Independent and Combined Effects of Ethanol
Self-Administration and Nicotine Treatment on Hepatic
CYP2E1 in African Green Monkeys□ S
C. S. Ferguson, S. Miksys, R. Palmour, and R. F. Tyndale
Centre for Addiction and Mental Health and Departments of Psychiatry, Pharmacology and Toxicology, University of Toronto,
Ontario, Canada (C.S.F., S.M., R.F.T.); and Department of Psychiatry, McGill University, Montreal, Quebec, Canada (R.P.)
Received May 2, 2011; accepted August 24, 2011
Cytochrome P450 2E1 metabolizes ethanol and also bioactivates
many toxins and procarcinogens. Elevated levels of hepatic
CYP2E1 are associated with an increased susceptibility to chem-
ical toxicity and carcinogenesis. This study investigated the induc-
tion of hepatic CYP2E1 by ethanol and nicotine, alone and in
combination, in a nonhuman primate model. Monkeys that self-
administered ethanol and that received subcutaneous injections of
nicotine (0.5 mg/kg b.i.d.), alone and in combination, were com-
pared with control animals (four groups, n ? 10/group). Chlorzoxa-
zone (CZN) was used as a probe drug to phenotype in vivo CYP2E1
activity before and after chronic ethanol and/or nicotine exposure.
CYP2E1 protein levels and in vitro chlorzoxazone metabolism were
assessed in liver microsomes. Average daily ethanol consumption
was ?3.0 g/kg (blood ethanol levels ?24 mM) and was unaffected
by nicotine treatment. Ethanol self-administration and nicotine
treatment, alone and in combination, significantly increased in vivo
CZN disposition compared with that in control animals. The effect
of ethanol was only observed at higher levels of intake. Ethanol and
nicotine increased CYP2E1 protein levels and in vitro CZN metab-
olism, with combined exposure to both drugs resulting in the
greatest increase. The effect of ethanol was also dependent on
level of intake. Chronic exposure to ethanol and nicotine induced
hepatic CYP2E1 activity and protein levels, particularly when both
drugs were used in combination and when ethanol intake was high.
These results have important implications for public health, given
the association between elevated CYP2E1 and disease, and the
large proportion of individuals who are exposed to ethanol and
nicotine, often in combination.
Cytochrome P450 2E1 is a drug-metabolizing enzyme that is re-
sponsible for the biotransformation of numerous low-molecular-
weight compounds, including ethanol, several commonly used indus-
trial solvents, environmental pollutants, and various clinical drugs
(Lieber, 1997). Many of these substrates are procarcinogens or cyto-
toxins that are bioactivated by CYP2E1. CYP2E1 is also known to
generate high levels of reactive oxygen species that can cause cell
damage via lipid peroxidation and DNA strand breaks (Caro and
Elevated levels of CYP2E1 are associated with increased suscep-
tibility to chemical toxicity and carcinogenesis. Several polymor-
phisms have been identified in the human CYP2E1 gene. Individuals
with the CYP2E1 RsaI c2 allele, associated with increased CYP2E1
transcriptional activity (Hayashi et al., 1991), are more susceptible to
toxicity from industrial chemicals bioactivated by CYP2E1 such as
vinyl chloride (Wang et al., 2010) and n-hexane (Zhang et al., 2006).
This and other CYP2E1 genetic variants have been associated with
increased risk for hepatocellular (Munaka et al., 2003), colorectal
(Morita et al., 2009), and esophageal cancer (Liu et al., 2007).
Hepatic CYP2E1 can be induced by a variety of compounds, many
of which are substrates. Ethanol is an inducer of hepatic CYP2E1
protein and activity in humans and monkeys (Lieber, 1997; Ivester et
al., 2007). Smoking increases CYP2E1 activity in humans (Benowitz
et al., 2003), and chronic nicotine treatment increases hepatic
CYP2E1 protein levels and activity in monkeys (Lee et al., 2006b).
Approximately 90% of smokers also consume alcohol (Shiffman
and Balbanis 1995), yet little is known about the combined effects of
ethanol and nicotine on hepatic CYP2E1 levels. It was previously
shown that rats exposed to both ethanol and nicotine had significantly
greater levels of hepatic CYP2E1 protein compared with rats exposed
to either drug alone (Yue et al., 2009). This enhancement of CYP2E1
protein was partially attributed to a nicotine-stimulated increase in
This study was supported by the Centre for Addiction and Mental Health;
Canadian Institute of Health Research [MOP97751]; Canadian Foundation for
Innovation [20289 and 16014]; Ontario Ministry of Research and Innovation;
Canada Research Chair in Pharmacogenetics (to R.F.T.); Canadian Liver Foun-
dation and Scholarship Program for Interdisciplinary Capacity Enhancement.
R.F.T. has shares in Nicogen Research Inc. Funds were not received from
Nicogen for these studies, nor was the manuscript reviewed by individuals
associated with Nicogen.
Article, publicationdate,and citation
S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: AGM, African green monkeys; CZN, chlorzoxazone; 6OHCZN, 6-hydroxychlorzoxazone; ANOVA, analysis of variance; TBS,
Tris-buffered saline; BEL, blood ethanol level; AUC, area under the plasma concentration-time curve.
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:2233–2241, 2011
Vol. 39, No. 12
Printed in U.S.A.
ethanol consumption. There was no indication as to how the observed
increases in CYP2E1 protein levels would affect in vitro and in vivo
CYP2E1 activity. Understanding the impact of ethanol and nicotine
on CYP2E1-mediated metabolism is crucial, given the large propor-
tion of the population that is exposed to both drugs and the potential
for elevated CYP2E1 to cause toxicity and disease.
The African green monkey (AGM) has been established as an
excellent animal model of human CYP2E1 expression and activity
(Lee et al., 2006b). Chlorzoxazone (CZN), a clinically used muscle
relaxant that is metabolized by CYP2E1 to 6-hydroxychlorzoxazone
(6OHCZN), is a validated probe drug for the measurement of
CYP2E1 activity in both humans and monkeys (Ernstgård et al., 2004;
Lee et al., 2006b). As in other species, CZN 6-hydroylation in monkey
liver microsomes can be inhibited by anti-CYP2E1 antibodies and
selective chemical inhibitors of CYP2E1, providing evidence that
CYP2E1 is the primary enzyme involved in the 6-hydroxylation of
CZN (Amato et al., 1998). AGMs are also useful in modeling human
alcohol consumption. These monkeys will voluntarily self-administer
alcohol at levels comparable to human consumption and are therefore
routinely used in alcohol research (Palmour et al., 1997).
