DRUG METABOLISM REVIEWS

Vol. 36, Nos. 3 & 4, pp. 511–529, 2004

The Discovery of the Microsomal Ethanol Oxidizing System

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and Its Physiologic and Pathologic Role

Charles S. Lieber, M.D., M.A.C.P.*

Mount Sinai School of Medicine, Section of Liver Disease and Nutrition and
Alcohol Research Center, Bronx Veterans Affairs Medical Center, USA

ABSTRACT
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Oxidation of ethanol via alcohol dehydrogenase (ADH) explains various metabolic

effects of ethanol but does not account for the tolerance. This fact, as well as the
discovery of the proliferation of the smooth endoplasmic reticulum (SER) after
chronic alcohol consumption, suggested the existence of an additional pathway which
was then described by Lieber and DeCarli, namely the microsomal ethanol oxidizing
system (MEOS), involving cytochrome P450. The existence of this system was
initially challenged but the effect of ethanol on liver microsomes was confirmed by
Remmer and his group. After chronic ethanol consumption, the activity of the MEOS
increases, with an associated rise in cytochrome P450, especially CYP2E1, most
conclusively shown in alcohol dehydrogenase negative deer mice. There is also cross-
induction of the metabolism of other drugs, resulting in drug tolerance. Furthermore,
the conversion of hepatotoxic agents to toxic metabolites increases, which explains the
enhanced susceptibility of alcoholics to the adverse effects of various xenobiotics,
including industrial solvents. CYP2E1 also activates some commonly used drugs
(such as acetaminophen) to their toxic metabolites, and promotes carcinogenesis. In
addition, catabolism of retinol is accelerated resulting in its depletion. Contrasting
with the stimulating effects of chronic consumption, acute ethanol intake inhibits the
metabolism of other drugs. Moreover, metabolism by CYP2E1 results in a significant
release of free radicals which, in turn, diminishes reduced glutathione (GSH) and other

*Correspondence: Charles S. Lieber, M.D., M.A.C.P., Professor of Medicine and Pathology,

Mount Sinai School of Medicine, Bronx Veterans Affairs Medical Center, USA.

511

DOI: 10.1081/DMR-200033441 0360-2532 (Print); 1097-9883 (Online)

Copyright D 2004 by Marcel Dekker, Inc. www.dekker.com
 512 Lieber

defense systems against oxidative stress which plays a major pathogenic role in
alcoholic liver disease. CYP1A2 and CYP3A4, two other perivenular P450s, also
sustain the metabolism of ethanol, thereby contributing to MEOS activity and possibly
liver injury. CYP2E1 has also a physiologic role which comprises gluconeogenesis
from ketones, oxidation of fatty acids, and detoxification of xenobiotics other than
ethanol. Excess of these physiological substrates (such as seen in obesity and diabetes)
also leads to CYP2E1 induction and nonalcoholic fatty liver disease (NAFLD), which
includes nonalcoholic fatty liver and nonalcoholic steatohepatitis (NASH), with
pathological lesions similar to those observed in alcoholic steatohepatitis. Increases of
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CYP2E1 and its mRNA prevail in the perivenular zone, the area of maximal liver
damage. CYP2E1 up-regulation was also demonstrated in obese patients as well as in
rat models of obesity and NASH. Furthermore, NASH is increasingly recognized as a
precursor to more severe liver disease, sometimes evolving into ‘‘cryptogenic’’
cirrhosis. The prevalence of NAFLD averages 20% and that of NASH 2% to 3% in the
general population, making these conditions the most common liver diseases in the
United States. Considering the pathogenic role that up-regulation of CYP2E1 also
plays in alcoholic liver disease (vide supra), it is apparent that a major therapeutic
challenge is now to find a way to control this toxic process. CYP2E1 inhibitors oppose
alcohol-induced liver damage, but heretofore available compounds are too toxic for
clinical use. Recently, however, polyenylphosphatidylcholine (PPC), an innocuous
mixture of polyunsaturated phosphatidylcholines extracted from soybeans (and its
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active component dilinoleoylphosphatidylcholine), were discovered to decrease

CYP2E1 activity. PPC also opposes hepatic oxidative stress and fibrosis. It is now
being tested clinically.

