Arch Toxicol (2014) 88:20132032

DOI 10.1007/s00204-014-1371-y

REVIEW ARTICLE

The role ofepoxide hydrolases inhealth anddisease

AhmedA.ElSherbeni AymanO.S.ElKadi

Received: 11 August 2014 / Accepted: 11 September 2014 / Published online: 24 September 2014
Springer-Verlag Berlin Heidelberg 2014

Abstract Epoxide hydrolases (EH) are ubiquitously

expressed in all living organisms and in almost all organs
and tissues. They are mainly subdivided into microsomal
and soluble EH and catalyze the hydration of epoxides,
three-membered-cyclic ethers, to their corresponding dihydrodiols. Owning to the high chemical reactivity of xenobiotic epoxides, microsomal EH is considered protective
enzyme against mutagenic and carcinogenic initiation. Nevertheless, several endogenously produced epoxides of fatty
acids function as important regulatory mediators. By mediating the formation of cytotoxic dihydrodiol fatty acids on
the expense of cytoprotective epoxides of fatty acids, soluble EH is considered to have cytotoxic activity. Indeed, the
attenuation of microsomal EH, achieved by chemical inhibitors or preexists due to specific genetic polymorphisms,
is linked to the aggravation of the toxicity of xenobiotics,
as well as the risk of cancer and inflammatory diseases,
whereas soluble EH inhibition has been emerged as a promising intervention against several diseases, most importantly
cardiovascular, lung and metabolic diseases. However, there
is reportedly a significant overlap in substrate selectivity
between microsomal and soluble EH. In addition, microsomal and soluble EH were found to have the same catalytic
triad and identical molecular mechanism. Consequently,
the physiological functions of microsomal and soluble EH
are also overlapped. Thus, studying the biological effects of
microsomal or soluble EH alterations needs to include the
effects on boththe metabolism of reactive metabolites, as
well as epoxides of fatty acids. This review focuses on the

A.A.ElSherbeni A.O.S.ElKadi(*)
Faculty ofPharmacy andPharmaceutical Sciences, 2142J
Katz GroupRexall Centre forPharmacy andHealth Research,
University ofAlberta, Edmonton, AB T6G 2E1, Canada
e-mail: aelkadi@ualberta.ca

multifaceted role of EH in the metabolism of xenobiotic and

endogenous epoxides and the impact of EH modulations.
Keywords Epoxides Xenobiotics Reactive
metabolites Epoxyeicosatrienoic acids
Epoxyoctadecenoic acids Polymorphism

Introduction
Epoxides are three-membered cyclic ethers that are either
produced in or introduced to biological systems. Epoxides
are the primary metabolites of aromatic and alkenic compounds by xenobiotic-metabolizing enzymes, most importantly cytochrome P450 (P450) (Snyder etal. 1993). Also,
epoxides are directly introduced into our bodies as an environmental pollutant (Herrero etal. 1997). The importance of
epoxides is originated from their high reactivity and electrophilicity due to their strained cyclic structure and the polarization of electrons on the CO bond. Therefore, epoxides are
known to cause cancer and organ damage by interacting with
nucleophilic endogenous macromolecules, notably DNA and
proteins (Nebert and Dalton 2006). They react with the electron-rich DNA to produce DNA adducts and/or DNA strand
breaks, causing DNA mutations and eventually carcinogenesis
(Costa etal. 2012). They are also reported to cause enzyme
inhibition by epoxideprotein adduct formation (Thomas etal.
1969), and to bind to critical protein targets leading to several
toxic effects (Zheng etal. 1997). Recently, the biological functions of several epoxides, most prominently the epoxides of
fatty acids, have been reported to mediate several signaling
pathways. Alterations in the levels of these epoxides have been
linked to many pathological conditions.
Epoxide hydrolases (EH) are a group of enzymes
responsible for the rapid biotransformation of epoxides.

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Arch Toxicol (2014) 88:20132032

Fig.1Substrates and inhibitors

of EH. a The configuration of
cis and trans-epoxides. b Xenobiotic substrate spectrum of EH.
c Examples of epoxide- and
amide-based inhibitors of mEH

Therefore, EH play a significant role in in vivo detoxification of reactive metabolites, by catalyzing the hydrolysis of
epoxides to more soluble and easily excretable metabolites.
Moreover, EH regulate several physiological and pathological functions through controlling tissue levels of biologically active epoxide mediators. There are at least five members of EH in mammals (Fretland and Omiecinski 2000);
however, two EH are of the ultimate significance, namely
microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH). The other three EH have narrow substrate specificity and merely oriented toward endogenously
formed epoxides, which are the hepoxilin, cholesterol and
leukotriene A4 epoxide hydrolase. In this review, we aimed
to discuss the multifaceted role of EH and the impact of
their modulations on biological systems.

Microsomal epoxide hydrolase

Microsomal epoxide hydrolase displays high affinity
toward broad spectrum of epoxide substrates, which can be

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classified to aromatic- and alkenic-based substrates (Fig.1)

(Arand etal. 2003). Therefore, mEH is well recognized as
the main enzyme oriented toward the metabolism of xenobiotic epoxides. mEH belongs to the larger family of /
hydrolase fold enzymes, where the / hydrolase fold links
N-terminal with C-terminal (Zou etal. 2000). Its molecular weight is about 51kDa (Porter etal. 1986), formed of
455 amino acid residues (Friedberg etal. 1994b). The high
evolutionary conservation of mEH, 80% sequence homology between human, rat and rabbits, indicates its essential
biological function. The N-terminal functions as membrane
anchor (Friedberg etal. 1994b), while the C-terminal contains the catalytic site (Zou etal. 2000). mEH is characterized by its compact structure that makes the enzyme highly
resistant to thermal inactivation, as well as proteolytic
digestion (Ioannides 2002). The exact quaternary structure
of mEH is still not available (Ioannides 2002); fortunately,
the quaternary structure of a closely homologues enzyme
was determined, which is the EH from the fungus Aspergillus niger (Zou etal. 2000). However, it lacks the N-terminal membrane anchor and presents as a soluble homodimer,


2014

Arch Toxicol (2014) 88:20132032

Fig.1Substrates and inhibitors

of EH. a The configuration of
cis and trans-epoxides. b Xenobiotic substrate spectrum of EH.
c Examples of epoxide- and
amide-based inhibitors of mEH

Therefore, EH play a significant role in in vivo detoxification of reactive metabolites, by catalyzing the hydrolysis of
epoxides to more soluble and easily excretable metabolites.
Moreover, EH regulate several physiological and pathological functions through controlling tissue levels of biologically active epoxide mediators. There are at least five members of EH in mammals (Fretland and Omiecinski 2000);
however, two EH are of the ultimate significance, namely
microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH). The other three EH have narrow substrate specificity and merely oriented toward endogenously
formed epoxides, which are the hepoxilin, cholesterol and
leukotriene A4 epoxide hydrolase. In this review, we aimed
to discuss the multifaceted role of EH and the impact of
their modulations on biological systems.