We investigated the independent and combined effects of ethanol
self-administration and nicotine treatment on in vivo CZN disposition,
hepatic CYP2E1 protein levels, and in vitro CZN metabolism. We
hypothesized that ethanol and nicotine would independently induce
CYP2E1 levels, resulting in increased CZN clearance in vivo and in
vitro CZN metabolism. In addition, combined ethanol and nicotine
exposure was hypothesized to result in greater induction of CYP2E1
protein and in vitro CZN activity compared with those of either drug
alone, owing to both the direct effects of the inducers and a nicotine-
mediated increase in alcohol consumption. The induction of CYP2E1
by ethanol is dose-dependent in humans (Millonig et al., 2011);
therefore, we also investigated whether the effects of ethanol were
dependent on the level of alcohol intake.
Materials and Methods
Materials. CZN and 2-benzoxazolinone were purchased from Sigma-Al-
drich Canada Ltd. (Oakville, ON, Canada). All other chemicals were obtained
from standard commercial sources. Protein estimation was performed with dye
reagent purchased from Bio-Rad Laboratories (Hercules, CA). Prestained
molecular weight protein markers were purchased from MBI Fermentas (Flam-
borough, ON, Canada). Hybond nitrocellulose membrane was purchased
from Pall Corporation (Pensacola, FL). Human cDNA-expressed CYP2E1,
CYP2A6, CYP2A1, CYP2A2, CYP2D6, CYP3A4, and CYP2B6 were pur-
chased from BD Biosciences (San Diego, CA). Polyclonal anti-rat CYP2E1
antibody was purchased from Fitzgerald Industries (Acton, MA). Horseradish
peroxidase-conjugated anti-sheep secondary antibody was purchased from
Millipore Corporation (Billerica, MA). Chemiluminescent substrate was pur-
chased from Thermo Fisher Scientific (Mississauga, ON, Canada). Autoradio-
graphic film was purchased from Ultident (St. Laurent, PQ, Canada).
Animals. Adult male African green monkeys (vervets, Chlorocebus ae-
thiops) were housed outdoors in social groups at Caribbean Primates Ltd. (St.
Kitts). They were acquired from a large, isolated, and nonendangered Carib-
bean population (Palmour et al., 1997). Monkeys were given standard rations
of Purina monkey chow supplemented with fresh fruit and vegetables twice a
day and were allowed to feed ad libitum. Drinking water was also available ad
Drug Treatment. The study timeline is shown in Fig. 1. The first 14 days
of the study consisted of an ethanol preference screening phase, where mon-
keys were given access to 10% v/v alcohol in 0.5% w/v sucrose solution for 4
h/day. Forty monkeys that voluntarily consumed more than 1 g of ethanol/kg
per day were selected and randomized into four groups based on daily ethanol
consumption (n ? 10/group). The following 14 days (days 15–28) consisted of
a washout period, during which monkeys had no exposure to ethanol or
nicotine. During the second phase of the study, from days 29 to 42, monkeys
in the ethanol-only (group 2) and ethanol ? nicotine (group 4) groups were
allowed to self-administer 10% alcohol in 0.5% sucrose solution for 4 h/day,
whereas the other groups (1 and 3) consumed 0.5% sucrose solution on the
same schedule. During the third phase of the study, from days 43 to 63, in
addition to alcohol (or sucrose), monkeys in the nicotine-only (group 3) and
ethanol ? nicotine (group 4) were given subcutaneous injections of nicotine
bitartrate (milligram base in saline, pH 7.0) twice daily at a dose of 0.05 mg/kg
on day 43, 0.1 mg/kg on day 44, 0.25 mg/kg on day 45, and 0.5 mg/kg for
subsequent days. The first injection was given 30 min before the alcohol (or
sucrose) access period. The second injection was given 10 h later. Monkeys in
the ethanol-only (group 2) and control (group 1) groups were given saline
injections (as a vehicle control for nicotine bitartrate) on the same schedule. On
day 50, nicotine treatment and alcohol access were suspended to conduct
pharmacokinetic testing. Monkeys received 7 mg/kg CZN intragastrically
under ketamine anesthesia, and blood samples were drawn at t ? 10 min (10
min before CZN administration) and at 10, 20, 30, 60, 120, 240, and 360 min
after CZN administration. Blood samples (2 ml) were drawn immediately after
the alcohol access period on days 38 and 59 to determine blood ethanol levels.
All blood samples were centrifuged, and the plasma was removed and frozen
for subsequent drug analyses. A commercial enzymatic assay kit (Sigma-
Aldrich, St. Louis, MO) was used to determine plasma blood levels of ethanol.
Body weights at the start of the study (average ? 5.9 ? 0.5 kg) were not
significantly different from body weights at sacrifice (average ? 5.7 ? 0.5 kg)
(paired t test, p ? 0.5). There were no significant differences in body weights
among groups at the start of the study [one-way ANOVA, F(3, 36)? 0.3784,
FIG. 1. Overview of the study timeline.
Monkeys were randomized into four study
groups (n ? 10/group); Study groups con-
sisted of a no-drug control group (group 1),
an ethanol (EtOH)-only group (group 2), a
nicotine-only group (group 3), and an eth-
anol ? nicotine group (group 4). Groups
were named based on drug exposure during
the last phase of the study (phase III).
Blood ethanol levels (BELs) were mea-
sured near the end of phase II and phase III.
In vivo CZN metabolism was assessed dur-
ing the washout period before phase II and
at the end of phase III. On day 50, nicotine
treatment and alcohol access was sus-
pended to conduct pharmacokinetic testing.
FERGUSON ET AL.
p ? 0.05] or at sacrifice [one-way ANOVA, F(3, 36)? 1.860, p ? 0.05].
Animals were sacrificed by exsanguination via the femoral artery under
ketamine anesthesia, and livers were immediately dissected and flash-frozen in
liquid nitrogen and stored at ?80°C until further use. Upon visual inspection
of the livers, there were no apparent signs of injury or cirrhosis. The experi-
mental protocol was reviewed and approved by the Institutional Review Board
of the Behavioral Sciences Foundation and the University of Toronto Animal
Care Committee. All the procedures were conducted in accordance with the
guidelines of the Canadian Council on Animal Care.
In Vivo CZN Plasma Assessments. Plasma CZN levels were assayed on
the basis of the methods in Lee et al. (2006b). In brief, plasma was centrifuged
at 3000g for 10 min, after which 100 ?l of the supernatant was removed and
incubated with ?-glucuronidase (10 mg/ml, 50 ?l) overnight at 37°C. After
incubation, an internal standard of 2-benzoxazolinone (2.0 mM, 25 ?l), 10%
perchloric acid (120 ?l), and ethyl acetate/hexane (3 ml) was added, the
sample was shaken for 30 min and centrifuged at 3500g for 15 min, and the
organic phase was evaporated to dryness at 37°C. The sample was reconsti-
tuted into 110 ?l of mobile phase consisting of 50 mM ammonium acetate
(adjusted to pH to 4.0 with 1 M glacial acetic acid)-acetonitrile (65:35). CZN
was measured by high-performance liquid chromatography with UV detection
at 287 nm. An Agilent Zorbax SB-C18 column (5 ?m, 4.6 ? 250 mm; Agilent
Technologies, Santa Clara, CA) was used to separate CZN and 2-benzoxazo-
linone using a flow rate of 0.7 ml/min. The retention times for CZN and
2-benzoxazolinone were 18.5 and 10.1 min, respectively.