Key Words: Microsomal ethanol oxidizing system; CYP2E1; Alcohol

dehydrogenase; Nonalcoholic steatohepatitis; Nonalcoholic fatty liver disease.

INTRODUCTION

The discovery of the microsomal ethanol oxidizing system (MEOS) (Lieber and
DeCarli, 1968), a new pathway of ethanol metabolism, was part of a fundamental
change in the scientific approach to alcoholic liver disease. For many decades, the
dogma had prevailed, as clearly expressed by Charles Best, the co-discoverer of insulin,
that ‘‘there is no more evidence of a specific toxic effect of pure ethyl alcohol upon
liver cells than there is for one due to sugar’’ (Best et al., 1949). The basis of this
dogma was the observation that rats fed alcohol in the drinking water did not develop
liver lesions unless their diet was deficient. We confirmed these findings but observed
that, in view of the rat’s aversion for alcohol, blood levels of alcohol in these rats were
below those observed in patients who develop alcoholic liver disease. When the rat’s
aversion for alcohol was overcome by incorporating ethanol into totally liquid diets,
higher blood levels, comparable to those seen in alcoholics, were achieved. Under those
conditions, the first stage of alcoholic liver disease, namely fatty liver, was reproduced
(Lieber et al., 1963). As a result, the concept of alcohol as a hepatotoxic drug evolved
and prompted a number of studies to elucidate the mechanism of the toxicity. It was
known that alcohol was metabolized mainly by the hepatic ADH with a minor
contribution of hepatic catalase. In the ADH pathway, the oxidation of ethanol to
acetaldehyde is coupled with the reduction of NAD+ to NADH, and the resulting redox
 The Discovery of the Microsomal Ethanol Oxidizing System 513
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Figure 1. Hepatic, nutritional, and metabolic abnormalities after ethanol abuse. Malnutrition,
whether primary and secondary, can be differentiated from metabolic changes or direct toxicity,
resulting partly from ADH-mediated redox changes, effects secondary to microsomal induction, or
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acetaldehyde production. MEOS, microsomal ethanol oxidizing system; GSH, glutathione [From
Lieber (1998)].

change produces a number of metabolic effects (Fig. 1), including alterations of lipid
oxidation and synthesis, as reviewed elsewhere (Lieber, 2000). However, not all the
effects of ethanol could thereby be understood, including the fact that chronic alcohol
consumption results in a metabolic adaptation, namely an increased capacity to get rid
of the alcohol (Hawkins et al., 1966; Lieber and DeCarli, 1970). This could not be
explained by the ADH pathway, which is not inducible at the blood levels achieved in
vivo in humans and the corresponding concentrations in experimental animals. This
suggested the possibility of an additional pathway for ethanol metabolism, a hypothesis
which had also been raised by the morphologic finding that chronic administration of
ethanol to rats results in a proliferation of the smooth endoplasmic reticulum (SER)
(Iseri et al., 1964, 1966) (Fig. 2), an observation similar to the one made by Remmer
and Merker (1964) after the administration of variety of other drugs, including
hepatotoxic ones. The proliferation of the SER after ethanol was also shown in human
livers (Lane and Lieber, 1966). A number of studies, including prominently those by
Remmer and Merker (1965), had shown that proliferation of this SER seen after the
administration of various other drugs was the counterpart of an adaptive increase of the
activity of the enzymes metabolizing these drugs and which were located on these
proliferating membranes. These observations lead us to conclude that the proliferation
of the SER we had discovered after ethanol administration was ‘‘not to be considered
specific for alcohol toxicity since proliferation of the SER has been seen after the
administration of a number of toxic agents,’’ and we formulated the hypothesis that
‘‘the changes observed may reflect the induction of alcohol-metabolizing enzymes’’
(Lane and Lieber, 1966). This hypothesis was then verified by the discovery of a new
 514 Lieber
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Figure 2. A. Portion of a hepatocyte of rat fed ethanol-containing diet for 16 days. Large clusters
of smooth endoplasmic reticulum (SER) are prominent. The Golgi apparatus (G) near the nucleus
has many vesicles filled with pale granules.  17,200 [From Iseri et al. (1966)]. B. Rat fed the
sucrose control diet. Portion of a hepatocyte immediately adjacent to a central vein. It shows the
usual prominent rough endoplasmic reticulum but the smooth endoplasmic reticulum is much less
abundant than in A.  17,000 [From Iseri et al. (1966)].

pathway of ethanol metabolism located in the endoplasmic reticulum, namely the

MEOS, both in rats and in man (Lieber and DeCarli, 1968).

