Current Drug Metabolism, 2004, 5, 225-234
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Cytochrome P450 in Neurological Disease
M. Liu, P.D. Hurn and N.J. Alkayed*
Department of Anesthesiology & Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore,
Maryland 21287, USA and Department of Anesthesiology & Peri-Operative Medicine, Oregon Health & Science
University, Portland, Oregon 97239, USA
Abstract : Advances in a multitude of disciplines support an emerging role for cytochrome P450 enzymes and their
metabolic substrates and end-products in the pathogenesis and treatment of central nervous system disorders, including acute
cerebrovascular injury, such as stroke, chronic neurodegenerative disease, such as Alzheimer's and Parkinson's disease, as
well as epilepsy, multiple sclerosis and psychiatric disorders, including anxiety and depression. The neural tissue contains its
own unique set of P450 genes that are regulated in a manner that is distinct from their molecular regulation in peripheral
tissue. Furthermore, brain P450s catalyze the formation of important brain signaling molecules, such as neurosteroids and
eicosanoids, and metabolize substrates as diverse as vitamins A and D, cholesterol, bile acids, as well as centrally acting
drugs, anesthetics and environmental neurotoxins. These unique characteristics allow this family of proteins and their
metabolites to perform such vital functions in brain as neurotrophic support, neuroprotection, control of cerebral blood flow,
temperature control, neuropeptide release, maintenance of brain cholesterol homoeostasis, elimination of retinoids from
CNS, regulation of neurotransmitter levels and other functions important in brain physiology, development and disease.
Key Words: Cytochrome P450, eicosanoids, EETs, neurosteroids, estrogen, cerebral ischemia, preconditioning, neuroprotection.
INTRODUCTION
Cytochrome P450 enzymes (P450) are heme-containing
family of proteins [1] that catalyze the formation of
biologically important lipid signaling molecules, including
eicosanoids and steroid hormones, and play important roles in
cellular adaptation to oxidative stress [2] and environ- mental
toxins. The P450 enzymes are membrane-bound proteins that
can be classified depending on cellular localiza- tion into
microsomal P450s, which reside in the endoplasmic reticulum,
and mitochondrial P450s, which are associated with the inner
mitochondrial membrane. Microsomal P450s use molecular
oxygen to oxidize substrates by transferring one atom of
oxygen to their substrate and the other to water. This action
requires electron transfer from nicotinamide adenine
dinucleotide phosphate (NADPH) via two co- factors, the
flavoprotein cofactor P450-reductase and cytochrome b5.
Mitochondrial P450s require two additional cofactors,
adrenodoxin and adrenodoxin reductase, also referred to as
ferredoxin and ferrodoxin reductase. The highest
concentrations of P450s are in liver, adrenals, gonads and
placenta. Microsomal P450s in the liver are primarily
involved in drug metabolism and detoxification of foreign
chemicals (xenobiotics). P450s in adrenals, gonads, and
placenta are primarily involved in steroid hormone synthesis.
The last decade witnessed major advancements in the
genetics and molecular biology of cytochrome P450, which
*Address correspondence to this author at the Department of Anesthesiology &
Peri-Operative Medicine, Oregon Health & Science University, 3181 S.W.
Sam Jackson Park Rd., UHS-2, Portland, Oregon 97239-3098, USA;
Tel: (503) 418-5502; Fax: (503) 494-4588; E-mail: alkayedn@ohsu.edu
1389-2002/04 $45.00+.00
allowed the identification of new members of the P450
superfamily and the assignment of P450-catalyzed biochemical reactions to specific genes. These advances have also
lead to the development of gene-specific molecular tools and
sensitive assays that expanded our knowledge of tissue
distribution of P450s and transformed our understanding of
the physiological function of P450 proteins and their role in
disease state [3]. One of the most significant advances in the
P450 field was the discovery that P450 enzymes are
expressed in brain parenchymal cells and cerebral blood
vessels and that many P450 products are endogenous constituents of brain and play important roles in brain function and
neurological disease.
The overall level of P450 enzymes in brain is reported to
be between 0.5% and 10% [4, 5] of their level in liver.
However, levels in specific neuronal populations may be
equivalent or even higher than corresponding levels in
hepatocytes [6]. P450 enzymes in brain are capable of
sustaining some of the same activities as those described in
liver, adrenals and gonads, such as drug metabolism and
steroidogenesis. However, these reactions play different
roles in the central nervous system (CNS) compared to
peripheral tissue. Hepatic P450 enzymes metabolize and
impact overall drug levels in plasma, and steroid hormones
formed in gonads, adrenals and placenta are secreted into the
circulation and affect distant targets in an endocrine fashion.