Microsomal Membrane Preparation. Monkey liver tissue was homoge-
nized in 100 mM Tris, 0.1 mM EDTA, 0.1 mM dithiothreitol, and 0.32 M
sucrose (pH 7.4) for immunoblotting or in 1.15% w/v KCl for in vitro
metabolism assessments and then centrifuged at 12,500g for 30 min at 4°C.
The supernatant was then centrifuged at 110,000g for 90 min at 4°C, and the
pellet was resuspended in 100 mM Tris, 0.1 mM EDTA, 0.1 mM dithiothreitol,
1.15% w/v KCl, and 20% v/v glycerol for immunoblotting or 1.15% w/v KCl
for in vitro metabolism assays. The protein content of liver microsomes was
assayed with the Bradford (1976) technique using a Bio-Rad Protein Assay kit.
Microsomes were stored at ?80°C.
In Vitro CZN and 6OHCZN Assessments. CZN 6-hydroxylation was
assayed according to the protocol established by Leclercq et al. (1998), in
which the protein concentration and incubation times were optimized for linear
formation of 6OHCZN. Monkey hepatic microsomal protein (0.4 mg) was
mixed with 0.1 M Tris buffer at pH 7.6, 10 mM magnesium chloride, 5 mM
NADPH, and 950 ?M CZN to a final volume of 500 ?l. The reaction mixture
was incubated for 20 min at 37°C. Zinc sulfate (15% w/v, 0.2 ml) was added
to stop the reaction, and 6.4 ?g of the internal standard, 2-benzoxazolinone in
Tris buffer, was added per reaction. After centrifugation for 10 min at 12,700g,
the supernatant was injected onto an Agilent Zorbax SB-C18 column (5 ?m,
4.6 ? 250 mm; Agilent Technologies) with UV detection at 287 nm. The
mobile phase consisted of 50 mM ammonium acetate (adjusted to pH to 4.0
with 1 M glacial acetic acid)-acetonitrile (65:35) with a flow rate of 0.7
ml/min. The retention times for CZN, 6OHCZN, and 2-benzoxazolinone were
18.5, 7.5, and 10.1 min, respectively. The absolute and relative recoveries of
6OHCZN were 90.4 and 99.4%, respectively.
Immunoblotting. Monkey liver microsomal protein was serially diluted to
generate a standard curve and to establish the linear detection range for the
immunoblotting assays. To determine cross-reactivity of the primary antibod-
ies, cDNA-expressed human CYP2E1, CYP2A6, CYP1A1, CYP2A2,
CYP2D6, CYP3A4, and CYP2B6 were used as positive or negative controls.
Liver microsomal proteins (4 ?g) were separated by SDS-polyacrylamide
gel electrophoresis (4% stacking and 8% separating gels) and then were
transferred overnight onto nitrocellulose membranes. Gels were stained with
Coomassie Blue R-250 to ensure equal loading of protein among lanes. To
detect hepatic CYP2E1, the membranes were first blocked with 1% skim milk
in 50 mM Tris-buffered saline (TBS) containing 0.1% w/v bovine serum
albumin and 0.1% v/v Triton X-100 for 1 h. Membranes were then incubated
with anti-CYP2E1 antibody diluted 1:1000 for 2 h, followed by three 5-min
washes with TBS containing 0.1% v/v Triton X-100. The membranes were
then blocked again with the initial blocking solution for 1 h and incubated with
peroxidase-conjugated rabbit anti-sheep antibody diluted 1:5000 for 1 h, fol-
lowed by three 5-min washes with TBS containing 0.1% v/v Triton X-100.
Proteins were visualized using chemiluminescence followed by exposure to
autoradiography film. Immunoblots were analyzed using MCID Elite software
(InterFocus Imaging Ltd., Linton, UK), and the relative density of each band
was expressed as arbitrary density units after background was subtracted.
Isolation, cDNA Synthesis, and mRNA Quantification. Liver tissue (50–
100 mg) was homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA), and
total RNA was isolated according to the TRIzol reagent protocol. RNA
concentrations were determined spectrophotometrically and total RNA integ-
rity was confirmed by electrophoresis on a 1.2% agarose gel (Onbio, Inc.,
Richmond Hill, ON, Canada) stained with ethidium bromide and inspection of
the 28S and 18S ribosomal bands. cDNA was synthesized using 1 ?g of total
RNA, random hexamers (Invitrogen), RiboLock RNase inhibitor (Fermentas,
Burlington, ON, Canada) and MMLV Reverse Transcriptase (Invitrogen)
according to protocols provided by the manufacturers. Primers for real-time
PCR amplification of CYP2E1 and ?-actin were as follows: CYP2E1 forward
primer (CYP2E1ex1), 5?-CCG CTT CCC ATC ATC GGG AAC-3?; CYP2E1
reverse primer (CYP2E1ex1), 5?-GCG CTT TCA CCG CCT TGT A-3?;
?-actin forward primer (ACTBFex3), 5?-CAG AGC AAG AGA GGC ATC
CT-3?; and ?-actin reverse primer (ACTBRex4/), 5?-GGT CTC AAA CAT
GAT CTG GGT C-3?. The sequence of CYP2E1 in African green monkey is
not known; primer specificity was based on human CYP2E1 and rhesus
macaque (Macaca mulatta) CYP2E1. Amplification and fluorescence detec-
tion were performed using the Applied Biosystems ViiA7 Real-Time PCR
system (Invitrogen). The real-time PCR amplification mixture (20 ?l) con-
tained 1 ?l of synthesized cDNA, 10 ?l of 2? Fast SYBR-Green Mix
(Invitrogen), and 0.3 ?M concentrations of each primer. Cycling conditions
consisted of an initial activation of AmpliTaq Fast DNA polymerase followed by 40
CYP2E1 mRNA levels were obtained by normalization to ?-actin and use of the
comparative CT method for relative quantification as described by the manufacturer
(Real-Time PCR Chemistry Guide; Invitrogen).
Statistics. Differences in alcohol (or sucrose) consumption and blood eth-
anol levels (BELs) were assessed by one-tailed Student’s t test, unpaired tests
for between-group comparisons, and paired tests for within-group compari-
sons. All pharmacokinetic statistical analyses were performed using SAS
software (version 8.2; SAS Institute, Cary, NC). One-tailed paired Student’s t
tests were used to assess differences in in vivo CZN pharmacokinetic param-
eters measured before and after drug administration. A one-tailed unpaired
Student’s t test was used to compare the change in CZN AUC0–6 hamong
groups. One-way ANOVA followed by post hoc tests (Kruskal-Wallis test and
test for linear trend) were used to determine differences in CYP2E1 protein
levels, mRNA levels, and in vitro CZN metabolism between groups. Correla-
tions were calculated with Pearson correlation coefficients.