ISOLATION AND CHARACTERIZATION OF THE MEOS

Its Differentiation from ADH and Catalase

For many decades, the dogma had prevailed that ADH was the only significant
pathway for ethanol metabolism. Indeed, the multiple forms of this cytosolic enzyme
catalyze the conversion of ethanol to acetaldehyde, coupled with the reduction of
 The Discovery of the Microsomal Ethanol Oxidizing System 515

NAD+ to NADH. This results in a strikingly altered NAD/NADH ratio and an

associated redox change, shown to be responsible for many metabolic effects of ethanol
(Fig. 1) (Lieber, 1992, 1998) but not all (see Introduction). The proposal that the newly
discovered MEOS pathway plays a significant role in ethanol metabolism and toxicity
initiated a decade of lively debate with several groups objecting to it: some invoked
ADH contamination of the liver microsomes (Isselbacher and Carter, 1970), whereas
others claimed that the MEOS was due to a hydrogen peroxide-dependent reaction
promoted by contaminating catalase (Ziegler, 1972). Specifically, it had been
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postulated that the combination of H2O2 generation from NADPH oxidase and catalase
could account for microsomal ethanol oxidation (Roach et al., 1969; Tephly et al.,
1969), because an H2O2-generating system (glucose – glucose oxidase) can be
substituted for NADPH. Actually, the latter is not unexpected, because not only do
microsomes contain catalase, but commercial glucose oxidase (used to generate
NADPH) is contaminated with catalase. It had also been reported that microsomes from
acatalasemic mice fail to oxidize ethanol (Vatsis and Schulman, 1973) but this claim
was subsequently retracted (Vatsis and Schulman, 1974). Indeed, the catalatic activity
of hepatic microsomes of acatalasemic mice subjected to heat inactivation was
decreased but NADPH-dependent MEOS remained active and unaffected (Lieber and
DeCarli, 1974; Teschke et al., 1975a).
Eventually, MEOS was solubilized and separated from ADH and catalase activities
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by diethylaminoethyl cellulose column chromatography (Teschke et al., 1972, 1974a),

as confirmed by others (Mezey et al., 1973). Furthermore, whereas catalase reacts
peroxidatically primarily with methanol and ethanol but not with alcohols of longer
aliphatic chains (Chance, 1947), the NADPH-dependent MEOS was capable of
metabolizing n-propanol as well as n-butanol. This was shown in hepatic microsomal
preparations and in reconstituted systems that contained the microsomal components
cytochrome P450, NADPH-cytochrome P450 reductase and phospholipids, and
exhibited no ADH or catalase activity (Chance, 1947; Mezey et al., 1973; Teschke
et al., 1972, 1974a, 1974b, 1975a, 1975b). Such a system was also reconstituted from
acatalasemic mice (Teschke et al., 1975a). Finally, successful reconstitution was
accomplished with either partially purified or highly-purified microsomal cytochrome
P450 from alcohol (Ohnishi and Lieber, 1977) or phenobarbital (Miwa et al., 1978)
treated rats. Based on these and various other studies, and despite the original claim to
the contrary (Thurman et al., 1972), it was finally agreed by the principal contenders
involved that catalase cannot account for the microsomal ethanol oxidation (Teschke
et al., 1977; Thurman and Brentzel, 1977), and that the MEOS is distinct from ADH
and catalase and dependent on cytochrome P450, as reviewed elsewhere (Lieber,
1997a). This pathway was also described in humans (Lieber and DeCarli, 1968, 1970;
Song et al., 1986; Wrighton et al., 1986) and, in a new nomenclature system, it was
proposed that the ethanol-inducible cytochrome P450 be designated as CYP2E1
(Nelson et al., 1993). This CYP2E1 was found to be increased 4- to 10-fold in liver
biopsies of recently drinking subjects (Tsutsumi et al., 1989), with a corresponding rise
of its mRNA (Takahashi et al., 1993).

