Mutation Research 569 (2005) 101–110

Review

Role of cytochromes P450 in chemical toxicity and oxidative

stress: studies with CYP2E1
Frank J. Gonzalez∗
Laboratory of Metabolism, National Cancer Institute, Bethesda, MD 20892, USA

Received 15 March 2004; received in revised form 14 April 2004; accepted 18 April 2004
Available online 18 November 2004

Abstract

Cytochromes P450 are responsible for metabolism of most xenobiotics and are required for the efficient elimination of foreign
chemicals from the body. Paradoxically, these enzymes also metabolically activate biologically inert compounds to electrophilic
derivatives that can cause toxicity, cell death and sometimes cellular transformation resulting in cancer. To establish the role
of these enzymes in toxicity and carcinogenicity in vivo, gene knockout mice have been developed. To illustrate the role of
P450s in toxicity, CYP2E1-null mice were employed with the commonly used analgesic drug acetaminophen. CYP2E1 is the
rate-limiting enzyme that initiates the cascade of events leading to acetaminophen hepatotoxicity; in the absence of this P450,
toxicity will only be apparent at high concentrations. Other enzymes and nuclear receptors are also involved in activation or
inactivating chemicals. CYP2E1 is induced by alcohol and the primary P450 that carries out ethanol oxidation that can lead to the
production of activated oxygen species and oxidative stress that elevate ERK1/2 phosphorylation through EGRF/c-Raf signaling.
Paradoxically, activation of this pathway inhibits apoptotic cell death stimulated by reactive oxygen generating chemicals but
accelerates necrotic cell death produced by polyunsaturated fatty acids. CYP2E1 is thought to contribute to liver pathologies
that result from alcoholic liver disease and non-alcoholic steatohepatitis.
© 2004 Elsevier B.V. All rights reserved.

Keywords: Cytochromes P450; CYP2E1; Gene knockout mice; Acetaminophen; Metabolic activation; Carcinogens; Reactive oxygen species;
Oxidative stress ERK; MAPK

Abbreviations: ASH, alcoholic steatohepatitis; P450, cytochromes P450; ERK1/2, extracellular signal-regulated kinase 1/2; NAPQI, N-
acetyl-p-benzoquinone imine; ROS, reactive oxygen species; GST, glutathione S-transferase; PPAR␣, peroxisome proliferators-activated receptor
␣; PUFA, polyunsaturated fatty acid; EGFR, epidermal growth factor receptor
∗ Tel.: +1 301 496 9067; fax: +1 301 496 8419.

E-mail address: fjgonz@helix.nih.gov.

0027-5107/$ – see front matter © 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.mrfmmm.2004.04.021
102 F.J. Gonzalez / Mutation Research 569 (2005) 101–110

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
1.1. Cytochromes P450. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
1.2. Methods for analysis of xenobiotic metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
2. Xenobiotic metabolism null mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
2.1. Acetaminophen hepatotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
3. Role of CYP2E1 in oxidative stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
3.1. Mechanism of CYP2E1 induced ROS and toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