In contrast, P450 products generated in brain remain
contained within the brain and specifically impact brain
function. For example, drug metabolizing P450s in brain
specifically metabolize centrally acting drugs, and steroids
formed in brain remain and act in brain in a paracrine
manner. Furthermore, as will be reviewed below, brain
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P450s perform functions that are unique to brain such as
neuroprotection, control of cerebral blood flow, formation of
neuroactive steroids, temperature control [7], neuropeptide
release [8], maintenance of brain cholesterol homoeostasis,
elimination of retinoids from CNS, regulation of neurotransmitter levels and other functions important in brain
physiology, development and disease. Finally, P450 genes are
regulated differently in CNS as compared to peripheral tissue.
For example, environmental toxins such as polycyclic
aromatic hydrocarbons are strong inducers of P450 isoforms
1A1 and 1B1 in liver, but little induction is observed in brain
[4]. Similarly, antiepileptic drug phenobarbital induces P450
2B, and steroids induce P450 3A in liver, but fail to do so in
brain [4]. Finally, some P450 enzymes are sexually dimorphic in liver, but not in brain. For example, P450 2C11 is
male-specific in liver, but is expressed in both male and
female brains [9].
Cytochrome P450 genes (CYP) are classified into
families and subfamilies based on sequence homology. In
humans, there are currently 57 known P450 genes arranged
into 18 families [3]. P450 isozymes belonging to families 1-4
(CYP1-4) are microsomal enzymes involved in drug and
arachidonic acid metabolism. Most of these isoforms have
been detected in brain, including P450 1A, 2B, 2C, 2D, 2E,
3A, 4A and 4F [4, 5]. Other P450 isoforms involved in
cholesterol metabolism and steroidogenesis have also been
detected in brain, including P450 7B, P450 11A1, P450 11B,
P450 26A and B, P450 17, P450 21 and P450 46 [4, 5].
Below, we summarize recent studies implicating P450s in
brain function and disease. Studies are grouped by P450
substrates or products, rather than specific P450 genes or
reactions since, as will be explained below, the formation
Liu et al.
and metabolism of some of these products require multiple
P450s, as well as non-P450 enzymes.
NEUROSTEROIDOGENESIS
Neurosteroidogenesis refers to steroid hormone synthesis
in brain [10]. As illustrated in Fig. (1), all five steroid hormone classes: progestagens, glucocorticoids, mineralocorticoids, androgens and estrogens, are derived from cholesterol
by the sequential action of steroidogenic enzymes. Fig. (1)
highlights the crucial role of P450 enzymes in neurosteroidogenesis. The first and rate-limiting step of steroidogenesis is
the conversion of cholesterol to pregnenolone in mitochondria catalyzed by cytochrome P450 side-chain cleavag,
which is encoded by CYP11A1. Progesterone is formed
from pregnenolone via the action of non-P450 enzyme 3hydroxysteroid dehydrogenase. Both progestagens undergo
sequential 17-hydroxylation and conversion to androstenedione and dehydroepiandrostenedeione (DHEA) by microsomal P45017A1. Androstenedione is converted to testosterone via the action of non-P450 17 hydroxysteroid
dehydrogenase (17HSD, also referred to as 17-ketosteroid reductase,
and testosterone is aromatized to 17-estradiol via
microsomal P45019A1. Progestagens can also be converted
to the glucocorticoids corticosterone and cortisol via the
sequential actions of 21-hydroxylase (P450 21A2) and 11hydroxylase (P45011B1). The mineralocorticoid aldosterone
is formed from corticosterone via the additional action of
aldosterone synthase (P450 11B2). Similar to P450 11A1,
P45011B1 and P45011B2 are mitochondrial enzymes. All
steroids are degraded and cleared via hydroxylation
primarily by microsomal P450 enzymes of families 1-3.
Below, we briefly summarize studies implicating specific
Fig. (1). Role of P450 enzymes in steroid synthesis and metabolism in brain. Steroid hormones and bile acids are derived from cholesterol by the
sequential action of steroidogenic enzymes, including cytochrome P450 enzymes, which are also involved in steroid metabolism and clearance
from brain. Modified from [4].
Cytochrome P450 in Neurological Disease
neurosteroids in brain function and disease, with special
emphasis on cerebrovascular physiology and disease.
