Rat Hepatic CYP2E1 Is Induced by Very Low Nicotine Doses:
An Investigation of Induction, Time Course, Dose Response,
ALINA L. MICU, SHARON MIKSYS, EDWARD M. SELLERS, DENNIS R. KOOP, and RACHEL F. TYNDALE
Departments of Pharmacology (A.L.M., E.M.S., R.F.T.) Medicine (E.M.S.), and Psychiatry (E.M.S.), and the Centre for Addiction and Mental
Health (S.M., E.M.S., R.F.T.), University of Toronto, Toronto, Ontario, Canada; and Department of Physiology and Pharmacology (D.R.K),
Oregon Health Sciences University, Portland, Oregon
Received March 27, 2003; accepted May 13, 2003
CYP2E1 is an ethanol- and drug-metabolizing enzyme that can
also activate procarcinogens and hepatotoxicants and gener-
ate reactive oxygen species; it has been implicated in the
pathogenesis of liver diseases and cancer. Cigarette smoke
increases CYP2E1 activity in rodents and in humans and we
have shown that nicotine (0.1–1.0 mg/kg s.c. ? 7 days) in-
creases CYP2E1 protein and activity in the rat liver. In the
current study, we have shown that the induction peaks at 4 h
postnicotine (1 mg/kg s.c. ? 7 days) treatment and recovers
within 24 h. No induction was observed after a single injection,
and 18 days of treatment did not increase the levels beyond
that found at 7 days. We found that CYP2E1 is induced by very
low doses of chronic (? 7 days) nicotine with an ED50value of
0.01 mg/kg s.c.; 0.01 mg/kg in a rat model results in peak
cotinine levels (nicotine metabolite) similar to those found in
people exposed to environmental tobacco smoke (passive
smokers; 2–7 ng/ml). Previously, we have shown no change in
CYP2E1 mRNA, and our current mechanistic study indicates
that nicotine does not regulate CYP2E1 expression by protein
stabilization. We postulated that a nicotine metabolite could be
causing the induction but found that cotinine (1 mg/kg ? 7
days) did not increase CYP2E1. Our findings indicate that nic-
otine increases CYP2E1 at very low doses and may enhance
CYP2E1-related toxicity in smokers, passive smokers, and
people treated with nicotine (e.g., smokers, patients with Alz-
heimer’s disease, ulcerative colitis or Parkinson’s disease).
CYP2E1 is a member of the cytochrome P450 superfamily
of heme-containing enzymes involved in the biotransforma-
tion of a variety of endogenous and exogenous compounds.
CYP2E1 is of interest due to its potentially important role in
the toxicity of a variety of chemicals and in the propagation
of hepatic diseases. A unique characteristic of CYP2E1 is its
high NADPH oxidase activity (Gorsky et al., 1984), which
results in increased production of reactive oxygen species,
cytochrome P450 isoforms (Gorsky et al., 1984). These reac-
tive intermediates can initiate lipid peroxidation, oxidative
stress, and Kupffer cell activation, thereby propagating cel-
lular injury and DNA strand breaks (Jarvelainen et al.,
?(superoxide radical) and H2O2relative to other
CYP2E1 activates many xenobiotics to hepatotoxic or car-
cinogenic products, including drugs such as acetaminophen
(Raucy et al., 1989), industrial solvents such as carbon tet-
rachloride (CCl4) (Manno et al., 1996), and nitrosamines such
as N-nitrosodimethylamine (Lin et al., 1998). These hepato-
toxicants and carcinogens are known to cause selective injury
predominantly in the perivenular area, providing further
evidence for the involvement of CYP2E1 as the presence and
induction of CYP2E1 occur predominantly in this zone of the
liver lobule (Tsutsumi et al., 1989). Furthermore, CYP2E1
activity strongly correlates with degree of tissue injury in-
duced by these toxicants (Lieber, 1997).