1. Introduction ing in cell toxicity and, in some cases, cell transfor-

mation leading to cancer. The O-acetylation and O-
Cytochromes P450 are a superfamily of heme- sulfation of arylamines, such as 4-aminobiphenyl and
containing monoxygenases that metabolize a large 2-acetylaminoflourene, and the heterocyclic amines
number of compounds. While a limited number of food mutagens can also result in unstable reactive
P450s are involved in the biosynthetic pathways of metabolites that can mutate DNA. Thus, xenobiotic
steroid and bile acid production, most P450s metabo- metabolism, while essential for catabolism and elim-
lize foreign compounds or xenobiotics including drugs, ination of drugs and other foreign compounds, can
toxicants and chemical carcinogens. P450s carry out produce activated toxicants and carcinogens. It has
the oxidation of carbon and nitrogen groups usually recently been recognized that under certain circum-
resulting in the addition of an alcohol. In some cases, stances, P450s can produce reactive oxygen species
an epoxide is added to an aromatic ring that is rapidly (ROS) that result in oxidative stress and cell death.
hydrolyzed to an alcohol by non-enzymatic addition of CYP2E1 is among the most active P450 producing
water, or converted to a trans dihydrodiol by epoxide ROS. This will be discussed in detail later.
hydrolases. Many metabolites generated by P450s are
subjected to conjugation with either glucuronic acid, 1.1. Cytochromes P450
sulfate, glutathione, or acetyl groups by phase 2 en-
zymes (see below). This can result in transformation P450s are complex and diverse in their regulation
of a hydrophobic molecule to a more water soluble and catalytic activities. Cloning and sequencing of
derivative that is easier to eliminate through renal and P450 cDNAs and, more recently, total genome se-
biliary excretion. quencing have revealed the existence of 102 putatively
Oxidation and conjugation of xenobiotics have his- functional genes and 88 pseudogenes in the mouse,
torically been referred to as phase 1 and phase 2 reac- and 57 putatively functional genes and 58 pseudo-
tions. The phase 1 enzymes include not only the P450s genes in humans [1]. These genes are grouped, based
but also the flavin-containing monoxygenases (FMO) on amino acid sequence similarity, into a large num-
and epoxide hydrolases (EH). The phase 2 enzymes ber of families. While several P450s are involved in
include the glutathione S-transferases (GST), UDP- the synthesis of steroid hormones and bile acids, and
glucuronosyltransferases (UGT), N-acetyltransferases the metabolism of retinoic acid and fatty acids, includ-
(NAT) and sulfotransferases (SULT). All of these en- ing prostaglandins and eicosanoids, a limited number
zymes consist of multiple forms or superfamilies sim- of P450s (15 in humans) are primarily involved in
ilar to the P450s (see below). While oxidation and xenobiotic metabolism. The xenobiotic-metabolizing
conjugation of xenobiotics usually lead to inert and P450s are found in families 1–4. Since a single P450
soluble derivatives, in some cases oxidation can lead can metabolize a large number of structurally diverse
to highly reactive electrophilic metabolites than can compounds these enzymes can collectively metabolize
covalently react with protein, RNA and DNA result- scores of chemicals found in the diet, environment and
 F.J. Gonzalez / Mutation Research 569 (2005) 101–110
administered as drugs. While metabolism of xenobi-
otics such as drugs is required to efficiently eliminate
them from the body, as noted earlier, certain chem-
icals are metabolically activated to reactive deriva-
tives that cause cell toxicity and cancer [2]. Among
these, the P450s that metabolically activate toxicants
and carcinogens are limited to a few forms including
CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2E1, and
to a limited extent the CYP3A subfamily. CYP1A1
and CYP1B1 metabolically activate polycyclic aro-
matic hydrocarbons such as benzo[a]pyrene and 7,12-
dimethylbenz[a]anthracene. CYP1A2 carries out the
N-oxidation of arylamines and heterocyclic amine food
mutagens and CYP2E1 metabolizes numerous low Mr
toxicants and cancer suspect agents (see below).
Xenobiotic-metabolizing P450s are found at highest