Estrogens
The role of estrogen in neuroprotection is well established [11]. Our previous work demonstrated that young
adult female rats sustain smaller infarcts after experimental
stroke induced by middle cerebral artery (MCA) occlusion
compared to age-matched males [12]. This sex difference in
ischemic brain injury in young adult rats disappeared after
surgical ovariectomy and was absent in middle-aged,
reproductively senescent rats [13], presumably due to loss of
ovarian function. We subsequently demonstrated that estradiol replacement in ovariectomized [14] and reproductively
senescent female rats [13] restores the protection observed in
young adult females against cerebral ischemia, suggesting
that estradiol is neuroprotective when administered at
physiologically relevant concentrations. The mechanism of
protection by estradiol is in part mediated via upregulation of
neuroprotective genes, such as bcl-2, in neurons within the
peri-infarct region [15]. To examine the effect of local tissue
estrogen formation on ischemic brain injury, mice with
targeted deletion of P450 aromatase (CYP19A1), which are
incapable of converting testosterone to estrogen, were
subjected to MCA occlusion and brain injury was compared
to wild type with intact tissue aromatase. Mice with targeted
disruption of exon 9 of the CYP 19A1 gene (aromatase
knock-out mice) sustained increased brain injury after
experimental stroke compared to wild-type mice with intact
P450 aromatase gene, suggesting an important role for P450
aromatase in protection from ischemic brain injury [16].
Estrogens are metabolized into catechol estrogens, 2- and 4hydroxy estrogens, via the actions of P450 1A and 1B. Both
genes are expressed in brain, [17] and catechol estrogens
have been detected in normal brain [18] and implicated in
neuronal cell death [19].
Androgens
As mentioned above, testosterone is produced in brain
from DHEA, which in turn is produced from pregnenolone
via the action of CYP17A1. Cytochrome P450 7B is the
major P450 isoform involved in terminal metabolism and
elimination of progesterone and androgens in brain. In
humans, DHEA and DHEA sulfate (DHEAS) are the most
abundant circulating plasma steroids during development and
puberty, and DHEA and its precursor pregnenolone are
detected in brain at higher levels than in plasma [5]. Their
levels decline with aging and stress, including stress related
to acute and chronic illness. Age- and stress-related decline in
DHEA levels prompted studies on the role of these
compounds in age-related decline in memory and cognitive
abilities [20] and protection from neurodegenerative disease
[21] and cerebral ischemia. In support of a trophic function,
DHEA and DHEAS increase neurite outgrowth and synapse
formation in the developing brain [10], promote neuronal
survival and differentiation [22], protect hippocampal neurons
against neurotoxicity induced by the glutamate agonists both
in vivo and in vitro [23] and improve recovery of function
after traumatic brain injury in rats [24]. These effects may in
part be mediated via N-methyl-D-aspartate (NMDA) receptor. For example, the effects of pregnenolone and DHEAS to
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enhance spatial memory in mice is thought to result from
their positive modulatory action at the NMDA receptor [10].
Finally, DHEAS, as well as progesterone and pregnenolone sulfate, bind sigma receptors with high affinity. Sigma
receptors are membrane-associated intracellular protein
receptors with unknown function. The sigma-1 receptor is
associated with endoplasmic reticulum membrane and is
mainly expressed in the central nervous system, but it can
also be found in steroidogenic tissue, such as the gonads and
adrenal gland, suggesting that it may be involved in steroid
metabolism or action. The natural ligand for sigma receptors is
unknown. However, a wide range of structurally diverse
chemicals has been shown to bind these receptors, most
notably neurosteroids. This action may be important to
neurological function and disease since the ability of
DHEAS to improve memory has been attributed to its action
on sigma receptors, and synthetic sigma-1 receptor ligand 4phenyl-1-(4-phenylbutyl) piperidine is protective against
cerebral ischemia, presumably by suppressing nitric oxide
release in response to NMDA [25].
Progesterone
We previously demonstrated that chronic progesterone
administration reduces cortical infarct in middle-aged,
reproductively senescent females, whose ovarian function has
naturally abated, and that the protection is not related to
effects of progesterone on cerebral blood flow [13].