Monkeys Voluntarily Self-Administered Ethanol. Monkeys con-
sistently self-administered high levels of 10% ethanol throughout the
study (Fig. 2A). Mean daily ethanol consumption during the 4-hour
ethanol access sessions in phases II and III ranged from 23.6 to 54.6
ml/kg (1.9–4.4 g ethanol/kg), resulting in average consumption of
38.12 ? 7.8 ml/kg (3.0 g ethanol/kg). A gradual increase in mean
daily ethanol consumption was observed as monkeys progressed from
phase II to phase III of the study. This increase was statistically
significant for both the ethanol-only (p ? 0.001) and the ethanol ?
nicotine groups (p ? 0.04) (Table 1). There was no significant
difference in mean ethanol consumption between the ethanol-only and
the ethanol ? nicotine groups (phase III; Table 1), indicating no effect
of nicotine treatment on voluntary alcohol consumption. Sucrose
consumption remained constant throughout the study.
Considerable individual variation in alcohol intake was observed.
Monkeys were divided into high and low ethanol consumers by
performing a median split based on mean daily consumption of
ethanol during phases II and II (Fig. 2B; Table 1).
BELs were measured on two occasions, once during phase II (day
38) and again during phase III (day 59). A paired test revealed no
significant difference in BELs measured during phase II and phase III
ETHANOL AND NICOTINE INDUCE HEPATIC CYP2E1
for either the ethanol-only and ethanol ? nicotine groups or for the
high and low consumer groups, despite increases in ethanol consump-
tion (Table 1).
In Vivo CZN Disposition Is Influenced by Nicotine Treatment
and the Level of Daily Ethanol Intake. A within-animals design was
use to assess changes in in vivo CZN metabolism due to animal
variation in CZN pharmacokinetics. A comparison of in vivo CZN
metabolism before and after drug administration indicated that the
nicotine-only group had a 34% decrease in CZN AUC (p ? 0.002)
and a 42% reduction in the maximum plasma CZN concentration
(Cmax) (p ? 0.001) compared with values before treatment (Table 2).
Mean values for all the assessed CZN pharmacokinetic parameters
were not significantly altered in the ethanol-only group; however,
several monkeys showed a substantially decreased CZN AUC after
ethanol self-administration (Fig. 3). In the ethanol ? nicotine group,
there was a significant reduction in CZN AUC by 27% (p ? 0.02),
Cmaxby 35% (p ? 0.003), and the time to maximum CZN concen-
tration (Tmax) by 35% (p ? 0.04). Kinetic parameters were not
significantly different among groups before drug administration (day
22), and control monkeys (group 1) did not show significant changes
in CZN pharmacokinetic parameters assessed on day 22 compared
with those on day 50 (Supplemental Fig. 1).
There was a significant correlation between post-ethanol CZN
AUC0–6 hand mean daily consumption of 10% ethanol (r ? 0.42, p ?
0.03) (Fig. 4A). The high ethanol consumers had a post-ethanol CZN
AUC0–6 hof 34 h ? ?g/ml, which was significantly lower than the
CZN AUC0–6 hof 64 h ? ?g/ml seen in the low ethanol consumers
(p ? 0.05) (Fig. 4B). Only in the high consumer group was there a
significant change in CZN AUC after ethanol consumption (p ? 0.05)
(Fig. 4C). Taken together, these results show a reduction in CZN AUC
by ethanol that is dependent on the level of intake.
Hepatic CYP2E1 Protein Levels Are Induced by Ethanol and
Nicotine, Particularly When Both Drugs Are Present in Combi-
nation and at Higher Ethanol Intakes. An immunoblotting assay
was established to measure CYP2E1 protein levels in monkey liver.
Detection of CYP2E1 in serially diluted liver microsomal protein
from a control monkey was shown to be linear from 2 to 20 ?g of
protein (Fig. 5A). All subsequent immunoblots were loaded with 5 ?g
of microsomal protein. The CYP2E1 antibody did not cross-react with
other cDNA-expressed human cytochromes P450 and monkey hepatic
CYP2E1 comigrated with cDNA-expressed human CYP2E1 (Fig. 5B).
Compared with control monkeys, ethanol self-administration alone
resulted in a 56% increase in CYP2E1 levels (p ? 0.05), nicotine
treatment alone resulted in a 55% increase (p ? 0.05), and combined
ethanol self-administration and nicotine treatment resulted in a 106%
increase (p ? 0.001), suggesting an additive effect (Fig. 6, A and B). In
comparing mean CYP2E1 levels across all the groups, there was a
significant linear trend: ethanol or nicotine exposure alone increased
CYP2E1 levels compared with no drug exposure, and CYP2E1 levels
were further increased with combined exposure to both drugs (ptrend?
0.0002). Ethanol and nicotine exposure, either alone or in combination,
did not significantly alter CYP2E1 mRNA levels in the liver [F(3, 33)?
2.423, p ? 0.083].
The effect of ethanol consumption level on CYP2E1 protein levels
was also examined. High ethanol consumers had a 97% increase in
CYP2E1 levels relative to those of control monkeys, whereas low
ethanol consumers had a 65% increase. In comparing mean CYP2E1
levels among monkeys in the control, low ethanol consumer, and high
ethanol consumer groups, there was a significant linear trend, indi-
Consumption of 10% ethanol increase over time with no increase in blood
Values are expressed as mean ? S.E. n ? 10/group.
Phase II Phase III
35.4 ? 1.2
37.8 ? 2.2
32.9 ? 1.6
37.3 ? 1.6†
mmol/l ml/kg per day mM/l
Group 3: EtOH-only
Group 4: EtOH ? nicotine
24.6 ? 3.9
23.8 ? 2.1
22.9 ? 3.8
25.5 ? 2.2
42.4 ? 1.9*
40.7 ? 3.1*
35.5 ? 1.6*
45.1 ? 1.9*‡
26.3 ? 9.3
29.7 ? 4.7
24.7 ? 4.0
31.7 ? 4.0
* p ? 0.05, significantly different from phase II.
†p ? 0.05 and‡p ? 0.001, significantly different from low consumer.
FIG. 2. Monkeys voluntarily self-administered ethanol. A, average daily consumption by monkeys self-administering alcohol in sucrose (squares) or sucrose alone (circles).