levels in the liver, the mammalian metabolic clearing-
house for both exogenous and endogenous chemicals.
However, some P450s are also expressed at lower levels
in extrahepatic tissues, most notably gut, lung, kidney
and hematopoetic tissue. Drugs administered orally or
intravenously, are subject to “first pass metabolism” in
the liver that is mediated in large part by P450s. This
usually results in their biological inactivation while a
drug that escapes first pass metabolism maintains its
biological activity. In the absence of P450s and hepatic
metabolic inactivation, drugs would stay in the body
for long periods of time, a situation that would lead to
an exaggerated and prolonged response to a drug. With
orally administered drugs, metabolism can also be ini-
tiated by P450s in the small intestine. Since metabolism
is the principle determinant of plasma concentrations
of a chemical, the composition and extent of expression
of P450s in gut and liver can have a major impact on
the efficacy and toxicity of a drug.
Drug toxicity can be affected by polymorphisms
in P450s and drug interactions. Humans polymor-
phisms occur with several of the xenobiotic metabo-
lizing P450s, including CYP2A6, CYP2C9, CYP2C19
and CYP2D6; with the exception of CYP2A6 that can
activate certain nitrosamines, these enzymes are pri-
marily involved in drug metabolism. No known func-
tional polymorphisms (that affects catalytic activity)
occur with P450s that metabolize toxins and carcino-
gens albeit there are rare mutant alleles for CYP1A2
[3] and CYP1B1. Mutants in CYP1B1 cause congenital
glaucoma [4]. In spite of the lack of genetic polymor-
phisms in toxin- and carcinogen-metabolizing P450s,
103
there is still a high degree of interindividual difference
in their expression as revealed by study of human liver
specimens. For example, CYP2A6 exhibits at least a
100-fold difference in expression among human liver
samples [5]. The molecular basis for these differences
is not known but may be due to induction of the P450
by endogenous hormonal and dietary compounds that
activate gene expression.
Drug interactions occur when two drugs are co-
administered and both are metabolized by the same
P450. One drug can then inhibit the metabolism of an-
other drug leading to high serum levels and extended
biological activity and sometimes toxicity. Drug inter-
actions are a major problem in human therapy espe-
cially with CYP3A4, a P450 involved in metabolism
of over 60% of all therapeutically used drugs [6]. In
addition, CYP3A4 activity is influenced by food and
natural products such as grapefruit juice and is a target
for induction by ligands for the nuclear receptor PXR.
1.2. Methods for analysis of xenobiotic
metabolism
Metabolism of xenobiotics can be assessed by use
of cell extracts including membrane fractions derived
from liver. However, it is not certain whether data ob-
tained using primary cultures of hepatocytes reflect the
intact liver. Indeed, in rat hepatocyte cultures it is well
established that the composition of P450s can change
[7]. A major limitation of this method to study human
xenobiotic metabolism is the general lack of availabil-
ity of human liver. However, there are some limited
commercial sources of human hepatocytes and liver
slices that have been used to determine whether a chem-
ical is metabolized by P450s and how it is metabolized
including the potential for production of an activated
metabolite [8]. Recombinant P450s can then be used
to determine which particular P450 enzyme carries out
metabolism of a chemical of interest.
A number of systems have been developed to pro-
duce catalytically active P450s. These enzymes are
particularly difficult to express because they are mem-
brane bound and contain a non-covalently bound heme.
In any case, bacteria, vaccinia virus, baculovirus, Cos
cells, and lymphoblastoid cells have been used to ex-
press human P450s [9]. Today, recombinant human
P450s are available from commercial sources with the
most widely used produced using baculovirus. To de-
104 F.J. Gonzalez / Mutation Research 569 (2005) 101–110
termine the potential effects of xenobiotics in humans,
these systems are especially important since marked