Microsomal epoxide hydrolase

Microsomal epoxide hydrolase displays high affinity
toward broad spectrum of epoxide substrates, which can be

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classified to aromatic- and alkenic-based substrates (Fig.1)

(Arand etal. 2003). Therefore, mEH is well recognized as
the main enzyme oriented toward the metabolism of xenobiotic epoxides. mEH belongs to the larger family of /
hydrolase fold enzymes, where the / hydrolase fold links
N-terminal with C-terminal (Zou etal. 2000). Its molecular weight is about 51kDa (Porter etal. 1986), formed of
455 amino acid residues (Friedberg etal. 1994b). The high
evolutionary conservation of mEH, 80% sequence homology between human, rat and rabbits, indicates its essential
biological function. The N-terminal functions as membrane
anchor (Friedberg etal. 1994b), while the C-terminal contains the catalytic site (Zou etal. 2000). mEH is characterized by its compact structure that makes the enzyme highly
resistant to thermal inactivation, as well as proteolytic
digestion (Ioannides 2002). The exact quaternary structure
of mEH is still not available (Ioannides 2002); fortunately,
the quaternary structure of a closely homologues enzyme
was determined, which is the EH from the fungus Aspergillus niger (Zou etal. 2000). However, it lacks the N-terminal membrane anchor and presents as a soluble homodimer,

Arch Toxicol (2014) 88:20132032

2015

Table1Xenobiotics reported to be metabolized through mEH-dependent pathways

Category

Xenobiotic

mEH substrate

Product

References

Drug

Carbamazepine
Phenobarbital
Phenytoin

Carbamazepine-10,11-oxide
Phenobarbital oxide
Phenytoin-3,4-oxide

Eugster etal. (1991)

Kerr etal. (1989)
Hartsfield etal. (1995)

Environmental
pollutant

Benzo(a)pyrene

Benzo(a)pyrene 4,5-, 7,8-,

and 9,10-oxide
Chrysene 1,2-, and 3,4-oxide

Carbamazepine-10,11-dihydrodiol
Phenobarbital dihydrodiol
5-(3,4-Dihydroxy-1,5-cyclohexadien1-yl)-5-phenylhydantoin
Benzo(a)pyrene 4,5-, 7,8-, and
9,10-dihydrodiol
Chrysene 1,2-, and 3,4-dihydrodiol

1,3-Butadiene

Dimethylbenz[a]
anthracene-3,4-oxide
1-Nitropyrene 4,5-, and
9,10-oxide
Butadiene 1,2-, and 3,4-oxide

Dimethylbenz[a]anthracene3,4-dihydrodiol
1-Nitropyrene 4,5-, and
9,10-dihydrodiol
Butadiene 1,2-, and 3,4-diol

Insecticide

4-Vinyl cyclohexene
Anthracene
Benzene
Chloroprene
Cis-stilbene
Epichlorohydrin
Ethylene oxide
Naphthalene
Styrene
Dieldrin

Epoxyethyl epoxycyclohexane
Anthracene oxide
Benzene oxide
3-Chlorobut-3-ene-1,2-oxide
Cis-stilbene oxide
Epichlorohydrin
Ethylene oxide
Naphthalene oxide
Styrene-7,8-oxide
Dieldrin

Dihydroxyethyl dihydroxycyclohexane
Anthracene dihydrodiol
Benzene dihydrodiol
3-Chlorobut-3-ene-1,2-diol
Cis-stilbene diol
3-Chloropropane-1,2-diol
Ethylene diol
Naphthalene dihydrodiol
Styrene-7,8-diol
6,7-Trans-dihydroaldrindiol

Bauer etal. (2003)

Munter etal. (2003)
Newman etal. (2005)
Rossi etal. (1983a)
Herrero etal. (1997)
Hall etal. (1988)
Carlson (1998)
Feil etal. (1970)

Toxin

Aflatoxin B1

Aflatoxin B1-8,9-oxide

Aflatoxin B1-8,9-dihydrodiol

Guengerich etal. (1998)

Chrysene
Dimethylbenz[a]
anthracene
Nitropyrene
Industrial
chemical

contrary to mEH (Zou etal. 2000). As for mEH gene,

EPHX1, it is located on the long arm of chromosome 1 and
is approximately 20kb in size (Hartsfield etal. 1998). It is
composed of eight introns and nine exons, of which exons
29 are coding (Hassett etal. 1994b).
Substrates
The modeled quaternary structure of mEH shows an active
site located at the end of straight substrate access tunnel
of mEH (Zou etal. 2000). Consequently, mEH is unable
to catalyze the hydrolysis of trans-epoxides (Meijer and
DePierre 1988; Newman etal. 2005). Also, mEH substrates
have been reported to be mono-, 1,1-di- or cis-1,2-disubstituted epoxides (Fig.1) (Fretland and Omiecinski 2000;
Newman etal. 2005). Trans-disubstituted and trisubstituted epoxides are poor substrates for mEH, or even act
as inhibitors (Arand etal. 2003) (Fig.1). Moreover, mEH
demonstrates high enantio-selectivity, as the enzyme attack
is preferentially made on the (S)-carbon in the epoxide
ring to yield (R,R)-diols for racemic epoxides (Armstrong
1999). Typical substrates for mEH include toxic and procarcinogenic contaminants, endogenous substances and
some common drugs. Examples for environmental contaminants metabolized by mEH are the epoxide derivatives

Sims etal. (1974)

Glatt etal. (1993)
Miyata etal. (1999)
Heflich etal. (1990)
Malvoisin and
Roberfroid (1982)
Cannady etal. (2002)

of polycyclic aromatic hydrocarbons (PAH), which possess potent genotoxic and carcinogenic effects (Shou etal.
1996). Other important environmental contaminants for
mEH include styrene oxide (Costa etal. 2012), as well as
the epoxide metabolites of 1,3-butadiene, benzene, aflatoxin B1, chrysene, nitropyrene, naphthalene and anthracene. Important clinical drugs metabolized by mEH include
epoxide metabolites of anticonvulsant drugs, phenytoin
and carbamazepine. Xenobiotics reported to be metabolized through mEH-dependent pathways are enumerated
in Table1. Also, several endogenous lipids were found to
be mEH substrates, such as androstene oxide (16,17epoxyandrosten-3-one) and estroxide (epoxyestratrienol)
(Vogel-Bindel etal. 1982). Furthermore, several epoxyfatty
acids, such as epoxystearic acid and epoxyeicosatrienoic
acids (EETs), are hydrolyzed by mEH; however, they are
relatively poor substrates for mEH compared with sEH
(Morisseau 2013; Zeldin etal. 1993).
Tissue andspecies expression
Microsomal epoxide hydrolase has been primarily isolated
and characterized in the microsomal fraction (Decker etal.
2009); however, mEH activity is occasionally detected in
the cytosol as well (Gill etal. 1983). The major location of

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mEH in the cell is the endoplasmic reticulum (von Dippe

etal. 2003), but it has also been detected in association
with the plasma membrane (De Berardinis etal. 2000).
mEH is attached to the endoplasmic reticulum membrane
with its N-terminal anchor in a way that the enzyme is oriented toward the cell cytosol (Holler etal. 1997). Nevertheless, for mEH attached to the plasma membrane, it was
found to be oriented toward the extracellular medium (Zhu
etal. 1999).
Microsomal epoxide hydrolase is expressed in almost
all organs and tissues of mammals; its expression is generally the highest in the liver, followed by adrenal gland,
lung, kidney and intestine in mammals (Oesch etal. 1977).
In humans, adrenal gland may have the highest mEH content (Papadopoulos etal. 1994), whereas in mice, the testis
has the highest content (Newman etal. 2005). Moreover,
the relative mEH contents have been reported to vary with
disease state, environmental exposures, sex and age (Newman etal. 2005).
Substantial variation in mEH expression has been
reported between different species. Therefore, the different
toxic effects of epoxides observed among different species
can be attributed to these variations in mEH expression.
For example, it has been reported that mEH content in rat
liver is about 0.75% of all microsomal liver protein, while
mouse mEH content is never above 0.2% (Oesch etal.
1977). Interestingly, styrene oxide, which is an epoxide
with strong electrophilic properties, was found in the blood
of mice in higher concentrations compared with rats, after
receiving the same dose of styrene oxide precursor, styrene
(Mendrala etal. 1993). Noteworthy, human liver mEH content is well above 1% of all microsomal protein; therefore,
human is expected to be comparatively resistant to styrene
oxide toxic effects (Oesch etal. 1977).
Molecular mechanism
Despite the quaternary structure of mEH is not available (Zou etal. 2000), there is a good understanding of its
molecular mechanism by studying close relative enzymes
(Fretland and Omiecinski 2000). mEH is characterized
by high substrate affinity and low turnover number, usually smaller than 1 molecule per second, with most of its
substrates (Oesch etal. 2004). mEH catalyzes the overall
reaction of the addition of a water molecule to an epoxide ring to ultimately yield a trans-dihydrodiol (Newman
etal. 2005). It was found that labeled water molecules
in the reaction medium are incorporated into the enzyme
itself rather than the dihydrodiol (Lacourciere etal. 1993).
This was the first indication of an unusual mechanism for
epoxide hydrolysis by mEH. Thereby, it was concluded
that mEH molecular mechanism of hydrolysis involves
two chemical steps (Decker etal. 2009; Fretland and