Similarly, progesterone administration during the reperfusion
period reduces ischemic injury in ovariectomized, young
adult female rats [26]. Progesterone has also been shown to
facilitate cognitive recovery after traumatic brain injury,
presumably by reducing edema and secondary neuronal loss
[27]. Finally, progesterone promotes myelination of injured
nerves, likely by promoting oligodendrocyte differentiation
[10]. In peripheral nerves, progesterone is produced from
pregnenolone by Schwann cells, and blocking its local
synthesis or action after cryolesioning of the male mouse
sciatic nerve impairs remyelination of the regenerating axons,
whereas administration of progesterone to the lesion site
promotes the formation of new myelin sheaths [28]. In
addition to these trophic effects, progesterone has also been
shown to exhibit acute effects. Progesterone reduces membrane lipid peroxidation after traumatic brain injury, likely
through membrane-stabilizing antioxidant effect [29].
Progesterone is also known to modulate-aminobutyric acid
(GABA) receptor channel activity and expression [30] and
attenuate excitatory neuronal responses [31], which may
underlie progesterone's anxiolytic [32] and antiepileptic [33]
properties. A synthetic analogue of pregnanolone sulfate
inhibits NMDA-induced currents and cell death in primary
cultures of rat hippocampal neurons [34]. Progesterone metabolites allopregnanolone and pregnenolone are endogenous
agonists of GABA(A) receptor. GABA is the major inhibitory neurotransmitter in the mammalian CNS, and rapid
synaptic inhibition is mediated through activation of GABA
(A) receptors. Upon administration, these steroids exhibit
anesthetic, sedative and anxiolytic actions, which may have
implications in CNS disorders as diverse as epilepsy [35],
anxiety [36], insomnia [37], migraine and cluster headache
[38]. Consistent with a memory-enhancing function,
pregnenolone sulfate injection into the amygdala enhanced
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post-training memory processes [39]. Depending on subunit
composition, anchoring or regulatory proteins, and posttranslational modifications, neurosteroids act on GABA(A)
receptor to elicit either agonistic or antagonistic activity [40].
For example, pregnenolone sulfate is an excitotoxin that has
been shown to exacerbate NMDA-induced death of
hippocampal neurons [41] and retina [42], presumably via
cytochrome c release and caspase activation [43].
Corticosteroids
To examine the effect of psychological stress on stroke
outcome, we previously modeled social stress by exposing
mice repeatedly to an aggressive male mouse before
induction of a controlled ischemic insult [44]. Stressed mice
sustained larger infarcts after cerebral ischemia compared to
unstressed mice, which was associated with lower expression
of bcl-2 mRNA. In addition, social stress had no effect on
infarct size in transgenic mice that constitutively express
increased neuronal bcl-2, suggesting that stress may
exacerbate injury by suppressing bcl-2 expression. In this
study, we measured postischemic concentrations of plasma
corticosterone, the major adrenal gland-derived stress
hormone in mice, which correlated with larger infarcts in
wild-type mice. However, as illustrated in Fig. (1), glucocorticoids are also produced endogenously in brain, which
may play important roles in psychiatric disorders and neurodegenerative disease. For example, stress increases brain
concentrations of the neurosteroid allotetrahydrodeoxycorticosterone (THDOC), an allosteric modulator of GABA(A)
receptor, which has been implicated in epilepsy, post-traumatic stress disorder and depression [45]. THDOC has also
been shown to attenuate the behavioral and neuroendocrine
consequences of repeated maternal separation during early
life [46], which may have negative consequences on
neuronal vulnerability and survival.
CHOLESTEROL METABOLITES, BILE ACIDS AND
VITAMINS D
Unlike peripheral tissue, where excess cholesterol is
cleared via lipoprotein particles, the blood brain barrier
prevents cholesterol removal from brain via this mechanism.
In brain, cholesterol is removed after hydroxylation to 24Rand 24S-hydroxycholesterol via cholesterol 24-hydroxylase
(P45046A1) [47]. CYP46A1 is expressed at 100-fold higher
levels in brain than in liver [47]. Elimination of cholesterol
products is an important protective mechanism since these
products possess neurotoxic effects and have been implicated in the pathogenesis of Alzheimer's disease [48].
Deletion of cholesterol 24-hydroxylase did not alter brain
growth or myelination, but reduced sterol excretion from the
CNS [49].
Cytochrome P450 46A1 is also part of the bile acid
synthetic pathway. Bile salts are polar derivatives of cholesterol, formed via the action of P450 enzymes. Tauroursodeoxycholic acid (TUDCA), a hydrophilic bile acid, is
protective against ischemic [50] and hemorrhagic [51] stroke,
likely via inhibition of mitochondrial dysfunction and
subsequent caspase activation. TUDCA also improves
survival and function of nigral transplants in a rat model of
Parkinson's disease [52] and reduces striatal neuropathology
in a transgenic animal model of Huntington's disease [53].