‘‘Ethanol-Specific’’ Cytochrome P450

Despite the discovery of CYP2E1 and its prevailing role in microsomal ethanol
oxidation, the term MEOS was maintained because cytochromes P450 other than
 516 Lieber

CYP2E1, as well as hydroxy radicals, can contribute to ethanol metabolism in the

microsomes. Indeed, in rat liver microsomes, ethanol is oxidized not only by CYP2E1
but also by CYP1A2 (Kunitoh et al., 1993). In humans also, there were some
indications of the involvement of P450s other than CYP2E1 (Lasker et al., 1987).
Specifically, it was demonstrated that human CYP2E1 and CYP1A2 (expressed in
b-lymphoblastoid cells) can oxidize ethanol (Asai et al., 1996). Furthermore, human
CYP2E1, CYP1A2, and CYP3A4 were heterologously expressed in HepG2 cells, and
their ethanol oxidation was assessed using corresponding selective inhibitors (Salmela
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et al., 1998). All three P450 isoenzymes metabolized ethanol. Selective inhibitors also
decreased microsomal ethanol oxidation in the livers of 18 organ donors. The P450-
dependent ethanol oxidizing activities correlated significantly with those of the specific
mono-oxygenases and the immunochemically determined microsomal content of the
respective P450. The mean CYP2E1-dependent ethanol oxidation was twice that of
CYP1A2 or CYP3A4. Thus, CYP2E1 plays the major role in the ethanol oxidation by
human liver microsomes, but since the combined activity of CYP1A2 and CYP3A4 is
comparable to that of CYP2E1, these P450s can significantly contribute to microsomal
ethanol oxidation and may, therefore, also be involved in related pathophysiology.
Accordingly, the term MEOS was maintained to characterize total microsomal ethanol
oxidation, not only that catalyzed by CYP2E1.
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POLYMORPHISM OF CYP2E1

Polymorphic sites in the 5’-flanking region of the human cytochrome 2E1 gene
have been reported with several restriction fragment length polymorphisms that may
affect transcriptional regulation or the functional activity of the expressed protein
(Hayashi et al., 1991; Hirvonen et al., 1993; Persson et al., 1993; Uematsu et al., 1991,
1994; Watanabe et al., 1994) as reviewed in detail elsewhere (Lieber, 1999). Indeed, in
humans, interindividual variability of CYP2E1 levels is great but it is probably due to
differing induction levels as a result of environmental factors, since no relationship was
found between any of the three known polymorphisms of CYP2E1 and its activity
(Carriere et al., 1996). Thus, although CYP2E1 is involved in the activation of
various carcinogens, at the present time none of the polymorphic sites determined in
the CYP2E1 gene can be used as a marker for increased risk (Rannug et al., 1995).
Specifically, RsaI polymorphism did not affect lung cancer risk (Watanabe et al., 1995).

REGULATION OF CYP2E1 EXPRESSION

The regulation of CYP2E1 expression is complex, involving transcriptional,

posttranscriptional, and posttranslational events (Lieber, 1999).
The rat hepatic CYP2E1 gene is transcriptionally activated within 1 day after birth
(Ueno and Gonzalez, 1990). This activation is accompanied by a demethylation of
cytosine residues located within the 5’-flanking region of the gene, suggesting that
methylation of specific residues in the 2E1 gene is responsible for the lack of
transcription of the 2E1 gene in fetal liver. CYP2E1 remains relatively stable during
 The Discovery of the Microsomal Ethanol Oxidizing System 517

the remainder of the life span. One of the most characteristic features that distin-
guishes CYP2E1 from other ethanol-metabolizing pathways is its striking inducibility,
demonstrated in several species, including humans (Tsutsumi et al., 1989) (Fig. 3).
Several studies supported the role of increased CYP2E1 mRNA and enhanced de novo
enzyme synthesis in the induction process. Kubota et al. (1988) found that induction of
CYP2E1 protein in hamsters by ethanol and pyrazole was associated with an increase
in translatable CYP2E1 mRNA, while Kim and Novak (1990) observed increased rates
of [14C] leucine incorporation into CYP2E1 protein after treatment of rats with pyri-
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dine, a phenomenon attributed to the enhancement, by pyridine, of CYP2E1 mRNA