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Omiecinski 2000). mEH forms an ester intermediate with

the epoxide substrate; thereafter, the final hydrolysis of the
ester intermediate to the dihydrodiol is occurred (Decker
etal. 2009) (Fig.2).
mEH active site is composed of a catalytic triad consisting of a His431, Asp226 and Glu404, in addition to two
tyrosine residues that provide an essential support to the
catalytic triad (Fretland and Omiecinski 2000; Oesch etal.
2004). mEH recognizes the substrate that is positioned in
the active site by trapping the epoxide oxygen between the
two tyrosine residues via hydrogen bonding (Decker etal.
2009; Fretland and Omiecinski 2000). Due to the position
of the catalytic triad at the end of the substrate access tunnel, hydrophobic interactions between the substrate and the
enzyme dictate certain stereo- and regio-selectivity(Decker
etal. 2009; Zou etal. 2000). The first chemical step is
very rapid and involving a nucleophilic substitution reaction between the epoxide ring activated by hydrogen bonding and the catalytic nucleophile, which is the aspartic acid
(Fretland and Omiecinski 2000). An ester is formed between
aspartate and the substrate to displace the CO bond in the
epoxide ring; simultaneously, one of the two tyrosine residues gives a proton to the resulted activated oxygen (Decker
etal. 2009; Zou etal. 2000) (Figure2). In the second step,
the ester formed between substrate and mEH is subsequently hydrolyzed by so-called activated charge relay system composed of a histidine, as well as aspartate or glutamate residues (Decker etal. 2009; Zou etal. 2000).
Evidently, the first step, ester formation, proceeds in a
rate much faster than the second step, ester hydrolysis (Fretland and Omiecinski 2000). Therefore, the rate-limiting step
of the overall reaction is the hydrolysis of the ester intermediate and release of a dihydrodiol (Fretland and Omiecinski
2000). It is worth to note that the formation of ester intermediate is reversible, contrary to the hydrolysis (Armstrong
1999). Also, it was observed that detoxification efficiency
of the enzyme was underestimated based on the determined
overall rate of dihydrodiol formation (Oesch etal. 2004,
2000). In fact, the toxic epoxides are rapidly trapped and
deactivated by forming covalent ester intermediate with
the aspartate residue of mEH, without any significant formation of dihydrodiol (Oesch etal. 2004, 2000). Hence,
the reactive epoxides are eliminated much faster than the
dihydrodiols are formed by mEH (Oesch etal. 2000). Noteworthy, mEH presents in a high concentration in the tissues,
for example mEH concentration is 1050mol per kg wet
weight human liver tissue (Thomas etal. 1990).
Regulation
The regulation of mEH occurs at the transcriptional or
translational levels. Several different noncoding first exons
for EPHX1 have been reported, which allows several

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2017

Fig.2Enzymatic mechanism
of EHagonist interaction. a
The mechanism of epoxide
hydrolysis by EH. The epoxide
ring is positioned by hydrogen
bonding with tyrosine residues,
then, the nucleophilic aspartic
acid residue attacks the epoxide
ring, leading to the intermediate, which is hydrolyzed by
histidineaspartate/glutamate
residues of EH. b The mechanism of EH inhibition by carbamide-based inhibitors, where
X is CH2, O or NH. Hydrogen
bonding is established between
the carbonyl group of the inhibitor and tyrosine residues of EH,
and between the amino group
of the inhibitor and aspartate
residue of EH, occupying the
catalytic triad

independent promoters for transcriptional control of mEH

(Ioannides 2002). Thereby, several compounds are found to
modulate mEH expression. mEH inducers include 2-acetylaminofluorene, methylcholanthrene, stilbene oxide, peroxisome proliferator-activated receptor (PPAR) agonists,
phenobarbitone, imidazoles and heavy metals, whereas
mEH inhibitors include acriflavine, growth hormone, gadolinium chloride, lipopolysaccharide and glucocorticoids.
mEH is closely associated with P450 during xenobiotic
metabolism, and this association is facilitated by the direct
inductive effect of P450 enzymes on mEH activity (Taura
Ki etal. 2002).
Physiological functions
The straightforward function of mEH is detoxifying the
electrophilic epoxides; however, mEH has been reported

to bio-activate a number of potent carcinogens. Noteworthy, the true extent of mEH contribution in xenobiotic
metabolism is underestimated due to the short half-life of
epoxide intermediates, making the direct measurements of
these epoxides very difficult. For example, the half-life of
aflatoxin B1-8,9-oxide was reported to be approximately
1s (Guengerich etal. 1996). Additionally, mEH has been
proven to exhibit some physiological functions by metabolizing endogenous epoxides (Newman etal. 2005).
Detoxification ofreactive epoxides
Due to its very broad substrate selectivity and its high
expression in the liver and other metabolizing organs
(Arand etal. 2003; Oesch etal. 1977), mEH is effectively involved in the cytoprotective mechanism against
epoxides. As aforementioned, epoxides are generally