CYP2E1 activity is induced by a variety of compounds
many of which are also substrates of this enzyme. Ethanol is
the best characterized CYP2E1 substrate and inducer. It is
estimated that CYP2E1 is responsible for approximately 20%
of ethanol metabolism at pharmacologically relevant blood
alcohol concentrations (Matsumoto et al., 1996). Moreover,
CYP2E1 is increased 4- to 10-fold in liver biopsies of recently
alcohol drinking patients (Tsutsumi et al., 1989) and
CYP2E1 is thought to contribute to the increased ethanol
metabolism and subsequent metabolic tolerance that devel-
Financial support for this study was provided by Canadian Institutes of
Health Research (CIHR) grant MT 14173, a Canadian Research Chair in
Pharmacogenetics and the Centre for Addiction and Mental Health. A prelim-
inary report of this study was presented at the following conference and
published in abstract form [Micu AM, Howard LA, Miksys S, Sellers EM, and
Tyndale RF (2002) Induction of ethanol-inactivating and pro-carcinogen acti-
vating CYP2E1 by Nicotine. Proc Soc Res Nicotine Tob].
Article, publication date, and citation information can be found at
ABBREVIATIONS: CHO, Chinese hamster ovary; ETS, environmental tobacco smoke.
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics
JPET 306:941–947, 2003
Vol. 306, No. 3
Printed in U.S.A.
at ASPET Journals on November 3, 2015
ops in alcoholics (Lieber, 1999). Induction of CYP2E1 by
agents such as ethanol can lead to a more pronounced for-
mation of toxic and reactive metabolites, which could lead to
a higher risk of organ damage and carcinogenicity in people
exposed to substrate chemicals and solvents. This may ex-
plain the increased vulnerability to hepatic toxicity of alcohol
abusers upon exposure to therapeutically and industrially
used xenobiotics such as acetaminophen and CCl4(Seeff et
In addition, CYP2E1 activity can be induced by endoge-
nous substrates and by pathophysiological states that result
in the accumulation of ketones, such as fasting, diabetes, and
obesity (Lieber, 1997, 1999). This physiological role may ex-
plain CYP2E1’s high functional and regulatory conservation
across species and within the human population (Lieber,
The molecular regulation of CYP2E1 induction is complex,
involving transcriptional, post-transcriptional, and post-
translational mechanisms (Lieber, 1997). The mechanism
involved in CYP2E1 induction may depend on the dose, du-
ration, and/or route of inducer treatment, as well on the
specific structurally diverse inducing agent. For example,
CYP2E1 induction by ethanol seems to occur by a two-step
mechanism: post-translational mechanisms at low ethanol
levels and transcriptional mechanisms at high ethanol levels
(Badger et al., 1993; Ronis et al., 1993).
Cigarette smoke induces CYP2E1 activity in rodents and
in humans (Villard et al., 1998; Benowitzet al., 1999b). By
inducing the activity and expression of CYP2E1, cigarette
smoke may enhance the production of carcinogenic and toxic
metabolites, further increasing the risk for cancer and organ
damage in smokers. These findings could perhaps explain the
fact that alcohol abuse and smoking interact synergistically
as etiological factors for cancers (Garro and Lieber, 1990).
Furthermore, because CYP2E1 substrates are commonly
found in the industrial workplace (Raucy et al., 1993; Wang
et al., 1996), smokers in these environments may be more
sensitive to chemical injury than nonsmokers.
Our laboratory has demonstrated that nicotine (0.1–1.0
mg/kg s.c. ? 7 days), which is the most abundant chemical in
cigarette smoke, increases CYP2E1 protein and activity in
the rat liver (Howard et al., 2001). Because the observed level
of CYP2E1 induction (1.5–1.8-fold) by nicotine was saturated
at the doses tested in this initial study, we hypothesized that
lower nicotine doses can induce hepatic CYP2E1. Such lower
doses may be important in terms of passive smokers or peo-
ple on nicotine replacement therapy who may also be at risk
of liver damage associated with increased CYP2E1 enzyme.
The purpose of the current study was to further characterize
this effect in terms of the dose-response relationship, time
course for induction, recovery time course of induction, mech-
anisms of regulation, and potential involvement of the nico-
tine metabolite cotinine in CYP2E1 induction.