Liu et al.
Cholesterol is also the precursor of vitamin D, which
plays an important role in normal bone growth, calcium and
phosphorus metabolism, and tissue differentiation. Synthesis
of active vitamin D (1,25-dihydroxycholecalciferol, calcitriol)
from its endogenous precursor, 25-hydroxyvitamin D3 (25OHD3), is catalyzed by P450 27B1 (25-OHD3 1-hydroxylase), a mitochondrial P450 enzyme inducible by parathyroid
hormone [54]. A mutation in CYP27B1 gene is associated
with Vitamin D-dependent rickets type I [3]. Because
P45027B1 [55] is expressed in brain, and Vitamin D
receptors are expressed in both brain and immune cells,
Vitamin D may have an immune modulator function and
may play a role in autoimmune disease [56]. In agreement
with this idea, Vitamin D administration has been shown to
block the progression of relapsing encephalomyelitis in
animal models of multiple sclerosis [57]. CYP27A1 has been
suggested to be an anti-atherogenic enzyme because of its
important role in converting cholesterol into polar metabolites [58]. A genetic defect in P45027A1 leads to the
neurological disorder cerebrotendinous xanthomatosis, a rare
autosomal recessive lipid-storage inherited disease characterized by abnormal bile synthesis and deposition of cholesterol
and its 5-reduced derivative cholestanol in various tissues,
including neural tissue, leading to progressive CNS neuropathy marked by dementia, spinal cord dysfunction, and
cerebellar ataxia [59].
Finally, vitamin A (retinoid) metabolite retinoic acid
(RA) is degraded by P45026A and 26B. Genetic linkage
analysis maps Alzheimer's disease to genetic loci containing
P450 genes involved in RA degradation [60]. Furthermore,
deletion of P450 reductase, an electron donor to all microsomal P450s, in transgenic mice is associated with severe
inhibition of vasculogenesis, hematopoiesis and severe
developmental defects in brain, which was associated with
elevated levels of retinoic acid and reduced levels of retinal.
The phenotype was partially reversed by limiting exposure to
retinoic acid [61].
DRUG METABOLISM, TOXINS AND ANESTHETICS
Cytochrome P450 enzymes are highly inducible in
response to environmental toxins, and play important roles in
drug metabolism and bioactivation, foreign chemical
detoxification and cellular adaptation to oxidative stress [2,
62]. However, as mentioned above, brain P450s play specific
roles in local metabolism and inter-individual variations in
responses to centrally acting drugs, alcohol, toxins and
anesthetics [63]. Furthermore, brain and liver P450s respond
differently to the same inducers, likely reflecting differences
in molecular regulation. For example, the classical inducers
of liver P450 enzymes,-naphtoflavone and phenobarbital,
have little effect on P450 levels in brain [4]. On the other
hand, brain P450 levels are affected by such solvents as
ethanol and toluene as well as nicotine, antiepileptic drug
phenytoin, neuroleptic drugs clozapine and sulpiride, and
antidepressant drug mianserin [4-6]. Many drug-metabolizing P450s are expressed at the blood-brain interface and in
brain regions not protected by the blood brain barrier, such as
the choroid plexus, the median eminence, area postrema and
the posterior pituitary. This lead to the notion that the
presence of P450 in these locations represent an 'enzymatic
Cytochrome P450 in Neurological Disease
barrier' evolved to protect the brain from toxic chemicals [64,
65].
Specific drug-metabolizing P450s deserve a special
discussion because of their importance in metabolizing
centrally acting drugs and toxins. P450 2E1 is inducible by
ethanol and nicotine and metabolizes ethanol to acetaldehyde, a highly reactive neurotoxin [6]. This isoform is also
capable of generating reactive oxygen species (ROS) that can
induce oxidative stress and cytotoxicity [66] and has been
implicated in the development of fetal alcohol syn- drome
[67] characterized by cognitive and behavioral deficits.
P450s in general represent a significant source of ROS that in
peripheral tissue may contribute to ischemia reperfusion
injury [68].