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reactive electrophilic mutagens, carcinogens and/or cytotoxins (Zakim and Vessey 1985). The mEH catalyzes
the hydrolysis, and therefore, the detoxification of the
epoxy metabolites of some commonly used anticonvulsant drugs, carbamazepine, phenytoin and phenobarbitone (Hartsfield etal. 1995), to the more polar dihydrodiols (Armstrong 1987). In the body, these anticonvulsant
drugs are bio-activated by P450 enzymes to give reactive
epoxymetabolites (Kerr etal. 1989; Pisani etal. 1993).
The major metabolite of carbamazepine, carbamazepine10,11-oxide, is produced mainly by CYP3A4 (Decker
etal. 2009). These epoxymetabolites are reactive and
proven to be harmful both in vitro and in vivo (Kerr etal.
1989; Strickler etal. 1985). Simultaneous administration
of mEH inhibitor with carbamazepine, phenytoin and
phenobarbitone was found to increase their epoxymetabolites in humans (Kerr etal. 1989; Rambeck etal. 1990);
consequently, the incidence of serious toxicities was
increased (Rambeck etal. 1990). Also, it was proposed
that mEH inhibitors may increase the teratogenic effect
of carbamazepine and phenytoin (Kerr and Levy 1989).
Interestingly, another group of anticonvulsant drugs were
found to inhibit mEH, such as valpromide, valproate and
progabide (Kerr etal. 1989).
Also, several reports have been published regarding
the industrial contaminant, styrene, discussing the role
of mEH in its metabolism (Carlson 1998). Styrene enters
human bodies predominantly as an occupational exposure,
especially for the workers in polyester-plastic industries
(Costa etal. 2012; Sumner and Fennell 1994). By P450,
styrene undergoes metabolic transformation to an epoxide, namely styrene-7,8-oxide, which represents about
90% of the total styrene dose (Sumner and Fennell 1994).
Evidently, styrene oxide possesses a potent genotoxic and
carcinogenic effects, compared with the less genotoxic and
carcinogenic styrene (Costa etal. 2012; Sumner and Fennell 1994). Oesch etal. have used styrene oxide to prove
that the cytoprotective effect of mEH is characterized by
a certain threshold, i.e., the genotoxicity of styrene oxide
increases steeply after certain dose limit (Oesch etal.
2004, 2000). They showed that mEH in Chinese hamster
fibroblast cells can provide protection against styrene
oxide-induced genotoxicity to a concentration of at least
100M (Oesch etal. 2004, 2000). Above this concentration, a steep rise occurs in the genotoxic effect of styrene
oxide (Oesch etal. 2004). Also, several other xenobiotics
are oxidized, mainly by P450, to yield reactive epoxides
that are effectively detoxified by mEH. These xenobiotics
include 4-vinyl cyclohexene, which causes ovarian toxicity
in mice (Cannady etal. 2002), and known potent carcinogens, such as 1,3-butadiene, chloroprene and aflatoxin B1
(Guengerich etal. 1998; Munter etal. 2003; Ward etal.
1996).

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Formation ofreactive metabolites

Several xenobiotics can be bio-activated by a mEH-dependent cascade of bio-transformations (Decker etal. 2009).
The cascade is initiated by the formation of aromaticbased epoxides and arene oxides and mainly by P450. One
example is the formation of the highly toxic benzoquinone
and semiquinone radical from benzene (Fig.3) (Bauer
etal. 2003). It was reported that CYP2B1 and CYP2E1
catalyze the transformation of benzene to benzene oxide
(Snyder etal. 1993), which is then converted to catechol
by dehydrogenases and mEH (Snyder etal. 1993). Catechol is a hydroquinone that is easily convertible to benzoquinone and semiquinone radical linked to leukemia in
humans (McHale etal. 2012). Therefore, animals deficient
in mEH were found to be resistant to benzene-induced
toxicity (Bauer etal. 2003). Another example is the bioactivation of PAH to dihydrodiol epoxides metabolites,
which are characterized by their potent carcinogenic effect
and their resistance to detoxification by cellular defenses
(Friedberg etal. 1994a; Holder etal. 1974). For example
benzo[a]pyrene, it is mainly metabolized by CYP1A1 and
CYP1A2 to generate benzo[a]pyrene 4,5-, 7,8- and 9,10oxide, which are reactive epoxides by themselves (Fig.3)
(Sims etal. 1974); subsequently, it is rapidly deactivated by
mEH to the dihydrodiol metabolite (Fig.3) (Decker etal.
2009; Sims etal. 1974). The dihydrodiol metabolite is a
good substrate for P450 and again it is oxidized to form
the highly toxic dihydrodiol epoxides, notably benzo[a]
pyrene-7,8-dihydrodiol-9,10-oxide (Fig.3) (Friedberg etal.
1994a; Sims etal. 1974). These final metabolites cannot
undergo spontaneous rearrangement via NIH shift, due to
lacking of aromaticity (Friedberg etal. 1994a), or be inactivated by mEH, since dihydrodiol epoxides are very poor
substrate for the enzyme (Friedberg etal. 1994a). Here,
glutathione conjugation is truly the last line of defense,
which is proven, in many cases, to be insufficient. Other
PAH, such as, chrysene and 7,12-dimethylbenz[a]anthracene, are also activated by the same cascade to yield the
highly reactive dihydrodiol epoxides (Liu etal. 2008).
It was reported that mEH knockout mice are resistant to
7,12-dimethyl benz[a]anthracene-induced carcinogenicity
and immunosuppression (Miyata etal. 1999). Presumably,
the trans-dihydrodiols are of lower reactivity compared
with their precursor, epoxides. However, for certain xenobiotics, this is not true and the dihydrodiol is more reactive, for example, epichlorohydrin, a highly reactive epoxide, which is used for producing elastomers, pesticides
and pharmaceuticals (Rossi etal. 1983b). The hydrolyzed
derivative of epichlorohydrin is 3-chloropropane-1,2-diol,
which is a potent carcinogen (Cho etal. 2008). Nevertheless, one study investigated the effect of mEH inhibition
on epichlorohydrin-induced carcinogenicity, showing that

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Fig.3Bioactivation of benzene
(a) and benzo(a)pyrene (b) by
mEH-dependent pathway

mEH is important for epichlorohydrin deactivation (Rossi

etal. 1983b). In the same context, it was reported that mEH
knockout mice are resistant to 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) neurotoxicity (Liu etal. 2008).
The mechanism is not fully elucidated, but it was observed
that dopaminergic neurons were less susceptible to MPTP
insult in mEH knockout mice (Liu etal. 2008).
Regulation ofphysiological functions
Microsomal epoxide hydrolase catalyzes the hydrolases
of several biologically active epoxide mediators, such
as epoxides from estradiol and testosterone (Disse etal.
1980); therefore, it was proposed that mEH is involved in
testosterone to estradiol conversion (Morisseau 2013). This
is supported by a report describes a decrease in estradiol
formation upon treatment with mEH inhibitor in human
ovary (Hattori etal. 2000). Several other epoxysteroids are
also hydrolyzed by mEH; however, it has not yet confirmed
in vivo (Vogel-Bindel etal. 1982). Despite the relatively
lower affinity of mEH compared with sEH toward epoxides

of fatty acids (Decker etal. 2009), mEH may have a significant kinetic advantage due to its co-localization with P450
in the endoplasmic reticulum (Decker etal. 2012). Noteworthy, the formation of epoxides of fatty acids is mainly
catalyzed by endoplasmic reticulum-attached enzymes,
such as P450 (Imig 2012). The epoxides of fatty acids that
can be hydrolyzed by mEH include epoxystearic acid (Zeldin etal. 1996), EETs (Decker etal. 2012) and anandamide epoxides (Snider etal. 2007). Moreover, an increase
in mEH level was associated with Alzheimers disease in a
study conducted in humans (Liu etal. 2006).
Modulations
Inhibition
A number of mEH inhibitors have been developed, which
can be divided into two classes (Fig.1). The first class is
the epoxide-based mEH inhibitors; they inhibit mEH by
acting as substrates for mEH that rapidly bind to mEH
but very slowly dissociate from it, i.e., uncompetitive