translational efficiency (Kim et al., 1990). Elevated levels of hepatic CYP2E1 mRNA
were also described in alcohol-treated rats (Diehl et al., 1991; Ingelman-Sundberg et al.,
1988) and enhanced levels of both hepatic CYP2E1 protein and mRNA were found in
actively drinking patients (Takahashi et al., 1993). The latter study also showed that in
control subjects, CYP2E1 protein distribution, assessed by the immunohistochemical
staining of liver sections with anti-CYP2E1 antibodies, was found to be analogous to
that of the mRNA. Cellular levels of CYP2E1 protein and its mRNA were significantly
correlated (Tsutsumi et al., 1993), which indicated that mRNA stabilization and/or
transcriptional activation are involved in ethanol-mediated CYP2E1 induction in
humans. By measuring both the synthesis and the degradation of radiolabeled CYP2E1
protein at induced high steady-state levels in the rat, ethanol appeared to stimulate rates
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of de novo CYP2E1 synthesis but had no effect on rates of enzyme degradation

(Tsutsumi et al., 1993). Therefore, it is reasonable to assume that the enhancement of
de novo CYP2E1 synthesis noted under these conditions in ethanol-treated rats results
from increased steady-state levels of CYP2E1 message and/or the increased efficiency
with which this mRNA is translated. CYP2E1 induction appears to occur via two steps:

Figure 3. Hepatic cytochrome P4502E1 levels in alcoholics and in nondrinkers. Cytochrome

P4502E1 was quantitated by scanning Western blots of microsomes obtained from percutaneous
liver biopsies using anti-CYP2E1 antibodies [From Lieber (1997a)].
 518 Lieber

a post-translational mechanism at low ethanol concentrations and an additional

transcriptional one at high ethanol levels (Badger et al., 1993; Ronis et al., 1993).

PHYSIOLOGICAL ROLE OF CYP2E1

As reviewed elsewhere (Lieber, 1999), CYP2E1 is highly conserved within the

human population, suggesting significant physiological functions. Indeed, there appears
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to be a dual physiological role of CYP2E1 (Fig. 4), namely one of detoxification and one
of nutritional support. That CYP2E1 contributes to the defense mechanisms of the body
against the penetration of toxic xenobiotics is suggested by its location and inducibility at
port of entries into the body, and by its broad substrate specificity. Indeed, consistent
with such a role is the high concentration of CYP2E1 in the nose and oropharynx
(exposed to airborne xenobiotics) and in the liver, which filters all the portal circulation
and traps xenobiotics entering the body through the gastro-intestinal tract. The high
inducibility of CYP2E1 at these sites with their adaptive response to xenobiotics is
suggestive of a protective role. Thus, one can speculate that CYP2E1 may have resulted
from the evolutionary advantage of having a system in place that is characterized by a
very broad substrate specificity or even a relative absence of such specificity, which then
allows it to presumably detoxify a variety of newly emerging xenobiotic agents. On the
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downside of this, however, is the enhanced activation of carcinogens and the

corresponding increased carcinogenicity in these tissues (see ‘‘Role of Free Radical
Generation and Xenobiotic Activation in Hepatotoxicity and Carcinogenicity’’).
Regarding xenobiotics such as alcohol, CYP2E1 may play a dual detoxification
role: through its location in the liver, it may oppose the ethanol resulting from
gastrointestinal fermentation to enter the systemic circulation. Moreover, through its
marked inducibility at high ethanol concentration, it may prevent alcohol that has
entered the circulation from reaching excessive levels. This inducibility was shown not
only in experimental animals (Lieber and DeCarli, 1972) but also in man (Salaspuro
and Lieber, 1978). In volunteers, chronic administration of substantial amounts of
ethanol resulted in a progressively increased rate of clearance of alcohol from the
blood. This accelerated blood alcohol disappearance occurred exclusively at relatively