Materials and Methods
Materials. Nicotine bitartrate and cotinine were purchased from
Sigma-Aldrich Canada Ltd. (Oakville, ON, Canada). Recombinant
viral-expressed rat CYP2E1 in lymphoblastoid cells was obtained
from BD Gentest (Woburn, MA). The protein assay dye reagent
concentrate was purchased from Bio-Rad (Hercules, CA). Prestained
molecular markers were purchased from MBI Fermentas (Flambor-
ough, ON, Canada). Rabbit anti-rat CYP2E1 polyclonal antibody was
generously provided by Magnus Ingelman-Sundberg (Department of
Physiological Chemistry, Karolinska Institute, Stockholm, Sweden).
Horseradish peroxidase-conjugated goat anti-rabbit IgG and Super-
Signal West Pico chemiluminescence substrate were purchased from
Pierce Chemical (Rockford, IL). Hybond ECL nitrocellulose mem-
brane was purchased from Amersham Biosciences (Toronto, ON,
Canada). All other chemical reagents were obtained from standard
commercial sources. GM0637 cells were obtained from National In-
stitute of General Medical Sciences Human Genetic Mutant Cell
Repository (Camden, NJ), and CHO-K1 cells were obtained from
American Type Culture Collection (Rockville, MD).
Animals. Adult male Wistar rats (250–300 g; Charles River,
St-Constant, PQ, Canada) were used throughout these experiments.
Upon arrival in the Animal Care Facility the animals were housed
two per cage and allowed to adapt to the novel environment for 1
week. The animals were kept in a controlled environment with a
12-h artificial light/dark cycle (light on at 6:00 AM). The animals
received rat chow and water ad libitum throughout the study period.
All procedures described in the present study were conducted in
accordance with the guidelines for the care and use of laboratory
animals and were approved by the Animal Care Committee of the
University of Toronto.
Drug Treatment. The rats received s.c. injections of either nico-
tine bitartrate or cotinine dissolved in sterile saline. Solutions of
nicotine containing 1 mg/ml (6.16 ?mol/ml) nicotine base were pre-
pared fresh daily by adding 28.51 mg of nicotine salt to 10 ml of
sterile saline (0.9% NaCl). This stock solution was titrated to pH 7.4
with 1 N NaOH and then was serially diluted to achieve desired
nicotine concentrations. All nicotine doses cited are calculated as the
base and were given per kilogram of body weight. Cotinine solution,
at a concentration of 1.086 mg/ml (6.16 ?mol/ml) cotinine, was also
prepared daily by adding 4.5 mg of cotinine to 4.14 ml of saline.
Storage bottles were kept wrapped in aluminum foil due to the light
sensitivity of both nicotine and cotinine. Control animals were
treated with vehicle (saline) using an identical administration pro-
tocol. Rats were sacrificed by decapitation 4 h after the last drug
treatment unless specified otherwise. Livers were rapidly removed,
frozen immediately in liquid nitrogen and stored at ?80°C until used
for microsomal preparation.
Recovery Time-Course Study. In a time-course study, rats
were treated with 0 or 1 mg/kg nicotine (n ? 3/group) for 7 consec-
utive days and were sacrificed at 0.5, 2, 4, 8, 12, 18, and 24 h after the
last drug treatment. Saline controls were included for each time
point to eliminate any possible contribution of a diurnal cycle effect
on CYP2E1 levels (Bruckner et al., 2002).
Induction Time-Course Study. Rats (n ? 4/group) received a
single, acute injection of 0 or 1.0 mg/kg nicotine s.c. and were sacri-
ficed 4 h later; these findings were contrasted to animals treated for
5, 7, and 18 days.