13


2020

reversible inhibitors, notably 1,1,1-trichloropropene2,3-oxide and cyclohexene oxide (Pacifici and Rane 1983).
The second class is carbamide-based mEH inhibitors; the
first mEH inhibitor of this class was identified by studying the drug interaction between carbamazepine and valpromide, revealing that valpromide exhibits mEH inhibitory activity (Meijer etal. 1984). Examining the chemical
structure of valpromide showed that several amides are
tightly bound mEH inhibitors with low nanomolar dissociation constants (Morisseau 2013). Consequently, a
series of amide-based EH inhibitors were developed, and
they are proven to be more potent than the epoxide-based
mEH inhibitors (Morisseau etal. 2001). The mechanism
of inhibition of amide- and urea-based EH inhibitors is by
establishing hydrogen bonding between the carbonyl group
of the inhibitor and tyrosine residues, as well as between
the amino group of the inhibitor and aspartate residue, and
therefore occupies the catalytic triad (Fig.2) (Morisseau
2013). Their mEH inhibition kinetics is an apparent mix
of competitive and non-competitive kinetics (Morisseau
etal. 2001). An example for these inhibitors is elaidamide
(Morisseau etal. 2008). Furthermore, divalent heavy metals were also found to exhibit significant inhibitory activity against mEH (Draper and Hammock 1999). Mercury
and zinc are potent inhibitors to mEH with IC50s in the
M range, while nickel and lead are moderate inhibitors
(Draper and Hammock 1999). Inhibition of mEH activity
in rat liver has been reported in streptozotocin-induced diabetes that was corrected by insulin administration (Thomas
etal. 1989). Starvation decreases mEH activity by about
60% (Thomas etal. 1989), and total parenteral nutrition
also produced a similar effect (Wildhaber etal. 2003).
Several compounds are known to suppress mEH activity
include acriflavine, growth hormone, gadolinium chloride,
lipopolysaccharide and glucocorticoids (Ioannides 2002;
Newman etal. 2005). mEH inhibition has been reported to
be beneficial in case on PAH exposure. 1,1,1-trichloropropene-2,3-oxide reduced the carcinogenicity of benzo[a]pyrene, as well as dimethylbenz(a)anthracene as assessed by
DNA binding, and the formation of fibrosarcoma in mice
(Kodama etal. 1980). Formation of reactive metabolites
through mEH-dependent pathway explains the increase
in PAH carcinogenicity by mEH inhibition. On the other
hand, the hepatotoxicity of vinyl chloride was aggravated
after mEH inhibition by 1,1,1-trichloropropene-2,3-oxide
in rats pretreated with polychlorinated biphenyl (Conolly
etal. 1979).
Induction
The induction of mEH has been observed as a consequence
of exposing to several compounds, include 2-acetylaminofluorene, methylcholanthrene and stilbene oxide (Cho and

13

Arch Toxicol (2014) 88:20132032

Kim 1998; Newman etal. 2005). Other inducers include

PPAR agonists, radiation, heavy metals, phenobarbitone
and imidazoles (Fretland and Omiecinski 2000; Ioannides
2002; Newman etal. 2005).
Polymorphism
Human EPHX1 has been characterized on chromosome
1q42.1 (Hartsfield etal. 1998), revealing that EPHX1
does not show genetic complexity of the other xenobioticmetabolizing enzymes (Hassett etal. 1994b). In 1994, Gaedigk etal. identified 9 single-nucleotide polymorphisms
(SNPs) in the coding (exonic) sequence; three were identified as non-synonymous SNPs (Gaedigk etal. 1994).
So far, several non-synonymous SNPs within EHPX1
exons have been identified; however, only two of them
are known to affect mEH enzyme activity, one in exon 3
(337T>C) and the other in exon 4 (415A>G). 337T>C SNP
leads to a tyrosine to histidine substitution at position 113
(Tyr113His), whereas 415A>G leads to a histidine to arginine substitution at position 139 (His139Arg). Tyr113His
and His139Arg confer slow and fast enzyme activity by
40 and 25%, respectively (Benhamou etal. 1998; Hassett
etal. 1994a). Due to their known effect on mEH activity,
these two SNPs have been studied extensively in human
population for potential association with xenobiotic toxicities and diseases. Alteration in mEH activity was associated with cancers of various tissues, viz. breast, colorectal, prostate, esophagus, stomach and liver, in addition to
other diseases and conditions, colorectal polyps, preeclampsia, Crohns disease, chronic obstructive pulmonary
disease (COPD), emphysema and oxidative stress. Four
recent meta-analyses stratified these factors for COPD (Li
etal. 2013), colorectal cancer (Liu etal. 2012), hepatocellular carcinoma (Zhong etal. 2013) and lung cancer (Liu
etal. 2013). With respect to COPD, slow mEH activity was
found to be a significant risk factor for COPD in Caucasian
population, but not in Asian population (Li etal. 2013). For
colorectal cancer, individuals with fast mEH activity were
found to be more resistant to colorectal cancer especially
for Caucasian individuals (Liu etal. 2012). For hepatocellular carcinoma, slow mEH activity was found to be a
significant risk factor (Zhong etal. 2013). In contrast, fast
mEH activity was a risk factor for lung cancer that underscores the bio-activating role of mEH in PAH metabolism
(Liu etal. 2013). It was found that SNPs associated with
increasing mEH activity potentiate the formation of PAHDNA adduct in prostate cancer patients (Nock etal. 2007)
and with the formation of hemoglobin adduct with acrylamide metabolite and glycidamide (Duale etal. 2009; Huang
etal. 2012). Also, the genotoxic effect of 1,3-butadiene was
found to be associated with polymorphic changes in mEH
activity (Abdel-Rahman etal. 2005; Wickliffe etal. 2003).

Arch Toxicol (2014) 88:20132032

Selected studies investigating Tyr113His and/or His139Arg

association with other diseases and conditions are enumerated in Table2. The significant association between
EPHX1 polymorphisms and several diseases underscores
the important role of mEH in the metabolism of reactive
molecules. Generally, slow mEH activity increase body
susceptibility to environmental challenges; however, this is
greatly altered by organ affected and ethnicity (Nock etal.
2007). Smoking, which leads to high exposure to PAH, can
make fast-EH-activity polymorphisms to be risk factor for
some diseases, especially lung diseases (Table2).

2021

2005, 2003). The best substrate is as yet known is threo10-hydroxy-9-phosphonooxy octadecanoic acid; however,
the molecular mechanism is still to be determined (Gomez
etal. 2004; Newman etal. 2003).
Tissue andspecies expression

There is a clear overlapping in substrate selectivity between

mEH and sEH; however, sEH is generally complementing
mEH in its xenobiotic-metabolizing activity. Comparatively, the regulating function of sEH in the metabolism of
fatty acid-derived epoxides mediators is considered more
biologically important (Decker etal. 2009). In contrast to
mEH, sEH presents as a homodimer in solution (Gomez
etal. 2004). sEH molecular weight is about 62kDa, formed
of 554 amino acid residues and belongs to the large family of / hydrolase fold enzymes (Grant etal. 1993). The
C-terminal of sEH is similar to mEH and provides the
epoxide hydrolase activity (Newman etal. 2005). Contrary
to mEH, the N-terminal of sEH does not act as a membrane
anchor; however, it carries a second catalytic site that was
found to be a functional phosphatase (Gomez etal. 2004;
Newman etal. 2005).

Interestingly, tissue distribution of sEH and mEH is completely independent. sEH is expressed in all organs and tissues but at different levels. The organ of highest sEH level
is generally liver, followed by kidney, heart, lung and brain
(Gill and Hammock 1980). sEH expression is confined to
vascular tissue of kidney, lung and brain, contrary to other
organs, such as liver and pituitary gland, where its expression is diffused in all tissues (Morisseau and Hammock
2005). It has been reported that sEH is exclusively localized in the cytosol in all organs, except for liver, where sEH
is localized in both cytosol and peroxisome (Enayetallah
etal. 2006). However, sEH activity has been commonly
observed in mitochondrial and microsomal fractions. For
long time, this activity was attributed to sEH trapped in
these fractions; only recently, Decker etal. identified two
new epoxide hydrolases, EH3 and EH4, that exhibit sEH
activity, but it is membrane-bound (Decker etal. 2012).
There are only sparse studies investigated the speciesdependent expression of sEH. Despite sEH gene expression in liver was comparable among mouse, rat and human,
there was a 100-fold difference in sEH activity between rat
and mouse liver (Hammock etal. 1997). Also, liver content of sEH has been reported to be in the following order,
mice>human>rat (Ioannides 2002).