species differences in P450s are known to occur. Since
rat, mouse and human liver enzymes can metabolize a
chemical quite differently, rodent model systems can-
not be used to reliably predict human safety upon expo-
sure to xenobiotics. Genetically modified mice could
be a solution to this problem.
2. Xenobiotic metabolism null mice
To determine the role of P450s in xenobiotic
metabolism and toxicity, gene knockout mice can be
used. Mice lacking several of the important toxin and
carcinogen metabolizing P450s have been made. These
include CYP1A1 [10], CYP1A2 [11], CYP1B1 [12]
and CYP2E1 [13]. Mice deficient in these enzymes or
even double null mice lacking two P450s [14] have no
deleterious phenotypes; they breed, develop normally
and exhibit no ill effects as a result of loss of P450 ex-
pression. A lack of phenotype was quite surprising in
the CYP2E1-null mice. The fact that there is no known
polymorphism in CYP2E1 and no report of any human
or human liver sample that completely lacks expres-
sion of the enzyme, would argue that this P450 has an
important role in mammalian development or physio-
logical homeostasis. In addition, CYP2E1 metabolizes
ketone bodies that are liberated during starvation indi-
cating a potentially important physiological function in
gluconoeogenesis [15]. However, CYP2E1-null mice
develop normally and show no overt deleterious phe-
notype indicating that this P450 does not have a critical
role in physiological homeostasis [13]. Taken together,
analysis of P450-null mice indicate that these enzymes
have no role in mammalian development or physiolog-
ical homeostasis but these observation do not exclude
potential subtle functions of P450s in the metabolism
of endogenous compounds [16]. However, P450-null
mice are resistant to toxicants and carcinogens [17].
Other xenobiotic-metabolizing enzymes have been
subjected to targeted gene disruption including the sol-
uble epoxide hydrolase (sEH) [18], microsomal epox-
ide hydrolase (mEH) [19], NAD(P)H:quinone oxidore-
ductase (NQO) 1 [20] and 2 [21], and glutathione S-
transferase (GST) P1 and P2 [22]. These mice also
did not display any phenotype again indicating that
other non-P450 xenobiotic-metabolizing enzymes are
not essential for reproduction, embryonic development
and physiological homeostasis. However, mice lacking
cEH, NQO1 and GSTP1/2 are resistant to toxicants and
carcinogens [23].
Mice lacking receptors that regulate the expression
of xenobiotic-metabolizing enzymes have been made
including aryl hydrocarbon receptor (AHR) [24–26],
constitutive androstane receptor (CAR) [27], and preg-
nane X receptor (PXR) [28,29]. With the exception of
AHR, these null mice also do not display any severe
phenotype suggesting that they are primarily involved
in regulating xenobiotic metabolism. However, PXR
[30–32] and CAR [33] are involved in the regulation
of bile acid metabolism and bilirubin degradation, re-
spectively [34]. While, mice lacking AHR are viable,
they have a severe liver phenotype characterized by
fibrosis [24,25,35,36]. All xenobiotic-metabolism and
receptor-null mice exhibit differences from wild-type
mice in their response to toxins and carcinogens [23].
2.1. Acetaminophen hepatotoxicity
CYP2E1 is responsible for the metabolism and
metabolic activation of a large number of low Mr chem-
icals, such as aliphatic, aromatic and halogenated hy-
drocarbons, many of which are solvents and indus-
trial monomers, and some of which are cancer sus-
pect agents [37]. Among the CYP2E1 substrates are
acetaminophen (AP), azoxymethane, benzene, chlor-
zoxazone, carbon tetrachloride, chloroform, methacry-
lonitrile, N-nitrosodimethylamine and vinyl chloride.
Thus, CYP2E1 may be an important determinant of
human susceptibility to toxicity and carcinogenicity of
industrial and environmental chemicals. Indeed, cat-
alytic activity of CYP2E1 has been associated with
susceptibility of toxicity under industrial exposure
to benzene [38]. However, while there are a num-
ber of SNPs in and around the gene, a polymor-
phism in CYP2E1 that leads to loss of expression
has not been found (http://www.imm.ki.se/CYPalleles/
cyp2e1.htm). Among the most well-studied CYP2E1
substrates is acetaminophen (AP), also called parac-
etamol.