Nicotine Dose-Response Study. Dose-response studies in-
cluded nine groups of rats (n ? 3/group), injected for 7 consecutive
days with 0, 0.001, 0.003, 0.005, 0.01, 0.02, 0.03, 0.1, or 1.0 mg/kg
nicotine s.c. We have previously demonstrated that treatment of rats
with 0.1 and 1.0 mg/kg nicotine results in a statistically significant
increase in CYP2E1 protein level in rat liver (Howard et al., 2001);
these doses were thus included as positive controls in the present
study. The nicotine doses used in this study are of behavioral and
pharmacological relevance. Subcutaneous nicotine administration of
1.6 and 1.2 mg/kg have been associated with central nicotinic recep-
tor adaptation, a pharmacodynamic change observed in brain re-
gions of smokers and hypothesized to be one pathway by which
nicotine exerts its behavioral effects such as tolerance (Rowell and
Li, 1997; Perry et al., 1999). This range also includes doses that rats
will self-administer (0.03 and 0.06 mg/kg/infusion or 0.3 and 0.6
mg/kg in 2 h) (Shoaib and Stolerman, 1999) and doses at which
nicotine exerts its discriminative stimulus in rats (ED50? 0.14
mg/kg s.c.) (Pratt et al., 1983). In addition chronic injections of 0.8 to
Micu et al.
at ASPET Journals on November 3, 2015
1.6 mg/kg nicotine increase ethanol self-administration by male
Wistar rats (Blomqvist et al., 1996).
Cotinine Study. Rats (n ? 3/group) were injected with cotinine at
1.086 mg/kg s.c. for seven consecutive days and were sacrificed at
0.5, 4, and 8 h after the last treatment.
Microsomal Preparation. Liver microsomes were prepared as
described previously (Miksys et al., 2000), aliquoted into small vol-
umes, and stored at ?80°C until use.
Western Blotting. Immunoblotting was performed as reported
previously (Howard et al., 2001), with minor modifications. In brief,
proteins (1.5 ?g/liver sample) were separated by SDS-polyacryl-
amide gel electrophoresis (4% stacking and 10% separating gels),
transferred overnight onto nitrocellulose membrane, and probed
with a polyclonal rabbit anti-rat CYP2E1 antibody (1:4,000 dilution
for 1 h). Microsomes from rat CYP2E1-expressing-lymphoblastoid
cells were used as positive control. A mini standard curve consisting
of 0, 1.0, 1.5, and 2.0 ?g of hepatic microsomal protein was included
in all experiments to allow the analysis of only those blots with
samples that fell within the linear region of detection of the standard
curve. Membranes were incubated with a peroxidase-conjugated sec-
ondary antibody (dilution 1:25,000 for 1 h) followed by detection by
chemiluminescence. Membranes were exposed to autoradiography
film (Ultident, St. Laurent, PQ, Canada) for 0.25 to 2 min. Digital
images of immunoblots were analyzed using MCID Elite software
(Imaging Research Inc., St. Catherines, ON, Canada). The relative
density of each band was corrected for the density of the film back-
ground and expressed as arbitrary density units.
CYP2E1 protein stabilization were performed according to published
protocols (Barmada et al., 1995; Huan and Koop, 1999). In brief,
Chinese hamster ovary cells (CHO-K1) that constitutively express
rabbit CYP2E1 protein (Barmada et al., 1995) and human fibroblast
cells (GM0637) that express rat CYP2E1 (Lin et al., 1998) were
treated with various concentrations of nicotine (5–0.00005 ?M).
CYP2E1 catalytic activity was assessed by the 6-hydroxylation of
chlorzoxazone. After overnight treatment with or without nicotine,
cells were incubated with 200 ?M chlorzoxazone for 6 h. Media were
then extracted and 6-OH-chlorzoxazone was determined by high-
performance liquid chromatography (Barmada et al., 1995). Cells
were allowed to lyse passively, insoluble debris was removed by
centrifugation, and the supernatants were frozen until use for im-
munoblot analysis. Western blotting was performed to measure the
levels of immunodetectable CYP2E1 protein. Methylpyrazole can
stabilize CYP2E1 in this in vitro model (Huan and Koop, 1999) and
was used here as a positive control.
Determination of Plasma Nicotine and Cotinine. Trunk
blood (6–8 ml) was collected at the time of sacrifice. Plasma was
prepared by centrifugation at 3000g for 10 min and was stored at
?20°C. Nicotine and cotinine plasma concentrations were measured
by standard high-performance liquid chromatography techniques
using 5-methylnicotine as the internal standard (Xu et al., 2002).
The limit of detection of the assay was 1.5 ng/ml, and there was a
linear relationship between detected chromatographic peak and nic-
otine and cotinine concentrations.