Substrates

Molecular mechanism

Soluble epoxide hydrolase is characterized by a bent substrate access tunnel (Argiriadi etal. 1999); therefore,
sEH interacts with trans-substituted epoxides, contrary to
mEH. For example, trans-stilbene oxide and trans-ethylstyrene oxide are effectively metabolized by sEH (Newman etal.2005). Noteworthy, bulky substrates are generally not a good substrate for sEH (Newman etal. 2005).
In fact, sEH displays its highest affinity toward epoxides
of fatty acids, which have relatively low molecular weight
(Morisseau and Hammock 2005; Newman etal. 2005).
sEH shows the highest affinity toward the epoxides of
-linolenic, eicosapentaenoic and docosahexaenoic acids,
and to a lower extent toward epoxide of arachidonic acid
(AA) and other fatty acids (Morisseau 2013). On the other
hand, the N-terminal domain is a functional phosphatase,
whose substrate is still unknown (Newman etal. 2003).
Based on the compact quaternary structure of the enzyme,
it has been suggested that the substrate will be lipophilic
in nature, possibly a phosphorylated lipid (Newman etal.

The molecular mechanism of epoxides hydration by sEH

and mEH is identical (Fig.2) (Morisseau and Hammock
2005); it involves two chemical steps, the first is ester formation between the epoxide substrate and the enzyme,
and the second is the release of the dihydrodiol substrate
(Borhan etal. 1995). The sEH catalytic site also contains
the essential histidine (His523) and aspartate (Asp334 and
Asp495) residues, in addition to two tyrosine (Tyr382 and
Tyr465) residues that provide an essential support to the catalytic triad (Gomez etal. 2004; Morisseau and Hammock
2005).

Soluble epoxide hydrolase

Regulation
Soluble epoxide hydrolase regulation is mediated by
PPAR-responsive element in the 5-flanking region of sEH
gene (Pinot etal. 1995). Therefore, it has been reported
that PPAR agonist, fenofibrate, induced sEH activity
by 13 folds in liver and twofolds in kidney (Schladt etal.

13


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Arch Toxicol (2014) 88:20132032

Table2The association of mEH polymorphism, Tyr113His and His139Arg, with different diseases, and conditions
Subjects Country

Polymorphism

Outcome

References

604

India
Czech/Tunisia

1,920

Australia

560

USA

Alcohol dependence risk by slow mEH

activity variant
Breast cancer risk by slow mEH activity
variant
Breast cancer risk by slow mEH activity
variant
No association with breast cancer

Bhaskar etal. (2013)

1,111

Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His

300

527

China/India

824

India/Taiwan

1,867

China/Netherlands

Crohns disease risk by fast mEH variant

Diabetes mellitus risk by slow mEH
activity variant
Esophageal carcinoma risk and progression by slow mEH activity variant
Esophageal carcinoma risk by fast mEH
activity variant in smokers
No association with esophageal cancer

de Jong etal. (2003)

262

Netherlands/
Caucasian
Egypt

730

USA

287

India

493

Italy

612
832

Netherlands/
Turkey
Australia

148

USA/Caucasian

Tyr113His

486

Tunisia/India

232

Finland

227

Finland

1,462
189
499

Egypt/Netherlands/
Turkey/Finland
South Africa
Slovakia/Caucasian

270

India

Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
Tyr113His
His139Arg
Tyr113His
His139Arg

918

USA/90% Caucasian His139Arg

Tyr113His
His139Arg
Tyr113His
Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
His139Arg
Tyr113His
Tyr113His

Fetal isolated orofacial clefts risk by

fast mEH activity variant in pregnant
smoker
Gastric cancer risk by fast mEH activity
variant
Liver cirrhosis risk by slow mEH activity variant
Lymphoblastic leukemia risk by slow
mEH activity variant
Ovarian cancer risk by fast mEH activity variant
Ovarian cancer risk by fast mEH activity variant
Oxidative stress by slow mEH activity
variant (in COPD patients)
Placental abruption risk by slow mEH
activity variant
Polycystic ovary syndrome risk by slow
mEH activity variant
Preeclampsia risk by fast mEH activity
variant
No association with preeclampsia risk
Prostate cancer risk by fast mEH activity variant
Prostate cancer risk by slow mEH activity variant
No association with prostate cancer risk

1987). On the other hand, PPAR agonist, rosiglitazone,

has been reported to downregulate sEH in cardiomyocytes
(Pang etal. 2011). Furthermore, a significant increase
in sEH expression has been reported in males of rats and
mice compared with females, suggesting hormonal domain
in the regulation of sEH (Newman etal. 2005). Recently,
angiotensin II, as well as homocysteine, was reported to
upregulate sEH expression (Zhang etal. 2012).

13

Khedhaier etal. (2008), Sarmanova etal.

(2004)
Spurdle etal. (2007)
de Assis etal. (2002)

Ghattas and Amer (2012)

Jain etal. (2008), Wang etal. (2003)
Ihsan etal. (2010), Lin etal. (2006)
Wang etal. (2006), Hassett etal. (1994a),
Zhang etal. (2003), Dura etal. (2012)
Ramirez etal. (2007)

Ghoshal etal. (2013)

Sonzogni etal. (2002)
Raijmakers etal. (2004), Tumer etal. (2012)
Spurdle etal. (2001)
Lancaster etal. (1996)
Lakhdar etal. (2011), Vibhuti etal. (2007)
Toivonen etal. (2004)
Korhonen etal. (2003)
Kamal etal. (2012), Zusterzeel etal. (2001),
Pinarbasi etal. (2007), Laasanen etal. (2002)
Gebhardt etal. (2004)
Sivonova etal. (2012)
Mittal and Srivastava (2007)
Nock etal. (2006)

Physiological functions
The potent biological activity reported for epoxides of fatty
acids and their significant levels found in all tissues underscore the important endogenous roles of sEH (Decker etal.
2009; Ioannides 2002). Extensive studies have been made
on sEH regulation of epoxides of AA, namely the EETs,
and, to a lower extent, on epoxides of linoleic acid (LA),

Arch Toxicol (2014) 88:20132032

2023

Fig.4The formation of dihydrodiols by EH-mediated hydrolysis of linoleic and arachidonic acid epoxides. The size of the arrow indicates the
affinity of the substrate to sEH, thereby, the rate of hydrolysis

namely the epoxyoctadecenoic acids (EpOMEs). These

epoxides were found to regulate several physiological functions, notably cardiovascular, pulmonary and renal functions. Also, sEH contribution to the metabolism of drugs
and other xenobiotics has been reported, but it is considered minor compared with mEH.

reported to exhibit a pro-inflammatory effect, and, in contrast to EETs, sEH is the main pathway for DHETs formation (Norwood etal. 2010). Therefore, it is believed that
sEH activity is deleterious by decreasing the cytoprotective
EETs and increasing the pro-inflammatory DHETs.
The epoxides ofLA