Figure 4. Physiologic and toxic roles of CYP2E1, the main cytochrome P450 of the microsomal
ethanol oxidizing system (MEOS). Many endogenous or xenobiotic compounds, including ethanol,
ketones and fatty acids, are substrates for CYP2E1 and induce its activity through various
mechanisms (see text), resulting in an array of beneficial as well as harmful effects [From
Lieber (1999)].
 The Discovery of the Microsomal Ethanol Oxidizing System 519

high concentrations of ethanol, the kinetics of which clearly indicate that a system is
involved with a Km of approximately 10mM, which is consistent with that of MEOS
(and CYP2E1) and at least 1– 2 orders of magnitude higher than that of ADH. Thus,
despite the fact that, at low ethanol concentrations, liver ADH has a much higher
capacity for ethanol oxidation than the CYP2E1 system, it is the MEOS, and
particularly its CYP2E1, which accounts for the metabolic adaptation to high
concentrations of ethanol that develops upon chronic ethanol consumption.
In terms of its nutritional role, CYP2E1 is inducible by fasting in the rat (Koop and
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Tierney, 1990). The increase may be due, at least in part, to ketones. Indeed, in rats
(Casazza et al., 1984), rabbits (Koop and Casazza, 1985), and humans (Reichard et al.,
1986), acetone appears to be actively utilized, being metabolized by a microsomal
acetone mono-oxygenase identified as CYP2E1 (Casazza and Veech, 1985; Johansson
et al., 1986). Acetone is both an inducer and a substrate of CYP2E1 (Koop, 1992; Yang
et al., 1990). The existence of a gluconeogenic pathway for acetone was shown by the
incorporation of [14C] acetone into glucose and amino acids during fasting and diabetic
ketoacidosis (Owen et al., 1982; Reichard et al., 1979, 1986) accounting for 10% of the
gluconeogenic demands. The conversion of acetone to acetol and then to methyl-
glyoxal, both intermediates in the gluconeogenic pathway, was demonstrated in vitro
(Casazza and Fu, 1985; Casazza et al., 1984; Koop and Casazza, 1985). The con-
tribution of CYP2E1 to the biotransformation of acetone was also shown in vivo by the
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increase in blood acetone after the CYP2E1 inhibitors diallyl sulfide, diallyl sulfoxide,
and diallyl sulfone (Chen et al., 1994).
The activity of CYP2E1 in fatty acid metabolism also illustrates the nutritional role
for CYP2E1. Indeed, CYP2E1, in addition to its ethanol oxidizing activity, catalyzes fatty
acid o-1 and o-2 hydroxylations (Adas et al., 1998; Amet et al., 1994; Laethem et al.,
1993). Ethanol feeding also results in an increased activity of CYP4A1 (Ma et al., 1993).
The CYP4A subfamily catalyzes o-hydroxylation at the terminal carbon of fatty acids.

ROLE OF MEOS AND ASSOCIATED CYTOCHROMES P450 IN

HEPATIC AND EXTRA HEPATIC PATHOLOGY

The mechanisms whereby alcohol causes liver injury are still not fully elucidated.
However, in a recent study on CYP2E1 induction among alcoholics, it was reported
that the oxidation of chlorzoxazone, a CYP2E1 substrate, was significantly higher in
patients with liver injury than in alcoholics without clinical and biochemical signs of
liver disease (Dupont et al., 1998), which suggests a link between the two. Indeed, PPC
and its active component dilinoleoylphosphatidylcholine were found to decrease
CYP2E1 activity (Aleynik and Lieber, 2001; Aleynik et al., 1999). Furthermore, PPC
was discovered to oppose oxidative stress (Lieber et al., 1997) and fibrosis (Lieber
et al., 1994) in alcohol fed baboons.

Role of Free Radical Generation and Xenobiotic Activation in

Hepatotoxicity and Carcinogenicity

In view of the capacity of CYP2E1 to generate reactive oxygen intermediates, such

as superoxide radicals (Dai et al., 1993) and the known toxicity of these free oxygen
radical species, it is to be expected that CYP2E1 plays a key role in the pathogenesis of
 520 Lieber