Phase I is the ethanol (EtOH) preference screening period (consumption levels at screening represent average consumption during this phase). During phase II monkeys
in the ethanol-only (group 2) and ethanol ? nicotine (group 4) groups self-administered ethanol in sucrose solution, whereas the other groups (1 and 3) consumed sucrose
solution only on the same schedule. During phase III in addition to alcohol (or sucrose), monkeys in the nicotine-only (group 3) and ethanol ? nicotine (group 4) groups
were given nicotine injections, whereas monkeys in the other groups (1 and 2) were give saline injections on the same schedule. B, a median split was used to categorize
monkeys into low and high ethanol consumers based on mean daily consumption of 10% ethanol during phases II and III. B, Open symbols represent monkeys in the
ethanol-only group (group 2), filled symbols represent monkeys in the ethanol ? nicotine group (group 4), and horizontal lines indicated group means. Significantly different
from low ethanol consumers: ?, p ? 0.05.
FERGUSON ET AL.
cating increasing CYP2E1 protein levels with higher levels of ethanol
consumption (ptrend? 0.0004) (Fig. 6C). CYP2E1 mRNA levels in
the liver were not significantly different among the high ethanol
consumer, low ethanol consumer, or control animals [F(2, 27)? 2.349,
p ? 0.1147].
In Vitro CZN Metabolism Is Induced by Ethanol and Nicotine,
Particularly When Both Drugs Are Present in Combination and
at Higher Ethanol Intakes. The rate of CZN metabolism to 6OH-
CZN was measured in vitro using monkey liver microsomes. There
was a significant positive correlation between hepatic CYP2E1 levels
and in vitro 6OHCZN formation velocity at 950 ?M (approximate
Vmax; r ? 0.45, p ? 0.002) (Fig. 7A). Compared with control
monkeys, ethanol self-administration alone and nicotine treatment
alone resulted in an 11% increase in 6OHCZN formation velocity,
although these increases were not significant after post hoc testing
(p ? 0.05) (Fig. 7B). Combined ethanol self-administration and
nicotine treatment resulted in a 21% increase (p ? 0.05), suggesting
an additive effect. In comparing mean CZN metabolism velocity
across all the groups, there was a significant linear trend, with mon-
keys in the ethanol and nicotine combined group having the highest
rate of CZN metabolism (ptrend? 0.003).
The impact of ethanol consumption level on in vitro CZN metab-
olism was investigated. High ethanol consumers had a 19% increase
in velocity of CZN metabolism compared with control monkeys,
whereas the low ethanol consumers had a 13% increase. In comparing
mean CZN metabolism velocity among monkeys in the control, low
ethanol consumer, and high ethanol consumer groups, there was a
significant linear trend, demonstrating that the rate of CZN metabo-
lism increases with higher levels of ethanol consumption (ptrend?
0.003) (Fig. 7C).
Humans and AGMs have similar hepatic CYP2E1 levels, in vivo CZN
2006b), and although the AGM CYP2E1 has not been sequenced,
CYP2E1s of both the cynomolgus monkey (Macaca fascicularis) and the
rhesus monkey (M. mulatta), which are closely related to the AGM, have
a greater than 90% amino acid homology to human CYP2E1. Thus,
AGMs offer a valuable model for predicting the impact of ethanol and
nicotine on CYP2E1 induction and metabolism in humans.
Monkeys consistently self-administered high levels of ethanol
throughout the study. Previous alcohol research classified AGMs
FIG. 3. Ethanol (EtOH) self-administration
and nicotine treatment reduced chlorzoxa-
zone AUC. A, mean CZN plasma concen-
tration over time curves for treatment
groups. n ? 10/group. B, lines represent the
change in plasma CZN AUC0–6 hfor indi-
vidual monkeys before drug administration
(day 22) and after drug administration (day
50). Diamonds with error bars represent
mean AUC0–6 h? S.E. for the group before
and after drug administration; for the etha-
nol-only group, the filled diamonds indicate
the mean including the monkey with a no-
ticeably high CZN AUC0–6 hboth before
and after ethanol exposure (indicated by the
dashed line) and the open diamonds indi-
cate the mean excluding this monkey. Sig-
nificantly different from same group of
monkeys before drug administration: ?, p ?
Kinetic parameters for CZN before drug administration (day 22) and after drug administration (day 50)
Values are expressed as mean ? S.E. n ? 10. Kinetic parameters were not significantly different among groups before drug administration.
EtOHNicotineEtOH ? Nicotine
BeforeAfterBeforeAfter Before After
AUC (hr ? ?g/ml)
54.8 ? 7.9
17.5 ? 2.7
1.3 ? 0.2
1.3 ? 0.1
56.6 ? 13.6
27.7 ? 5.7
0.9 ? 0.2
1.2 ? 0.1
44.8 ? 6.2
20.2 ? 3.6
1.1 ? 0.2
0.9 ? 0.1
29.5 ? 5.6*
11.7 ? 2.5*
1.3 ? 0.2
1.1 ? 0.1
56.8 ? 4.6
25.3 ? 3.0
1.1 ? 0.2
1.0 ? 0.1
41.6 ? 3.2*
16.4 ? 2.0*
0.7 ? 0.1*
1.8 ? 0.3
* p ? 0.05, significantly different from before drug administration within the same treatment group.
ETHANOL AND NICOTINE INDUCE HEPATIC CYP2E1
voluntarily self-administering between 0.8 and 3.5 g/kg ethanol/day as
moderate consumers (Palmour et al., 1997). In our study, average
daily ethanol intake was approximately 3.0 g/kg, identifying these
monkeys as moderate consumers. Moderate alcohol consumption in
humans has been described as intakes resulting in BELs ranging from
5 to 20 mM (Eckardt et al., 1998). In our study, monkeys achieved an
average BEL of 26 ? 2.55 mM, comparable to moderate consumption
in humans; use of BELs to compare ethanol consumption takes into
consideration the 2-fold greater rate of ethanol elimination in AGMs
compared with humans (Ervin et al., 1990).
Ethanol consumption steadily increased throughout the study,
whereas BEL remained unchanged over time, suggesting increased
rates of ethanol elimination in the monkeys. In humans, rats, and other
mammals, ethanol metabolism is primarily mediated by alcohol de-
hydrogenase and to a lesser extent by CYP2E1 (Matsumoto et al.,
1996; Lands, 1998). Human alcoholics have higher levels of CYP2E1,
no change in alcohol dehydrogenase, and an increased capacity to
eliminate ethanol compared with nonalcoholic humans, suggesting
that elevated CYP2E1 levels can affect ethanol metabolism (Vidal et
al., 1990). On the basis of this premise, induction of CYP2E1 in the
ethanol-consuming monkeys may have contributed to metabolic tol-
erance, allowing monkeys to consume more ethanol without a corre-
sponding rise in BELs.