The epoxides ofAA

Several P450 enzymes mediate the epoxygenation of AA to
5,6-, 8,9-, 11,12- and 14,15-cis-epoxyeicosatrienoic acids
in the presence of oxygen and NADPH (Capdevila etal.
2000). These P450 enzymes collectively named P450 arachidonate epoxygenases, typically, CYP2B, CYP2C and
CYP2J (Capdevila etal. 2000; El-Sherbeni and El-Kadi
2014) (Fig.4). sEH, then, catalyzes the hydration of EET
to dihydroxyeicosatrienoic acids (DHETs) (Imig 2012)
(Fig.4). EETs exhibit several beneficial effects on biological system, such as anti-inflammatory, analgesic, vasodilatory, anti-platelet, fibrinolytic and vascular smooth muscle
anti-migratory effects (Imig 2012; Sudhahar etal. 2010).
Because DHETs are less biologically active, EETs hydrolysis by sEH is considered a deactivation process. sEH mediates the main pathway for EETs deactivation, although
there are several other pathways, viz., -oxidation, chain
elongation, incorporation to plasma membrane, diffusion
and binding to plasma and tissue proteins, cyclooxygenase- and P450-mediated metabolism and chain elongation
(Imig 2012; Roman2002). Recently, DHETs have been

Linoleic acid is an essential fatty acid and represents the

major part of our dietary unsaturated fatty acids. It is
epoxidized in vivo to 9,10- and 12,13-EpOMEs. Historically, EpOMEs were first identified as neutrophil-derived
fatty acid epoxides, and therefore, EpOMEs are also
termed leukotoxins (Ozawa etal. 1988). The epoxidation can be enzyme-mediated, by P450 or cytochrome c,
or can occur by spontaneous reaction of LA with reactive
oxygen species. The most prominent P450-LA epoxygenase was reported to be CYP2C9 in human liver (Draper
and Hammock 2000) (Fig.4). On the other hand, sEH is
the enzyme responsible for the hydrolysis of EpOME to the
corresponding dihydroxyoctadecanoic acids (DiHOMEs)
(Fig. 4). In contrast to EETs, EpOMEs are cytotoxic as
proven in vivo and in vitro. They inhibit mitochondrial respiration leading to multiple organ failure such as cardiotoxicity, renal failure and adult respiratory distress syndrome
(Moran etal. 1997). However, several reports have suggested that the cytotoxic effect of EpOMEs is less potent
than of their dihydrodiol metabolites, DiHOMEs (Draper
and Hammock 2000; Mitchell etal. 2002). DiHOMEs

13


2024

also inhibit mitochondrial respiration, yet by different

mechanism, EpOMEs uncouple oxidative phosphorylation,
whereas DiHOMEs additionally inhibit the electron transport chain itself (Mitchell etal. 2002). DiHOMEs mediate the inhibition of Na+/K+-ATPase, and the induction of
nuclear factor kappa B (NF-B), in addition to mitochondrial dysfunction; consequently, they are potent cytotoxic
agents (Moran etal. 1997; Slim etal. 2001).
Modulations
Inhibition
Similar to mEH inhibitors, the first-generation sEH inhibitors worked as competitive reversible inhibitors by rapid
binding to sEH but slow dissociating from it (Morisseau
etal. 1998). An example of these epoxide-based inhibitors is chalcone oxide derivatives, such as 4-fluorochalcone oxide (Morisseau and Hammock 2005; Newman
etal. 2005). Morisseau etal. first reported that carbamide
compounds with appropriate substituents can exhibit
potent sEH inhibitory activity (Morisseau etal. 1999).
Now, several urea, amide and carbamate compounds
have been identified to exhibit nanomolar sEH inhibition
(Morisseau etal. 1999). One important urea-based sEH
inhibitor is 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU), which was taken to Phase IIA clinical trial
(Morisseau and Hammock 2013). Some modifications
were performed to the most successful sEH inhibitor
pharmacophore, urea, leading to benzamide and benzoxazole sEH inhibitors (Eldrup etal. 2009). These inhibitors have highly constrained structure sEH inhibitor,
therefore, are more specific to sEH (Shen and Hammock
2012). New sEH inhibitors exhibit much more potency,
and better pharmacokinetics than the old epoxide-based
inhibitors (Morisseau 2013). GlaxoSmithKline developed sEH inhibitor, GSK2256294, that has entered Phase
I clinical trial for COPD in male, obese and smoking
patients (Podolin etal. 2013). Very recently, the dimerization of sEH was found to be crucial for sEH activity,
introducing a novel target for allosteric sEH inhibitors
(Nelson etal. 2013). Evidently, sEH inhibitors have multiple beneficial effects on cardiovascular system, lung,
kidney, diabetes, inflammation and pain typically through
increasing EETs levels.
The effect ofsEH inhibition oninflammation Soluble
epoxide hydrolase promotes inflammation mainly by
transforming EETs to DHETs and EpOMEs to DiHOME
(Morisseau 2013). EETs have been reported to suppress
inflammation by multiple mechanisms; most importantly,
EETs inhibit tumor necrosis factor--induced activation
of NF-B, and the consequent cytokines expression and

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Arch Toxicol (2014) 88:20132032

potentiation of platelet-derived growth factor induced

cellular proliferation (Campbell 2000; Norwood etal.
2010). Also, EETs directly reduce the inflammatory
response of cytokines and inhibit the pro-inflammatory
activity of prostaglandin E2 (Campbell 2000; Morisseau 2013; Norwood etal. 2010). Furthermore, EETs
have been reported to be potent PPAR agonists (Wray
and Bishop-Bailey 2008). In contrast to EETs, DHETs
and DiHOMEs have pro-inflammatory activity, whereas
EpOMEs do not affect inflammatory cascade (Slim etal.
2001). DiHOMEs were found to induce NF-B in vascular endothelial cell cultures (Slim etal. 2001). Noteworthy, NF-B pathway regulates the expression of
several cytokines and pro-inflammatory proteins, such
as interleukin 6, vascular cell adhesion molecule-1,
cyclooxygenases and lipoxygenases. By reducing the
pro-inflammatory DiHOMEs and DHETs and inducing
the anti-inflammatory EETs, sEH inhibitors represent
a novel way to relieve inflammation (Morisseau 2013).
Therefore, sEH inhibitors have an advantage in treating
diseases with inflammatory etiology. It has been reported
that sEH inhibition increases the survival rate of mice
after lipopolysaccharide-induced acute systemic inflammation (Inceoglu etal. 2006).
The effect ofsEH inhibition oncardiovascular system Soluble epoxide hydrolase inhibitors have several
profound effects on the cardiovascular system attributed
to increasing EETs levels. EETs have a potent vasodilator activity via activating the large conductance calcium
activated K+channels on vascular smooth muscle cells
resulting in cell membrane hyperpolarization and vasodilation (Imig 2012). The first encouraging report was
the reduction in systolic blood pressure in male sEH-null
mice (Sinal etal. 2000). Therefore, sEH inhibitors have
received a lot of attention as a potential anti-hypertensive
agent, and it was found that potent sEH inhibitors have
the same reducing effect on blood pressure (Imig 2012).
EETs not only regulate blood pressure by vasodilation, but
also by inducing diuresis via promoting renal blood flow,
as well as sodium excretion (Morisseau and Hammock
2013). Controlling hypertension will relieve hypertension-related heart complications, such as cardiac hypertrophy and heart failure. Interestingly, it has been reported
that sEH inhibitors have direct cardioprotective effects;
sEH inhibitors have been successfully used for the prevention and control of cardiac hypertrophy and ischemia/
reperfusion injury (Aboutabl etal. 2011; Chaudhary etal.
2010; Imig 2012; Roman 2002). These cardioprotective
effects have been attributed to the reduction in reactive
oxygen species, apoptosis, fibrosis and inflammation, and
the induction of autophagy by EETs (Chaudhary etal.
2010; Imig 2012; Morisseau 2013; Roman 2002; Samokh-