Finally, P450 2E1 is responsible for the metabolism of
many volatile anesthetics such as halothane, isoflurane,
diethyl ether and chloroform [63]. Other anesthetics, including opioids, benzodiazepines and local anesthetics are
metabolized by CYP3A4 [63]. Members of the CYP3A
subfamily metabolize approximately 50% of drugs in therapeutic use [6]. Nicotine can induce CYP2B1, and CYP2B6 is
higher in brain regions of smokers than nonsmokers [6].
Both CYP2E1 and CYP2B6 activate tobacco smoke
procarcinogens, and CYP2B6 metabolizes and activates a
number of neurotoxins, such as methylenedioxymethamphetamine (MDMA, or "ecstasy"), cocaine and the insecticide
methyl-parathion [6]. Finally, P450 isoforms have been
implicated in the metabolism of neurotransmitters. For
example, CYP2D and CYP2E enzymes are expressed in
dopaminergic cells and have been implicated in dopamine
metabolism [6].
Toluene and clozapine induce P450 2D4, which may
protect against Toluene-induced rise in reactive oxygen
species and in the pharmacological actions of clozapine.
CYP2D isoforms may also play a role in Parkinson's disease.
Mutations in CYP2D6 are associated with increased incidence
of Parkinson's disease [69] and Alzheimer's
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229
disease variants with [70] and without Lewy bodies [71].
Furthermore, N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
(MPTP), the neurotoxic chemical capable of inducing
Parkinson's disease, is an inhibitor of P450 2D6 [72], and
P450 2D isoforms are also known to metabolize neurotoxic
and Parkinson-inducing dopamine metabolite tetrahydroisoquinoline [4]. CYP2D6 also metabolizes many centrally
active drugs, such as tricyclic antidepressants, selective
serotonin reuptake inhibitors, neuroleptics and anticonvulsants, as well as MPTP. Expression of CYP2D6 in brain and
its involvement in the metabolism of MPTP supported a role
for this enzyme as a susceptibility factor for Parkinsonism.
Higher expression of CYP2D6 in brain was detected in
alcoholics compared to non-alcoholics, possibly explaining
altered sensitivity of alcoholics to centrally acting drugs and
to the mediation of neurotoxic and behavioral effects of
alcohol [6]. Finally, CYP2D6 is the principal enzyme
involved in MDMA metabolism [73].
ARACHIDONIC ACID METABOLISM
As illustrated in Fig. (2), arachidonic acid (AA) is metabolized via 3 enzymatic pathways: cyclooxygenase (COX),
lipoxygenase (LOX) and P450 enzymes into an array of
biologically active eicosanoids. Arachidonic acid is metabolized by P450 epoxygenases and hydroxylases into
epoxyeicosatrienoic (EETs) and hydroxyeicosatetraenoic
acids (HETEs), respectively. The P450 metabolites of AA are
of special interest because of their vasoactive properties and
their involvement in vascular control and cerebro- vascular
disease, such as subarachnoid hemorrhage (SAH) and
ischemic stroke [74].
The brain and cerebral blood vessels express AAmetabolizing P450 enzymes, and both EETs and HETEs are
produced in brain parenchymal tissue and blood vessels [74].
The major P450 AA metabolite produced in blood vessels is
20-hydroxyeicosatetraenoic acid (20-HETE), a potent
vasoconstrictor in the cerebral circulation and a product of the
P450 4A family. 20-HETE plays an important role in the
Fig. (2). Enzymatic pathways of arachidonic acid metabolism. Arachidonic acid (AA) is metabolized via cyclooxygenase (COX) into
prostaglandins (PG), via lipoxygenase (LOX) into leukotriens (LT) and lipoxins (LX), and via P450 epoxygenase and hydroxylase into
epoxyeicosatrienoic acids (EETs) or hydroxyeicosatetraenoic acids (HETEs), respectively.
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Liu et al.
mechanism underlying autoregulation of cerebral blood flow
(CBF) and contributes to the vasodilator actions of nitric
oxide in the cerebral circulation [74]. Recently, 20-HETE has
been shown to play a role in the acute fall in CBF after SAH
[75], presumably by potentiating the vasoconstrictor
response of cerebral vessels to 5-hydroxytryptamine [76].
and in renal epithelial cell lines, EETs inhibit apoptosis
induced by hydrogen peroxide (H 2O2) and serum deprivation
[91].
EETs on the other hand are potent dilators of cerebral
blood vessels, and are produced in brain by astrocytes [77]
via the action of P450 epoxygenases, which insert an
epoxide group across the unsaturated carbon in any one of
four double bonds of AA, yielding 4 regioisomers of EETs:
5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET, depending on
which double bond is being replaced by an epoxide group.