Nicotine treatment did not affect voluntary consumption of 10%
ethanol in AGM. Some studies in rats have shown an increase in
ethanol consumption with chronic or repeated nicotine treatment,
whereas others have shown that nicotine treatment has no effect or
even decreases ethanol consumption (Blomqvist et al., 1996; Le ˆ et al.,
2000; Olausson et al., 2001; Sharpe and Samson, 2002). Differences
in rat strains, methods used to initiate ethanol consumption, and
duration of the ethanol access period may contribute to the inconsis-
tent results. A limited number of human studies have investigated the
effect of nicotine on alcohol consumption. Occasional smokers
(smoking an average of 10 cigarettes/week and only smoking on 2
days/week) consumed more alcohol when they smoked nicotine-
containing cigarettes compared with denicotinized cigarettes (Barrett
et al., 2006). A similar study in regular smokers (smoking more than
10 cigarettes/day) showed that smokers deprived of nicotine had a
greater urge to consume alcohol compared with nondeprived smokers
(Cooney et al., 2003). Therefore, the effect of nicotine on ethanol
consumption in either rats or humans is complex and requires further
investigation. Our study is the first to look at the effect of nicotine on
voluntary ethanol self-administration in monkeys in which we ob-
served no significant increase or decrease in ethanol consumption
from the nicotine administration under the conditions tested.
Here we show that variation in ethanol intake, within the range of
moderate drinking, can lead to very different CYP2E1 levels and
activity as measured by CZN metabolism. High ethanol-consuming
monkeys were only drinking 25% more ethanol per day than the low
ethanol consumers; however, they demonstrated nearly a 50% greater
reduction in postethanol CZN AUC in vivo. These results are inter-
esting from a public health perspective, because our results suggest
FIG. 5. Detection of CYP2E1 monkey hepatic protein was linear and specific. A, a
dilution curve of monkey hepatic microsomal protein showed linear detection of
CYP2E1 from 2 to 20 ?g. B, the anti-CYP2E1 antibody used for the CYP2E1
immunoblotting did not cross-react with cDNA-expressed human CYP2A6,
CYP2A1, CYP1A2, CYP2D6, CYP3A4, and CYP2B6. Monkey hepatic CYP2E1
comigrated with cDNA-expressed human CYP2E1.
FIG. 4. Correlation between ethanol (EtOH) consumption and chlorzoxazone AUC.
A, there was a significant negative correlation between mean consumption of 10%
ethanol (milligrams per kilogram per day) during phases II and III and CZN
AUC0–6 hafter drug administration (day 50). With the exclusion of the monkey with
noticeably high postethanol CZN AUC (circled), the correlation remains significant
(r ? 0.43, p ? 0.04). B, high ethanol consumers (Fig. 2B) had a significantly
reduced CZN AUC0–6 hcompared with low consumers (p ? 0.05). n ? 10/group.
C, lines represent the change in plasma CZN AUC0–6 hfor high or low ethanol
consumers before drug administration (day 22) and after drug administration (day
50). Diamonds with error bars represent mean AUC0–6 h? S.E. for the group before
and after drug administration with (filled) or without (open) the inclusion of a
monkey with a noticeably high CZN AUC0–6 hboth before and after ethanol
exposure (indicated by the dashed line). Significantly different from same group of
monkeys before drug administration: ?, p ? 0.05.
FERGUSON ET AL.
that among moderate consumers, those with higher daily ethanol
intakes may be at elevated risk for CYP2E1-associated diseases.
The average smoker has a total daily nicotine intake of 0.2 to 1.1
mg/kg (Benowitz and Jacob, 1984), resulting in plasma levels of 10 to
50 ng/ml during the day (Benowitz et al., 1990). The total daily
nicotine dose administered to the monkeys was at the high end of this
range (1.0 mg/kg per day) to compensate for the slightly faster
nicotine metabolism in AGMs (Lee et al., 2006a). Although the
pattern of nicotine intake in this study differs from smoking, the levels
and duration of nicotine in the plasma were estimated to be compa-
rable to those observed in human smokers (Benowitz et al., 1990; Lee
et al., 2006a). Nicotine increased CYP2E1 protein, CYP2E1 in vitro
activity, and in vivo CZN clearance in the monkeys, consistent with
the results of a previous study that assessed the effect of chronic
nicotine treatment on CYP2E1 and CZN disposition in African green
monkeys (Lee et al., 2006b). The increase in CZN clearance after
chronic nicotine exposure, without any change in CZN half-life,
suggests that nicotine increases CZN first-pass metabolism. It is
unlikely that the change in CZN clearance could be attributed to the
effect of nicotine on blood flow. Monkeys were administered CZN
more than 12 h after the last nicotine injection, at which point any
effect on hepatic blood flow would have subsided (Hashimoto et al.,
2004). In humans, smoking increases CZN clearance by 25% (Be-
nowitz et al., 2003). Nicotine-treated monkeys, likewise, showed a
34% decrease in CZN AUC, supporting a role for nicotine as the
CYP2E1-inducing agent in cigarette smoke. Our results are not con-
sistent with the conclusions from a recent study showing that admin-
istration of a 42-mg transdermal nicotine patch twice a day for 10 days
did not affect CZN clearance in humans (Hukkanen et al., 2010). This
discrepancy may be due to differences in the dose and duration of
nicotine achieved in the plasma with a transdermal nicotine patch
versus smoking, which our study was designed to model.
There was a trend for monkeys in the ethanol and nicotine com-
bined group having the highest CYP2E1 protein levels and fastest
rates of in vitro CZN metabolism compared with monkeys in either
the ethanol-only or nicotine-only groups. This trend was not present
in the in vivo CZN pharmacokinetic parameters, which may be due to
the timing of the CZN pharmacokinetic testing. The postdrug CZN
challenge was performed on day 50 and protein levels and in vitro
activity were assessed in tissue from monkeys sacrificed on day 64.
Ongoing induction of CYP2E1 by ethanol and nicotine may have
occurred between days 50 and 64 of the study, resulting in a lack of
correlation between in vivo and ex vivo assessment of CYP2E1
activity. In humans, moderate consumption of alcohol (40 g/day) over
a period of 4 weeks resulted in a gradual increase in CYP2E1 activity
measured by CZN clearance. CZN clearance was significantly faster
on day 28 than on to day 21 (Oneta et al., 2002). In rats, 7 days of
nicotine treatment are sufficient to induce CYP2E1 levels in the liver;
however, whether there is further induction of CYP2E1 beyond 7 days
is not known (Joshi and Tyndale, 2006). In a previous study, a
correlation between in vivo CZN clearance and CYP2E1 protein
levels was shown in nicotine-treated monkeys; however, the duration
between the in vivo and ex vivo testing was substantially shorter
compared with that in this study (7 versus 14 days) (Lee et al., 2006b).