Arch Toxicol (2014) 88:20132032

2025

Table3Polymorphisms detected in the EPHX2 that lead to change in activity

Location

Position

SNP

Outcome

References

Exon 2
Exon 3
Exon 4
Exon 8

55
103
154
287

Lys/Arg
Arg/Cys
Cys/Tyr
Arg/Gln

Exon 13

402403

insArg

Increase activity
Increase activity
Increase activity
Decrease activity and
protein stability
Decrease activity

Przybyla-Zawislak etal. (2003)

Koerner etal. (2007)
Przybyla-Zawislak etal. (2003)
Koerner etal. (2007), Przybyla-Zawislak etal. (2003),
Sandberg etal. (2000)
Sandberg etal. (2000)

Exon 16

470

Glu/Gly

Increase activity

Przybyla-Zawislak etal. (2003)

valov etal. 2013). Moreover, other studies described the

anti-arrhythmic and anti-atherosclerotic effects of sEH
inhibitors (Imig 2012).

and liver have a higher sEH content compared with female

mice (Pinot etal. 1995).
Polymorphism

The effect ofsEH inhibition onlung We mentioned before

that the only sEH inhibitor now in clinical trials is indicated
for COPD. sEH inhibitors effectively alleviate inflammation and improve lung functions in rat COPD model (Wang
etal. 2012). Acute tobacco-induced inflammation in rats
has also alleviated by sEH inhibitors administration (Smith
etal. 2005). This effect is believed to be exerted by sEH
inhibitor-dependent increase in pulmonary EETs (Morisseau 2013). However, a recent report suggested that the
anti-inflammatory effects of sEH inhibitors are mediated by
the decrease in DiHOMEs levels in tobacco-induced inflammation in spontaneous hypertensive rats (Davis etal. 2011).
Other pulmonary disease may also get advantage of sEH
inhibitors treatment including asthma, cystic fibrosis and
pulmonary artery hypertension (Morisseau 2013).
The effect ofsEH inhibition onpain One manifestation
of controlling inflammation by sEH inhibitors is relieving
inflammatory pain (Inceoglu etal. 2006). Interestingly, sEH
inhibitors were shown to block neuropathic pain as shown in
diabetic neuropathic rat model (Inceoglu etal. 2008). sEH
inhibitors achieve the antinociceptive effect by down-regulating cyclooxygenase II and up-regulating neurosteroids.
The antinociceptive effect of 14,15-EET seems to be also
mediated by the - and -opioid receptor pathway (Terashvili etal. 2008). sEH inhibitors may be exceptionally useful
as analgesic agents when used in combination with cyclooxygenase inhibitors (Schmelzer etal. 2006).

Several polymorphisms have been detected in sEH gene,

EPHX2, localized on chromosomal 8p21-p12 (PrzybylaZawislak etal. 2003). However, only six SNPs in EPHX2
have been reported to alter sEH activity as shown in
Table3. Fewer reports discussed the association of EPHX2
polymorphisms with pathological and physiological conditions in contrast to EPHX1. Of importance, a recent
report showed the increase in forearm vascular resistance
in human subjects with Lys55Arg genotype (sEH activity
increased) and the opposite effect with Arg287Gln (sEH
activity is decreased) (Lee etal. 2011). Likewise, Lys55Arg
genotype was linked to the increase in the risk of coronary
artery disease (Lee etal. 2006). Moreover, Arg287Gln genotype is associated with protection form ischemic injury in
cardiomyocytes (Morisseau 2013). Neuronal survival was
enhanced in Arg287Gln genotype after ischemic injury,
while Lys55Arg genotype increases cell death (Koerner
etal. 2007). Noteworthy, several studies reported the great
influence of factors, such as sex and race on the association
between EPHX2 polymorphism and disease risk. Intriguingly, the risk of coronary artery calcification is increased
in Arg287Gln genotype in two large clinical studies indicating the complex nature of sEH (Burdon etal. 2008; Fornage etal. 2004).

Hepoxilin, cholesterol andleukotriene A4 epoxide

hydrolases

Induction
The induction of sEH has been observed as a consequence
of exposing to PPAR agonists, such as clofibrate, fenofibrate and acetylsalicylic acid (Pinot etal. 1995). In contrast
to mEH, diabetes and starvation cause an increase in sEH
activity in rat liver that was corrected by insulin administration (Thomas etal. 1989). Moreover, male mice kidney

There are at least three more EH have been identified in

mammals, namely hepoxilin (Pace-Asciak and Lee 1989),
cholesterol (Watabe etal. 1983) and leukotriene A4 epoxide hydrolase (Thunnissen etal. 2001). They are of low
importance with respect to xenobiotic metabolism, because
their affinities to hydrolysis epoxyxenobiotics are very limited (Chen etal. 2004; Ioannides 2002; Pace-Asciak and

13


2026

Lee 1989; Watabe etal. 1983). Therefore, they have been

described to have very narrow substrate specificity that
confined to a number of endogenous epoxides (Ioannides
2002). Hepoxilin epoxide hydrolase only catalyzes the
hydrolysis of hepoxilins, which are the hydroxyepoxyeicosatrienoic acids (Decker etal. 2009). They are physiologically active AA metabolites that were found to be widely
distributed in mammals and are reported to be cytoprotective (Fretland and Omiecinski 2000). Interestingly, Cronin
etal. have found that hepoxilin epoxide hydrolase activity
in the liver is actually mediated by sEH, not by an independent enzyme (Cronin etal. 2011). For cholesterol epoxide hydrolase, it may provide a cytoprotective effect against
some toxic steroidal metabolites. It deactivates 5,6epoxycholestane-3-ol and 5,6-epoxycholestane-3-ol
that have some mutagenic effects (de Medina etal. 2010;
Fretland and Omiecinski 2000). On the other hand, leukotriene A4 hydrolase has been reported to biotransform the
less toxic leukotriene A4 to give leukotriene B4, which is a
potent proinflammatory and chemotactic agent (Chen etal.
2004; Decker etal. 2009; Rao etal. 2010). Leukotriene A4
hydrolase has been linked to atherosclerotic cardiovascular
heart diseases (Rao etal. 2010).

Conclusion remark
Epoxide hydrolases play multifaceted roles in the body.
While mEH protects the body from highly reactive compounds, sEH upregulation has been linked to several
pathological conditions. Through the same molecular
mechanism, mEH mediates the inactivation of xenobiotic
epoxides that can damage DNA and protein, and sEH mediates the biotransformation of the endogenous fatty acid
epoxides that are cytoprotective to their dihydrodiols that
are cytotoxic. Thus, there is a consensus that the inhibition
of mEH is generally detrimental, whereas the inhibition
of sEH is beneficial; however, the story is not that simple.
First, there is an overlap in substrate selectivity and therefore in physiological functions between mEH and sEH.
Thus, inhibiting sEH will affect the metabolism of reactive
metabolites, and inhibiting mEH will affect the metabolism
of epoxide of fatty acids. Second, due to the close homology and the identical molecular mechanism between mEH
and sEH, the structural requirements for their inhibitors
are closely similar. Therefore, confirming the selectivity of
an inhibitor toward either mEH or sEH is crucial point to
investigate. This point has been fulfilled in the early but not
in the recent studies developing sEH inhibitors. Lung has a
special advantage that the inhibition of mEH has beneficial
effects. The reason is that mEH has been reported to aggravate oxidative damages produced by aromatic xenobiotics,
such as PAH and benzene, present in cigarette smoke.

13

Arch Toxicol (2014) 88:20132032

Acknowledgments This work was supported by a grant from the
Canadian Institutes of Health Research (CIHR) [Grant MOP 106665].
AAE is the recipient of Egyptian Government Scholarship and
Alberta Innovates-Health Solutions studentship.

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