 2C11 (28) (Table 1), oxidation proceeded with an unprecedented high degree of enantiofacial selectivity for such an unbiased, acyclic molecule. The key role that specific forms of cytochrome P450 play in the control of the stereoselectivity of the epoxygenase is illustrated using the 11,12olefin of arachidonic acid. As revealed in Table 1, treatment of animals with selective P450 inducers can change the inventory of microsomal cytochrome P450s. This results in a 733
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TABLE
1. Enantioselective
properties
of the cylochrome
P450
cascade. Endogenous EETs have been detected in rat, human, and rabbit kidney, human urine, and rat brain (13, 14, 29, 30). A unique feature of the hepatic endogenous pools of EET is their presence as esters of cellular glycerolipids (31). ApEnzyme source % of totalEETS Il(R)12(S).,% Il(S)12(R)-,% proximately 92% of the total liver EETs were found esterified to phospholipids. Of the remaining EETs, 4% was present in Control microsomes 42 81 19 diglycerides, 4% in neutral lipids, and a small amount as -NF microsomes 43 75 25 free acids (less than 1% of the total). Analysis of the phosPB microsomes 32 17 83 pho!ipid pools demonstrated that the EETs were esterified at 58 95 5 CYT P450 IA! the sn-2 position (31). Most phospholipid-associated EETs CYT P450 1A2 22 13 87 were present in phosphatidylcholine (55% of the total), with CYT P450 2Bl 29 14 86 the hepatic phosphatidylinositol pools containing the organs CYT P450 2B2 37 16 84 highest relative EET concentrations (Table 2) (31). Studies CYT P450 2Cli 26 54 46 of the mechanism of EET-phospholipid formation indicate a The conditions used during incubations, product extraction, multistep process initiated by the cytochrome P450-catalyzed purification,and enantiomer resolution are detailed in ref 28. Values shown enantioselective epoxidation of arachidonic acid, ATPare means calculated from at least three different experiments with SE < dependent activation to the corresponding EET-CoA deriva10% of the mean. tive, and EET enantiomer-selective transfer to a lyso-lipid acceptor (31). This in vivo esterification process for enprofound alteration in the profile of positional and stereodogenous EETs appears to be unique. Although the in vitro chemical characteristics of the metabolites, e.g., treatment esterification of exogenously added HETEs has been demonwith PB results in an inversion in the absolute configuration strated (32), most bioactive eicosanoids are either secreted, of the epoxides vs. control or 3-NF treated animals (Table I) excreted, or undergo oxidative metabolism and excretion. (28). The concept of P450-enzyme-specific control of regioThe above data describe a new and potentially important and stereoselectivity of epoxidation was unequivocally demfunctional role for cytochrome P450 in the biosynthesis of onstrated using purified enzyme forms (28). Although cytounique membrane lipid pools. The EET-phospholipids are chrome P450 lA2 showed a high enantiotopic selectivity for biosynthetized, in vivo, under physiological conditions and the re,si-face of the lI,12-olefin (19:1 ratio of antipodes), from endogenous precursors. These data also indicate that, cytochromes P450 iAi, 2B1, and 2B2 catalyzed highly asymin contrast to most eicosanoids, cells have the potential for metric epoxidation at the opposite enantiotopic face of the free-EET generation through hydrolysis of the EET from phos11,12-olefin (sz,re-face), and 2C11 yielded almost racemic mixpholipids, independent of oxidative arachidonate metabolism. tures of enantiomers (28) (Table 1). Thus, the asymmetry of the epoxygenase reaction is under the control of a single protein catalyst. This suggests that the active site molecular PROSPECTS FOR THE FUTURE coordinates responsible for heme-fatty acid spatial orientation are remarkably rigid and structured, with cytochromes Membrane biology P450 1AI, 2B1, and 2B2 sharing a similar binding site geomThe formation and incorporation of epoxyarachidonoyl etry significantly different from that of cytochrome P450 1A2. lipids into membranes may underlie the mechanism responIn contrast to the lipoxygenase and cyclooxygenase memsible for some of the biological activities attributed to the bers of the arachidonic acid cascade, the stereoselectivity of EETs, and more important, indicate a functional role for the epoxygenase is under in vivo regulatory control. These cytochrome P450 in the control of membrane microenvironstudies also demonstrate the multienzyme nature of the ments and function (31). Evidence accumulated from studies epoxygenase reaction and they argue against the existence of of lipid peroxidation indicates that the presence of membrane a single protein responsible for catalysis of all epoxidations. phospholipids containing oxidized moieties has important consequences for cell membrane physicochemical properties, IN VIVO SIGNIFICANCE OF ARACHIDONIC ACID and consequently their structural and functional properties. These consequences include changes in membrane ion perEPOXIDES meability (33), alterations in the enzymatic activities of Even though in vitro studies are an important tool for the enmembrane enzymes (34), and membrane turnover (34) as zymatic characterization of metabolic pathways, they prowell as fluidity and fusogenic properties (35). Published revide only limite information concerning the in vivo significance of the metabolites and enzymes involved. An important step in defining a function for the epoxygenase reaction TABLE 2. Composition of the rat liver EET-containing was the demonstration that its products were endogenous 11, l2-epoxygenase”
constituents of several tissues (13, 14). Chiral analysis of the EET pools in rat liver and human kidney established their enantioselective formation, and unequivocally demonstrated their biosynthetic origin (29, 30). Animal treatment with phenobarbital resulted in a 3.7-fold increase in microsomal cytochrome P450 concentration and a concomitant 6.8- and 3.4-fold increase in the liver concentrations of 8(S),9(R)and i4(R),15(S)-EET, respectively. Both regioisomers were formed as nearly optically pure enantiomers (29). These results are important because they document a novel metabolic function for cytochrome P450, and also establish the arachidonate epoxygenase as a new member of the arachidonic acid metabolic
phospholipid
pools
Phospholipidpool EET-PC EET-PE EET-PI Concentrations
% Distribution
Concentration, smol/mol
55
70 85 106
32 12 are expressed as rmol
of either EET-PC,
EET-PE,
or EET-PI/mol of totalphosphatidylcholine (PC), phosphatidylethanolamine (PE), or phosphatidylinositol (P1)in ratliver.The different lipidpoo1swere isolatedand characterizedas detailedin ref31. Values are means calculated from at leastthreedifferentexperiments with SF.