AP is a widely used over-the-counter analgesic
that can cause hepatotoxicity, sometimes resulting in
death. This is usually caused by deliberate and acci-
dental overdosing the drug [39]. Over 30 years ago,
the hepatotoxicity of AP was found to be due to its
 F.J. Gonzalez / Mutation Research 569 (2005) 101–110
Fig. 1. Metabolism of acetaminophen by CYP2E1 and GST.
CYP2E1 produces the active metabolite from acetaminophen. The
active metabolite is a substrate for GST. Under conditions of high lev-
els of CYP2E1 and/or low levels of glutathione, the active metabolite
can bind to cellular macromolecules resulting in cell death.
metabolism and covalent binding to cellular macro-
molecules [40,41]. P450s and GSTs are largely respon-
sible for metabolism of the drug (Fig. 1). AP is con-
verted by P450s to a highly reactive quinone metabo-
lite, N-acetyl-p-benzoquinone imine (NAPQI) that can
produce reactive oxygen species and covalently bind
to cellular nucleophiles such as DNA, RNA and pro-
teins, resulting in cell death. Typically, this metabo-
lite is inactivated by glutathione conjugation to the
electrophilic P450-derived quinone metabolite. Toxi-
city of AP is enhanced by depletion of hepatic levels of
glutathione. Inhibition of glutathione synthesis exac-
erbates toxicity and administration of the glutathione
precursor N-acetylcysteine can be used for therapeutic
intervention by elevating the level of glutathione. Sul-
fotransferases can also neutralize the activated metabo-
lites formed by P450s. CYP2E1 is the principle P450
responsible for metabolism of AP [13]. This offered
a possible explanation why ethanol, an inducer of
CYP2E1, increased the LD50 for the drug in rats
[42] and why AP-induced hepatotoxicity is frequently
associated with alcohol consumption in humans
[42].
105
Fig. 2. The role of P450s in acetaminophen toxicity determined using
P450-null mice. AP was administered by intraperitoneal injection
to wild-type mice (䊉) and mice lacking CYP1A2 (), CYP2E1
(
) and both CYP1A2 and CYP2E1 (). The percent survival was
determined as described [14].
The CYP2E1-null mouse model has been used to
study a number of chemicals that are of concern for hu-
man risk assessment including susceptibility to AP hep-
atoxicity. Testing CYP2E1-null mice for sensitivity to
AP hepatotoxicity provided evidence that CYP2E1 was
responsible for initiating the cascade of toxic metabo-
lites from AP. Mice lacking CYP2E1 were highly resis-
tant to liver toxicity as compared with wild-type mice
[13,14]. Mice lacking both CYP2E1 and CYP1A2 were
almost totally resistant to liver toxicity at the highest
doses of AP tested (Fig. 2). The toxicity of AP is also
influenced by the genetic status of GSTP1/2 [43] and
the nuclear receptor CAR [44], an inducer of GSTPi.
3. Role of CYP2E1 in oxidative stress
Oxidative stress is the result of an increase in in-
tracellular prooxidant species such as H2 O2 , hydroxy
radicals (• OH) and superoxide anion radical (O2 − ).
High intracellular levels of these molecules, collec-
tively called reactive oxygen species (ROS), can lead
to damaged mitochondria, DNA modification, lipid
peroxidation, elevated cytokine production and even
cell death. In mammals, ROS can result in a num-
ber of pathological conditions including Alzheimer’s
disease, atherosclerosis, cancer, diabetes, chronic ob-
structive pulmonary disease, rheumatoid arthritis and
various neurological disorders [45]. Oxidative stress
106 F.J. Gonzalez / Mutation Research 569 (2005) 101–110
is also thought to be the principle cause of aging
[46].
Chronic consumption of alcohol can lead to hepato-
cyte cell death and cirrhosis. Indeed, alcohol-induced
liver damage is thought to be due to oxidative stress
that results from alcohol consumption [47]. Experi-
mentally, ethanol-induced oxidative stress can be in-
hibited by the administration of antioxidants including
precursors to glutathione, as described above for AP.
Metabolism of ethanol by alcohol dehydrogenase can
increase oxidative stress. CYP2E1, which can metab-
olize ethanol to acetaldehyde and 1-hydroxyethyl rad-
ical, produces ROS. On this basis, CYP2E1 is thought
to have a role in alcoholic steatohepatitis (ASH) that
leads to cirrhosis. As noted above, CYP2E1 also me-