Arachidonic acid epoxides, or EETs, are further metabolized
to less active dihydroeicosatrienoic acids via the action of
soluble epoxide hydrolase [8, 78].
One of the most intriguing findings in recent years is the
demonstration that certain P450 isoforms involved in AA
metabolism can be induced in brain, and that AA metabolites
produced by these isoforms may play a neuroprotective role
against ischemic cell death. This is best illustrated by
examining the effect on P450 gene expression of a neuroprotective strategy such as ischemic preconditioning (IPC),
whereby mild sublethal ischemic stress confers protection
against subsequent severe ischemic insult. As illustrated in
Fig. (3), ischemic preconditioning upregulates a battery of
defense mechanisms and switches cells to a more protected
phenotype, leading to enhanced tolerance against eminent
lethal ischemia. Using a rat model of focal cerebral ischemic
preconditioning, we previously demonstrated that mild
transient ischemia induces the expression of P450 2C11 AA
epoxygenase in brain [9]. Fig. (4) illustrates the experimental
procedure used to induce IPC by simulating the clinical
condition of transient ischemic attack (TIA), which often
precedes stroke [9]. Briefly, adult male rats were subjected to
three 10-min periods of middle cerebral artery occlusions
(MCAO) separated by 45-min intervals or sham surgery.
Three days later, rats were subjected to 2 hours of MCAO,
and infarct size was measured by 2,3,5-triphenyltetrazolium
chloride (TTC) at 24 hours of reperfusion. We also examined
whether ischemic tolerance is associated with higher tissue
perfusion during ischemia. Measurement of laser-Doppler
perfusion (LDP) during MCA occlusion on day 3 demonstrated that the relative change from baseline in LDP was not
different between sham and preconditioned groups, and
measurement of absolute blood flow rates within MCA
EETs are endogenous constituents of brain tissue and
exert a variety of physiological functions in brain, including
peptide hormone release from pituitary gland [8]. We
previously demonstrated that astrocytes express cytochrome
P450 2C11, and that astrocyte-derived EETs play an
important role in coupling neuronal activity to regional blood
flow [79] and maintenance of baseline CBF [80, 81].
Accumulating evidence suggests that EETs may inhibit cell
death in a variety of cell types, including brain parenchymal
cells. EETs are increased during myocardial ischemia [82]
and reduce ischemia-reperfusion injury in heart [83]. The
mechanisms underlying the anti-ischemic effect of EETs are
unknown, but a number of EETs' known properties can
potentially mediate this effect. As mentioned above, EETs are
vasodilators in the coronary [84, 85] and cerebral [86]
circulation and have been proposed to be an endotheliumdependent hyperpolarizing factor [87]. Furthermore, EETs
exert anti-thrombotic [88], anti-inflammatory [89], and antipyretic [7] effects. In endothelial cell culture, EETs directly
inhibit cell death induced by hypoxia-reoxygenation [90],
Effect of Ischemic Preconditioning on Infarct Size and
CBF
Fig. (3). The adaptive responses of brain to the deleterious effects of cerebral ischemia. Mild sublethal ischemic stress confers protection
against subsequent severe ischemic insult by upregulating defense mechanisms and switching brain cells to a new phenotype that is resistant to
apoptotic and necrotic cell death. Modified from [117].
Cytochrome P450 in Neurological Disease
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Fig. (4). Experimental protocol for induction of focal cerebral ischemic preconditioning in brain. Tolerance to ischemic injury, such as occurs
during stroke, is induced in brain via brief transient ischemic attack (TIA)-like episodes consisting of three 5-min periods of MCA occlusion,
separated by 45-min intervals. Tissue infarction is measured at 22 hours of reperfusion after stroke by triphenyltetrazolium chloride (TTC), and
P450 2C11 mRNA and protein expression is evaluated by RNase protection assay (RPA) and Western blotting, respectively.
territory using quantitative iodoantipyrine autoradiography
indicated that ischemic severity was equivalent between the
two groups despite the striking difference in infarct size,
suggesting that ischemic tolerance in this model is not
mediated via a blood-flow enhancing mechanism acting
during vascular occlusion. Cortical and striatal infarcts were
reduced by 50-60% in preconditioned as compared to shampretreated group. TIA alone, without subsequent stroke, did
not cause tissue infarction as determined by TTC.