alcohol-induced liver injury. Membranous lipids are one target of free radical attack
(Slater, 1984). In addition, the increased microsomal generation of active oxygen
derivatives could also contribute to ethanol toxicity through radical-mediated
inactivation of metabolic enzymes (Dicker and Cederbaum, 1988), including CYP2E1
itself (Koop and Tierney, 1990). Furthermore, CYP2E1 activates a score of xenobiotics
to highly hepatotoxic compounds. This pertains, for instance, to CCl4. It is known that
CCl4 exerts its toxicity after conversion to an active compound (most likely
trichloromethyl radical) in the microsomes, and alcohol pretreatment remarkably
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stimulates the toxicity of CCl4 (Hasumura et al., 1974), with perivenular predomi-
nance, which can be explained by the selective presence and induction of CYP2E1 in
that zone of the liver lobule (Ingelman-Sundberg et al., 1988; Tsutsumi et al., 1988,
1989). A large number of other organic compounds were found to show such a
selective injurious action in the liver as well as other tissues of the alcoholic. These
include industrial solvents, such as bromobenzene (Hetu et al., 1983) and vinylidene
chloride (Siegers et al., 1983), as well as anesthetics such as enflurane (Tsutsumi et al.,
1990) and halothane (Takagi et al., 1983). Ethanol administration also markedly
increased the activity of microsomal low-Km benzene-metabolizing enzymes (Nakajma
et al., 1987) and aggravated the hemopoietic toxicity of benzene. Thus, commonly used
industrial solvents and anesthetics, considered safe in normal subjects, may acquire
unusual hepatotoxicity in heavy drinkers. Enhanced metabolism (and toxicity) pertains
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also to a variety of prescribed drugs, including isoniazid and phenylbutazone (Beskid

et al., 1980).
The same mechanism of hepatotoxicity also applies to some over-the-counter
medications: therapeutic amounts of acetaminophen (2.5 to 4 g per day) can cause
severe hepatic injury in alcoholics (Seeff et al., 1986). In animals given ethanol for
long periods, hepatotoxic effects peak after alcohol withdrawal (Sato and Lieber, 1981)
when ethanol is no longer competing for the microsomal pathway but levels of the
toxic metabolites are at their highest. This observation has the important clinical
implication that alcoholics are most vulnerable to the toxic effects of acetaminophen
not while drinking, but shortly after cessation of chronic drinking, when the still
induced CYP2E1 activates acetaminophen to a highly toxic metabolite.
In addition, there is also an association between alcohol misuse and an increased
incidence of upper alimentary and respiratory tract cancers (Lieber et al., 1986). Many
factors have been incriminated, one of which is the effect of ethanol on the cytochrome
P450-dependent activation of procarcinogens to carcinogens. This has been demon-
strated with microsomes isolated from a variety of tissues, including the liver (the
principal site of xenobiotic metabolism) (Garro et al., 1981; Seitz et al., 1981), the
lungs (Garro et al., 1981; Seitz et al., 1981), the intestines (Seitz et al., 1985; Shimizu
et al., 1990) (the chief portals of entry for tobacco smoke and dietary carcinogens,
respectively), and the esophagus (Farinati et al., 1985) (where ethanol consumption is a
major risk factor in the development of cancer). Nitrosamines have been implicated:
they are metabolized by CYP2E1 even at low concentrations (Yang et al., 1990), with a
resulting increased mutagen production after CYP2E1 induction, which may contribute
to the enhanced carcinogenesis.
Most importantly, the main reaction of the MEOS is to oxidize ethanol to
acetaldehyde, which causes many of the toxic effects associated with excess ethanol
(Lieber, 1988, 1997b, 1998) (Fig. 1).
 The Discovery of the Microsomal Ethanol Oxidizing System 521

Pathologic Role of CYP2E1 in Kupffer Cells, Extrahepatic Tissues

and Nonalcoholic Steatohepatitis

Ethanol affects several Kupffer cell functions which may contribute to the
development of alcoholic liver injury, including up-regulation of Kuppfer cell CYP2E1
after chronic alcohol consumption (Koivisto et al., 1996). The induction was of the
same relative magnitude as in hepatocytes, but in absolute amounts, the CYP2E1
content was 10 times lower than in hepatocytes of the same animals.
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The induction of CYP2E1 in Kupffer cells by ethanol has several consequences,

including increasing the intracellular levels of acetaldehyde, the toxic metabolite of
ethanol. Indeed, Kupffer cells have been shown to metabolize ethanol (Wickramasinghe
et al., 1987) and the relative contribution of CYP2E1 to overall acetaldehyde
production of these cells is probably much higher than in hepatocytes. Indeed, in
murine Kupffer cells, cytochrome P450 inhibitors have been shown to reduce ethanol
metabolism by more than 50%, whereas ADH and catalase inhibitors decreased it only
by less than 10% (Wickramasinghe et al., 1987).
Increases of CYP2E1 content of rats fed ethanol with a high fat diet have been
detected in the pancreas by immunohistochemistry (Sohda et al., 1992) and Western
blotting (Norton et al., 1998), but catalytic activity of CYP2E1 in pancreas had not been
reported, probably because of methodological problems. In a more recent study,
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improvements in methodology of tissue preparation and enzyme measurements made it