tabolizes a large number of other low Mr chemicals to
reactive intermediates that themselves can bind to cel-
lular macromolecules such as DNA, RNA and protein
causing cell damage, hepatitis and cirrhosis. During
the course of the P450 catalytic cycle, P450s use H+
from NADPH to reduce O2 leading to the production
of H2 O2 and superoxide anion radical. The process of
uncoupling of the catalytic cycle can lead to escape of
O2 − . Thus, CYP2E1 metabolism of a number of its
substrates is known to lead to increased ROS. Interest-
ingly, even in the absence of substrate, expression of
CYP2E1 can generate ROS [48,49]. This phenomenon,
known as futile cycling, is due to P450-derived NADPH
oxidase activity that is independent of exogenous xeno-
biotic substrates [50].
Similar to ASH, induction of non-alcoholic steato-
hepatitis (NASH) is thought to be due to elevated ex-
pression of CYP2E1 and the resultant oxidative stress
[51–53]. ROS from CYP2E1 can produce lipid perox-
ides from polyunsaturated fatty acids (PUFA) and these
can react with other cellular macromolecules causing
cell toxicity and death. It is likely that the fatty livers
directly cause induction of CYP2E1 that contributes to
lipid peroxide production. In a rat model of NASH, pro-
duced by feeding a high fat diet, CYP2E1 is markedly
induced [54]. In a genetic model of methionine adeno-
syltransferase 1A (MAT1A) deficiency, NASH spon-
taneously develops and is associated with elevated
CYP2E1 expression and increased lipid peroxidation
that is decreased by addition of the CYP2E1 inhibitor
diallyl sulfide [55]. The increase in CYP2E1 in these
models is likely due to ␤-oxidation of fatty acids that
accumulate in liver resulting in high levels of ketone
bodies that are inducers of CYP2E1. Thus, increase in
hepatic fatty acids leads to an increase in ketone bod-
ies as a result of fatty acid ␤-oxidation in peroxisomes
and mitochondria. The ketone bodies in turn induce
CYP2E1 expression [56]. The elevated CYP2E1 can
then produce ROS that produces lipid peroxides. In hu-
mans CYP2E1 protein is spontaneously induced in the
livers of NASH patients [57]. To measure CYP2E1 ex-
pression in vivo, chlorzoxazone pharmacokinetics has
been used an in vivo metabolic probe. Obese humans
had elevated CYP2E1 activity and protein indicating
that CYP2E1 is related to or caused by hepatic pathol-
ogy resulting from morbid obesity; the level of P450
was correlated with the degree of steatosis [58]. As in
rodents a similar high level of CYP2E1 activity was
found in non-diabetic patients with NASH, indicating
that CYP2E1 is elevated in humans with this condi-
tion [59]. Chlorzoxazone metabolism in vivo was also
used to determine that Type II diabetics have elevated
CYP2E1 expression [60]. No significant difference was
found in Type I diabetics. While no significant dif-
ference was found between diabetics and controls in
CYP2E1 activity in vivo but an increase with obesity
was found [61].
It should be emphasized that the situation may dif-
fer between humans and rodents. Mice can be induced
to develop NASH by feeding a methionine and choline
deficient (MCD) diet. However, CYP2E1-null mice as
well as wild-type mice also develop the steatotic liver
phenotype and still produce significant lipid peroxi-
dase activity due to expression of other P450s such as
CYP4A10 and CYP4A14 [52]. The latter P450s are
induced through activation of the nuclear receptor per-
oxisome proliferators-activated receptor ␣ (PPAR␣).
Indeed, peroxisome proliferators-activated receptor ␣
(PPAR␣)-null mice are protected against diet induced
NASH probably due to the enhanced metabolism of
fatty acids and the removal of PUFAs from the liver
[62]. It should be recognized that a major species dif-
ference in PPAR␣ inducibility exist between rodent
models and humans. Mice and rats, CYP4A P450s are
readily induced whereas humans are refractive to in-
duction [63]. Thus, the results in animal models must
be interpreted with caution [64].
CYP2E1 also appears to be involved in kidney tox-
icity due to oxidative stress. Treatment of mice with
the anticancer drug cisplatin produces nephrotoxicity
indicated by elevated serum creatinine and urea, de-
 F.J. Gonzalez / Mutation Research 569 (2005) 101–110 107