Effect of IPC on P450 2C11 Expression
To determine if P450 2C11 expression in brain is
increased by IPC, rats were sacrificed and brains removed
and frozen on days 1, 2 or 3 after IPC or sham surgery.
Brains were then processed for quantification of P450 2C11
mRNA and protein using RNase protection assay (RPA) and
Western blotting, respectively. Induction of 2C11 mRNA was
apparent by day 2 in preconditioned vs. sham animals. By
day 3, mRNA levels were 2-3 times higher in ipsilateral
cortex and striatum in preconditioned vs. sham. The level of
2C11 protein on days 1 and 2 after IPC was not different than
sham. However, in agreement with RPA results, the level of
2C11 protein in ipsilateral hemisphere on day 3 was twofold
higher in preconditioned vs. sham animals [9].
Our preliminary work suggests that the reduction in
infarct size in TIA-preconditioned brain is causally linked to
P450 epoxygenase upregulation. Specifically, that the protection by IPC can be prevented by P450 epoxygenase
inhibition and mimicked by inhibition of EETs-metabolizing
epoxide hydrolase, suggesting that the epoxygenase pathway
plays an important role in mediating protection by IPC and
that EETs are protective against ischemic brain injury [92].
The molecular mechanisms utilized by EETs to promote
cell survival and inhibit cell death are unknown. EETs
activate multiple cell survival pathways [93], including the
phosphoinositide 3-kinase (PI3-K)/Akt [91], the mitogenactivated protein / extracellular signal regulated kinase [94]
and the cAMP/ protein kinase A pathways [95]. The PI3K/Akt pathway is a key neuronal survival signaling pathway
during brain development and neurodegenerative diseases [93,
96, 97], and phosphorylation of Akt has been shown to be
increased after cerebral [98, 99] and myocardial [100]
ischemia and to play an important role in mediating ischemic
tolerance in brain [101] and heart [102]. In addition, EETs
have other properties that can potentially mediate its
neuroprotective effect. These include EETs' antioxidant [90],
anti-inflammatory [89] and antipyretic [7] properties.
Furthermore, EETs regulate intracellular calcium concentration [103], inhibit platelet aggregation [88] and prevent
leukocyte adhesion to the vascular wall [89]. A related P450
metabolite, 16(R)-hydroxyeicosatetraenoic acid, suppresses
human leukocyte activation and reduces intracranial pressure
in a rabbit model of thromboembolic stroke [104]. EETs may
also exert their protective effect by tapping on other mechanisms of protection operative in IPC. For example, EETs have
been shown to activate ATP-sensitive K+ channels [105],
which are known to contribute to IPC [106]. EETs increase
cAMP [95], which may in turn induce expression of
neuroprotective genes known to be induced by IPC such as
bcl-2 [107]. EETs may exert neurotrophic effects via their
mitogenic actions [108], such as their role in mediating
epidermal growth factor signaling [109]. EETs may also
mediate the preconditioning effect by regulating intracellular
Ca2+ loads [103], a mechanism linked to IPC [110]. EETs and
P450 enzymes reciprocally regulate the cytokines
interleukin-1 [111], tumor necrosis factor [95] and the
sphingomyelin/ ceramide pathway [112], all of which have
been implicated in the mechanism of protection by IPC [113115]. Finally, EETs exert antioxidant activity [90], which has
also been linked to IPC [116].
CONCLUSIONS
The central role that P450 enzymes play in the metabolism of substrates as diverse as steroids, cholesterol, vitamins,
fatty acids, as well as drugs and anesthetics makes this group
232
Current Drug Metabolism, 2004, Vol. 5, No. 3
of enzymes an important player in normal CNS physiology
and pathobiology. While hepatic P450s specialize in systemic drug metabolism, and adrenal and gonadal P450 specialize in systemic steroid hormone production, brain P450
evolved as a local enzyme system involved in carrying out
specific brain functions, paracrine co-ordination of various
neuronal, glial and vascular activities, as well as providing
trophic support and adaptation to and protection from environmental stress, including oxidative and ischemic stress.
ACKNOWLEDGEMENT
Studies summarized above were supported in part by NIH
grants RO1 NS44313, RO1 NS33668 and PO1 HL59996.
The authors would like to acknowledge the excellent
editorial assistance of Robin Feidelson.
ABBREVIATIONS
25-OHD3 = 25-hydroxyvitamin D3
Liu et al.
TIA
TTC
= Transient ischemic attack
= 2,3,5-triphenyltetrazolium chloride
TUDCA = Tauroursodeoxycholic acid
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