Hepatic CYP2E1 mRNA levels were not significantly altered by
nicotine or ethanol exposure, suggesting that, at these doses and
durations of ethanol and nicotine exposure, induction of CYP2E1
protein occurs via a nontranscriptional mechanism. Consistent with
our findings, ethanol-treated rats with BELs similar to those of our
monkeys showed induction of hepatic CYP2E1 without a correspond-
ing increase in CYP2E1 mRNA levels (Ronis et al., 1993). Ethanol
induces CYP2E1 protein levels in rats by protecting the enzyme from
degradation (Roberts et al., 1995). Nicotine does not induce rat
hepatic CYP2E1 via transcription or by protein stabilization and may
FIG. 6. Ethanol (EtOH) self-administration and nicotine treatment,
alone and in combination, induced hepatic CYP2E1 protein levels.
A, representative immunoblot for hepatic CYP2E1 protein from
monkeys in the four study groups (showing n ? 4 of 10/group).
Coomassie Blue staining was used to confirm equal protein loading
among lanes. B, mean percentage increase in CYP2E1 protein
relative to control group. A significant linear trend was observed,
with animals in the ethanol ? nicotine group (group 4) having the
highest levels of CYP2E1. Significantly different from control
group: ?, p ? 0.05; ??, p ? 0.001. n ? 10/group. C, mean
percentage increase in hepatic CYP2E1 protein in high and low
ethanol consumers (Fig. 2B) relative to the control group. A sig-
nificant linear trend was observed, demonstrating increased
CYP2E1 levels with higher levels of alcohol consumption. Signif-
icantly different from control group: ?, p ? 0.05; ??, p ? 0.001.
n ? 10/group.
ETHANOL AND NICOTINE INDUCE HEPATIC CYP2E1
involve an increase in translational efficiency (Wu et al., 1997; Micu
et al., 2003). Thus, ethanol and nicotine may increase CYP2E1 in
monkey liver using post-transcriptional mechanisms similar to those
observed for rat liver CYP2E1.
In conclusion, ethanol and nicotine increased hepatic CYP2E1 protein
and in vitro CZN metabolism, leading to increased CZN clearance in
vivo. The effect of ethanol was dependent on the level of daily ethanol
intake and combined exposure to ethanol and nicotine resulted in the
highest levels of hepatic CYP2E1 protein and activity. Nicotine treatment
did not affect ethanol consumption. The induction of hepatic CYP2E1 by
nicotine and ethanol may mediate some of the negative health effects of
smoking and alcohol consumption via increased metabolic bioactivation
of many commonly used industrial chemicals, environmental pollutants,
and drugs into toxic metabolites (Lieber, 1997). Because a large propor-
tion of the population is exposed to ethanol, nicotine, or often both, these
findings have important implications for public health, health risk assess-
ment, and disease prevention.
We thank Dr. Bin Zhao and Ewa Hoffman for excellent technical assistance
and Jibran Khokhar for reviewing the manuscript.
Participated in research design: Miksys, Palmour, and Tyndale.
Conducted experiments: Ferguson, Miksys, and Palmour.
Performed data analysis: Ferguson.
Wrote or contributed to the writing of the manuscript: Ferguson and
Amato G, Longo V, Mazzaccaro A, and Gervasi PG (1998) Chlorzoxazone 6-hydroxylase and
p-nitrophenol hydroxylase as the most suitable activities for assaying cytochrome P450 2E1 in
cynomolgus monkey liver. Drug Metab Dispos 26:483–489.
Barrett SP, Tichauer M, Leyton M, and Pihl RO (2006) Nicotine increases alcohol self-
administration in non-dependent male smokers. Drug Alcohol Depend 81:197–204.
Benowitz NL and Jacob P 3rd (1984) Daily intake of nicotine during cigarette smoking. Clin
Pharmacol Ther 35:499–504.
Benowitz NL, Peng M, and Jacob P 3rd (2003) Effects of cigarette smoking and carbon
monoxide on chlorzoxazone and caffeine metabolism. Clin Pharmacol Ther 74:468–474.
Benowitz NL, Porchet H, and Jacob P 3rd (1990) Pharmacokinetics, metabolism, and pharma-
codynamics of nicotine, in Nicotine Psychopharmacology: Molecular, Cellular and Behav-
ioral Aspects (Wonnacott S, Russell MAH, and Stolerman IP eds) Oxford University Press,
Blomqvist O, Ericson M, Johnson DH, Engel JA, and So ¨derpalm B (1996) Voluntary ethanol
intake in the rat: effects of nicotinic acetylcholine receptor blockade or subchronic nicotine
treatment. Eur J Pharmacol 314:257–267.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254.
Caro AA and Cederbaum AI (2004) Oxidative stress, toxicology, and pharmacology of CYP2E1.
Annu Rev Pharmacol Toxicol 44:27–42.
Cooney JL, Cooney NL, Pilkey DT, Kranzler HR, and Oncken CA (2003) Effects of nicotine
deprivation on urges to drink and smoke in alcoholic smokers. Addiction 98:913–921.
Eckardt MJ, File SE, Gessa GL, Grant KA, Guerri C, Hoffman PL, Kalant H, Koob GF, Li TK,
and Tabakoff B (1998) Effects of moderate alcohol consumption on the central nervous
system. Alcohol Clin Exp Res 22:998–1040.
Ernstgård L, Warholm M, and Johanson G (2004) Robustness of chlorzoxazone as an in vivo
measure of cytochrome P450 2E1 activity. Br J Clin Pharmacol 58:190–200.
Ervin FR, Palmour RM, Young SN, Guzman-Flores C, and Juarez J (1990) Voluntary consump-
tion of beverage alcohol by vervet monkeys: population screening, descriptive behavior and
biochemical measures. Pharmacol Biochem Behav 36:367–373.
Hashimoto T, Yoneda M, Shimada T, Kurosawa M, and Terano A (2004) Intraportal nicotine
infusion in rats decreases hepatic blood flow through endothelin-1 and both endothelin A and
endothelin B receptors. Toxicol Appl Pharmacol 196:1–10.
Hayashi S, Watanabe J, and Kawajiri K (1991) Genetic polymorphisms in the 5?-flanking region
change transcriptional regulation of the human cytochrome P450IIE1 gene. J Biochem 110:
Hukkanen J, Jacob Iii P, Peng M, Dempsey D, and Benowitz NL (2010) Effects of nicotine on
cytochrome P450 2A6 and 2E1 activities. Br J Clin Pharmacol 69:152–159.
Ivester P, Roberts LJ 2nd, Young T, Stafforini D, Vivian J, Lees C, Young J, Daunais J,
Friedman D, Rippe RA, et al. (2007) Ethanol self-administration and alterations in the livers
of the cynomolgus monkey, Macaca fascicularis. Alcohol Clin Exp Res 31:144–155.
Joshi M and Tyndale RF (2006) Induction and recovery time course of rat brain CYP2E1 after
nicotine treatment. Drug Metab Dispos 34:647–652.
Lands WE (1998) A review of alcohol clearance in humans. Alcohol 15:147–160.
Le ˆ AD, Corrigall WA, Harding JW, Juzytsch W, and Li TK (2000) Involvement of nicotinic
receptors in alcohol self-administration. Alcohol Clin Exp Res 24:155–163.