creased creatinine clearance, and increased catalytic sitizes cells to exogenous agents such as menadione
iron [65]. This toxicity is markedly attenuated in mice and PUFAs as a result of activation of the EGFR/c-Raf
lacking CYP2E1. In vitro, kidney slices from CYP2E1- ERK1/2 signal transduction pathway. This can either
null mice treated with the drug produced considerably lead to protection against menadione-induced apopto-
less H2 O2 than those from wild-type mice. Cisplatin- sis or lead to an increase in PUFA-stimulated cell necro-
induced kidney cell death was due to apoptosis [65]. sis. It should be noted that these studies are largely
These studies show the role of CYP2E1 in ROS- derived from experiments using cultured cells where
induced disease is not restricted to kidney. CYP2E1 is over-expressed. It remains to be determined
whether this enzyme plays a role in non-xenobiotic me-
3.1. Mechanism of CYP2E1 induced ROS and diated hepatotoxicity and cell death leading to cirrhosis
toxicity in intact liver. The role of CYP2E1 in ERK1/2 signaling
in intact liver could be determined in the CYP2E1-null
A role for CYP2E1 in production of ROS and its mouse model [13].
consequences in intact cells has been investigated in The role of CYP2E1 in hepatoxicity can be illus-
cell lines stably expressing recombinant enzymes [66]. trated in the diagram shown in Fig. 3. CYP2E1 can me-
This system has been of great value in determining the tabolize substrates (R), and depending on the nature of
specific role of CYP2E1 in producing oxidative stress the compound, can produce electrophilic metabolites
in intact cells and the molecular consequence of the that can cause cell toxicity by reacting with cellular
oxidative stress [49]. Using a similar system, the mech- macromolecules. In the presence or even the absence
anism by which ROS produced by CYP2E1 affects of substrate, CYP2E1, with O2 and NADPH, can pro-
cell toxicity has recently been investigated [67]. Over-
expression of CYP2E1 in a hepatocyte-derived cell
line, results in an increase in phosphorylated extracel-
lular signal-regulated kinase 1/2 (ERK1/2), a mitogen-
activated protein kinase (MAPK) that is activated by
ROS. Treatment with menadione, a quinone that gen-
erates reactive oxygen, resulted in a markedly en-
hanced phosphorylation of ERK1/2 in the CYP2E1-
expressing cells as compared to control cells. Surpris-
ingly this is associated with protection of the cells from
menadione-induced apoptotic cell death. ERK1/2 in-
hibitors were used to demonstrate that the resistance
of CYP2E1-expressing cells to menadione-stimulated
cell death was due to the epidermal growth factor re-
ceptor (EGFR)c-Raf signal transduction pathway [68].
In contrast to the results with menadione, treatment of
CYP2E1-expressing cells with a PUFA such as arachi-
donic acid results in enhanced cell death that is inhib-
ited by antioxidants. PUFA treatment also caused an in-
crease in phosphorylation of ERK/1/2 in the CYP2E1-
expressing cells, but in contrast to menadione treat- Fig. 3. Schematic of the influence of CYP2E1 in generation of ROS
ment, inhibition of the ERK1/2 enhances cell death that and cell toxicity. Ethanol can induce expression of CYP2E1 result-
is due largely to necrosis, not apoptosis. PUFA-induced ing in elevated ROS. Fatty acids, most importantly PUFAs can be
cell death is thought to be the result of peroxidation of subjected to peroxidation leading to reactive species that can dam-
the unsaturated bonds in the fatty acids and their sub- age cellular macromolecules. Fatty acids can also be metabolized to
ketone bodies that can induce expression of CYP2E1 and be me-
sequent reaction with cellular macromolecules. These tabolized by CYP2E1 leading to even more ROS production. The
studies with CYP2E1-expressing cells suggests a low ROS increases phosphorylated ERK1/2 leading to gene activation
level of ROS produced by CYP2E1 expression sen- and modulation of apoptosis and necrosis.
108 F.J. Gonzalez / Mutation Research 569 (2005) 101–110
duce ROS that can cause cell toxicity by poisoning
mitochondria or reacting with PUFAs. Alcohol and a
fatty diet can affect the extent of toxicity by directly
and indirectly inducing levels of CYP2E1 with the re-
sultant increase in ROS. ROS stimulated cell toxicity
and cell death is influenced by a yet to be determined
mechanism through activation of the EGFR/c-Raf path-
way involving the MAPK ERK1/2. There remains the
prospect that inhibitors of CYP2E1 may be of value
as therapeutic aids for treatment of ASH and NASH in
intriguing [69].
4. Conclusions
P450s are required for the metabolism and inacti-
vation of drugs and to facilitate the removal of xeno-
biotics from the body. However, they are also required
for the metabolic activation of many toxicants and car-
cinogens. CYP2E1 is able to activate a large number
of chemicals and is required for the heptotoxicity of
toxins such as chloroform [70] and carbon tetrachlo-
ride [71]. CYP2E1 is also thought to play a role in
hepatic injury produced by ASH and NASH through
its ability of produced ROS. Therapeutic intervention
through inhibition of CYP2E1 activity for treatment
of liver toxicity could be possible since mice lacking
expression of the enzyme are normal.
Finally, CYP2E1 is able to produce oxidative stress
directly through NADPH oxidase activity and directly
through metabolism of xenobiotics. Metabolic activa-
tion of many of its substrates can lead to activated
metabolites that damage cellular macromolecules.
Both pathways can lead to DNA damage and, in same
cases, gene mutation and cell transformation. Thus, this
P450 is a critical mediator of toxicity such as that as-
sociated with ASH and NASH and carcinogenicity of
low Mr carcinogenic agents.
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