possible to detect the CYP2E1 and 1A1 activities in the pancreas and their increase after
chronic ethanol treatment (Kessova et al., 1998). This induction of CYP2E1 and CYP1A1
activities could play a role in the pathogenesis of pancreatitis and/or pancreatic cancer.
CYP2E1 was also detected on immunoblots of kidney microsomes (Ding et al., 1986),
and it was increased by chronic ethanol treatment (Ding and Coon, 1988). As reviewed
before (Lieber, 1992), alcohol abuse results in kidney damage, including fat accumulation
which maybe linked to the increase of kidney CYP2E1. As reviewed elsewhere (Lieber,
1999), CYP2E1 has also been described to a variable extent in a number of other tissues.
As reviewed before, ketones and fatty acids are also substrate and inducer of
CYP2E1. Accordingly, in obesity and diabetes, the excess of these substrates
commonly results in CYP2E1 up-regulation, demonstrated in an experimental model
of obesity (Raucy et al., 1991), as well as in patients with NASH (Weltman et al.,
1998) or in an experimental model of NASH (Lieber et al., 2004). NASH is
increasingly recognized as a precursor to more severe liver disease, leading to
‘‘cryptogenic’’ cirrhosis (James and Day, 1998). In the general population, the
prevalence of NAFLD averages 20% and that of NASH 2% to 3% (Yu and Keeffe,
2002). Thus, these conditions may be the most common liver diseases in the United
States. In addition, in view of the pathogenic role which up-regulation of CYP2E1 also
plays in alcoholic liver disease (vide supra), it is apparent that the major therapeutic
challenge is now to find a way to control this toxic process.

CONCLUSIONS

The studies of Remmer and Merker (1965) on the induction of the proliferation of
the SER by many xenobiotics had a major influence on the progress of pharmacology,
 522 Lieber

especially drug metabolism and toxicity, including our raising the hypothesis of a new
pathway of ethanol metabolism in the microsomes The studies of Remmer and his
group (Ariyoshi et al., 1970) also helped us to weather the storm that was raised with
our discovery of this new pathway. Remmer and co-workers confirmed our findings of
the effects of ethanol on enzymes activities and constituents of the liver microsomes
and extended them. Indeed, we had observed that the most striking CYP2E1 induction
occurs after chronic ethanol administration (Lieber and DeCarli, 1968) but, as shown
by the Remmer group (Ariyoshi et al., 1970), even one single acute dose can elicit
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changes in the microsomes.

The up-regulation of CYP2E1 plays a useful physiologic role when starvation and/
or low carbohydrate diets prevail because of its contribution to the metabolism of fatty
acids and its capacity to convert ketones to glucose (Lieber, 1997). Furthermore,
although it can activate some xenobiotics to toxic agents as well as carcinogens, it
helps detoxify other xenobiotics and it also clears alcohol from the blood when ethanol
reaches relatively high levels, particularly on a chronic basis, which triggers an
adaptive CYP2E1 response to the increased substrate concentration, as reviewed
elsewhere (Lieber, 1999). However, like many other useful adaptive systems, when the
adaptation ceases to be homeostatic and becomes excessive, adverse consequences
prevail. CYP2E1 leaks oxygen radicals as part of its stimulated operation and, when
this exceeds the cellular defense systems, it result in oxidative stress, with its various
For personal use only.

pathologic consequences. This is true not only when excess alcohol has to be
metabolized, as is the case in alcoholic steatohepatitis, but also when CYP2E1 is
confronted by an excess of ketones and fatty acids associated with diabetes and/or
obesity. These states also upregulate CYP2E1 and the resulting oxidative stress
generates or aggravates NASH. NASH has been recognized as an important precursor
of cryptogenic cirrhosis. The challenge is now to boost the cellular defense systems
against oxidative stress as well as to find effective but non-toxic inhibitors of CYP2E1
capable of down-regulating its activity in order to restore a proper balance between the
beneficial and the detrimental effects of the adaptive, but sometimes inappropriately
excessive, CYP2E1 response to exogenous stimuli.

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