Leclercq I, Horsmans Y, and Desager JP (1998) Estimation of chlorzoxazone hydroxylase
activity in liver microsomes and of the plasma pharmacokinetics of chlorzoxazone by the same
high-performance liquid chromatographic method. J Chromatogr A 828:291–296.
Lee AM, Miksys S, Palmour R, and Tyndale RF (2006a) CYP2B6 is expressed in African Green
monkey brain and is induced by chronic nicotine treatment. Neuropharmacology 50:441–450.
Lee AM, Yue J, and Tyndale RF (2006b) In vivo and in vitro characterization of chlorzoxazone
metabolism and hepatic CYP2E1 levels in African Green monkeys: induction by chronic
nicotine treatment. Drug Metab Dispos 34:1508–1515.
Lieber CS (1997) Cytochrome P-4502E1: its physiological and pathological role. Physiol Rev
Liu R, Yin LH, and Pu YP (2007) Association of combined CYP2E1 gene polymorphism with
the risk for esophageal squamous cell carcinoma in Huai’an population, China. Chin Med J
Matsumoto H, Matsubayashi K, and Fukui Y (1996) Evidence that cytochrome P-4502E1
contributes to ethanol elimination at low doses: effects of diallyl sulfide and 4-methyl pyrazole
on ethanol elimination in the perfused rat liver. Alcohol Clin Exp Res 20:12A–16A.
Micu AL, Miksys S, Sellers EM, Koop DR, and Tyndale RF (2003) Rat hepatic CYP2E1 is
induced by very low nicotine doses: an investigation of induction, time course, dose response,
and mechanism. J Pharmacol Exp Ther 306:941–947.
Millonig G, Wang Y, Homann N, Bernhardt F, Qin H, Mueller S, Bartsch H, and Seitz HK
(2011) Ethanol-mediated carcinogenesis in the human esophagus implicates CYP2E1 induc-
tion and the generation of carcinogenic DNA-lesions. Int J Cancer 128:533–540.
Morita M, Le Marchand L, Kono S, Yin G, Toyomura K, Nagano J, Mizoue T, Mibu R, Tanaka
M, Kakeji Y, et al. (2009) Genetic polymorphisms of CYP2E1 and risk of colorectal cancer:
the Fukuoka Colorectal Cancer Study. Cancer Epidemiol Biomarkers Prev 18:235–241.
Munaka M, Kohshi K, Kawamoto T, Takasawa S, Nagata N, Itoh H, Oda S, and Katoh T (2003)
Genetic polymorphisms of tobacco- and alcohol-related metabolizing enzymes and the risk of
hepatocellular carcinoma. J Cancer Res Clin Oncol 129:355–360.
Olausson P, Ericson M, Lo ¨f E, Engel JA, and So ¨derpalm B (2001) Nicotine-induced behavioral
disinhibition and ethanol preference correlate after repeated nicotine treatment. Eur J Phar-
Oneta CM, Lieber CS, Li J, Ru ¨ttimann S, Schmid B, Lattmann J, Rosman AS, and Seitz HK
FIG. 7. Ethanol (EtOH) self-administration and nicotine treatment increased in
vitro chlorzoxazone metabolism. A, hepatic CYP2E1 protein levels were positively
correlated with 6OHCZN formation velocity at 950 ?M CZN (approximate Vmax).
B, mean percentage increases in the 6OHCZN formation velocity relative to the
control group. A significant linear trend was observed, with animals in the ethanol ?
nicotine group (group 4) having the highest velocity. Significantly different from
control group: ?, p ? 0.05. n ? 10/group. C, mean percentage increases in velocity
of CZN metabolism in high and low ethanol consumers (Fig. 2B) relative to the
control group. A significant linear trend was observed, demonstrating increased
CZN velocity with higher levels of alcohol consumption. Significantly different
from control group: ?, p ? 0.05. n ? 10/group.
FERGUSON ET AL.
(2002) Dynamics of cytochrome P4502E1 activity in man: induction by ethanol and disap- Download full-text
pearance during withdrawal phase. J Hepatol 36:47–52.
Palmour RM, Mulligan J, Howbert JJ, and Ervin F (1997) Of monkeys and men: vervets and the
genetics of human-like behaviors. Am J Hum Genet 61:481–488.
Roberts BJ, Song BJ, Soh Y, Park SS, and Shoaf SE (1995) Ethanol induces CYP2E1 by protein
stabilization. Role of ubiquitin conjugation in the rapid degradation of CYP2E1. J Biol Chem
Ronis MJ, Huang J, Crouch J, Mercado C, Irby D, Valentine CR, Lumpkin CK, Ingelman-
Sundberg M, and Badger TM (1993) Cytochrome P450 CYP 2E1 induction during chronic
alcohol exposure occurs by a two-step mechanism associated with blood alcohol concentra-
tions in rats. J Pharmacol Exp Ther 264:944–950.
Sharpe AL and Samson HH (2002) Repeated nicotine injections decrease operant ethanol
self-administration. Alcohol 28:1–7.
Shiffman S and Balbanis M (1995) Associations between Alcohol and Tobacco. National Institute
on Alcohol Abuse and Alcoholism, Bethesda, MD.
Vidal F, Perez J, Morancho J, Pinto B, and Richart C (1990) Hepatic alcohol dehydrogenase
activity in alcoholic subjects with and without liver disease. Gut 31:707–711.
Wang W, Qiu YL, Ji F, Liu J, Wu F, Miao WB, Li Y, Brandt-Rauf PW, and Xia ZL (2010)
Genetic polymorphisms in metabolizing enzymes and susceptibility of chromosomal damage
induced by vinyl chloride monomer in a Chinese worker population. J Occup Environ Med
Wu D, Ramin SA, and Cederbaum AI (1997) Effect of pyridine on the expression of cytochrome
P450 isozymes in primary rat hepatocyte culture. Mol Cell Biochem 173:103–111.
Yue J, Khokhar J, Miksys S, and Tyndale RF (2009) Differential induction of ethanol-
metabolizing CYP2E1 and nicotine-metabolizing CYP2B1/2 in rat liver by chronic nicotine
treatment and voluntary ethanol intake. Eur J Pharmacol 609:88–95.
Zhang Y, Liu Q, Liu Q, Duan H, Cheng J, Jiang S, Huang X, Leng S, He F, and Zheng Y
(2006) Association between metabolic gene polymorphisms and susceptibility to periph-
eral nerve damage in workers exposed to n-hexane: a preliminary study. Biomarkers
Address correspondence to: Dr. Rachel F. Tyndale, Department of Pharma-
cology and Toxicology, Room 4326, 1 King’s College Circle, Toronto ON, Canada
M5S 1A8. E-mail: firstname.lastname@example.org
ETHANOL AND NICOTINE INDUCE HEPATIC CYP2E1