Statistical Analyses. Results are expressed as mean ? S.D.,
which represents the average values obtained from three different
animals per treatment group, from at least three separate experi-
ments. In all experiments, statistical significance of the difference
between control and treated groups was determined using unpaired
Student’s t test. For the dose-response study, one-way analysis of
variance was used for comparisons of multiple group means. Statis-
tically significant differences were determined at the 5% level (p ?
An immunoblotting assay was established to measure he-
patic CYP2E1. Detection of serially diluted CYP2E1 indi-
cated that the immunoblotting signal was linear up to 3.0 ?g
of microsomal protein from saline-treated animals; no signal
was detected in the absence of primary antibody (data not
shown). The specificity of the rabbit antibody raised against
rat CYP2E1 has been demonstrated previously (Hansson et
al., 1990), and we have reconfirmed this finding (Howard et
CYP2E1 Recovery Time Course. The return to basal
levels of CYP2E1 after induction, after 7 days of treatment
with 1.0 mg/kg nicotine, was assessed as a function of time
over 24 h. Western blot analyses revealed significant in-
creases in CYP2E1 levels of 1.2- (p ? 0.05), 1.3- (p ? 0.05),
1.6- (p ? 0.01), 1.4- (p ? 0.05), and 1.3-fold (p ? 0.01) at 0.5,
2, 4, 8, and 12 h post-treatment compared with their respec-
tive control groups (Fig. 1A). The higher levels of CYP2E1 in
nicotine-treated rats returned to the levels found in saline-
treated animals by 18 h post-treatment. In this study, the
concentration of plasma nicotine and its metabolite cotinine
were determined as a function of sacrifice time after the last
drug treatment. The plasma level of nicotine was highest at
30 min (first time tested) and declined by 4 h postinjection
(Fig. 1B). The level of cotinine peaked at 4 h and then slowly
declined to near control levels by 24 h post-treatment (Fig.
1B). The maximal increases in CYP2E1 protein were ob-
served at 4 h post-treatment, a time when nicotine levels
were essentially zero, whereas cotinine levels were at their
Fig. 1. Recovery time course of hepatic CYP2E1 induction by 1.0 mg/kg
s.c nicotine for 7 days. A, fold-induction in CYP2E1 levels relative to
saline- (Sal) treated animals over 24 h post-treatment. Each point rep-
resents the mean ? S.D. of at least three different experiments (n ? 3
rats). Significant difference from saline is indicated by ?, p ? 0.05. B,
corresponding plasma nicotine (Nic) and cotinine (Cot) levels over 24 h
Hepatic CYP2E1 Induction by Nicotine in the Rat
at ASPET Journals on November 3, 2015
peak. Neither nicotine nor cotinine was detected in vehicle-
Nicotine Induction Time-Course Study. Time-course
studies revealed that CYP2E1 induction by nicotine returned
to basal levels by 24 h. This finding prompted us to examine
whether the necessary changes leading to induction require a
chronic (7 days) treatment or whether these changes occur
after an acute (single) treatment. Western blot analyses (Fig.
2B) were carried out to determine the effect of a single, acute
dose of nicotine on the expression of hepatic CYP2E1. Treat-
ment of rats with 1.0 mg/kg nicotine for 1 day failed to alter
CYP2E1 protein levels (p ? 0.36) (Fig. 2A). However, in rats
treated chronically with the same dose for 5, 7, and 18 con-
secutive days, we observed a 1.4- (p ? 0.3), 1.7- (p ? 0.01),
and 1.5-fold (p ? 0.05) increase in CYP2E1 levels. Plasma
nicotine and cotinine levels were measured in this study at
the time of sacrifice (4 h post-treatment). We observed no
statistically significant (p ? 0.18 and p ? 0.06) accumulation
of nicotine (12, 14, 20, and 15 ng/ml) or cotinine (229, 250,
272, and 215 ng/ml) in rats treated for 1, 5, 7, or 18 days,
Dose-Dependent Induction of CYP2E1 by Nicotine.
Previous studies conducted in our laboratory have demon-
strated that treatment of rats with nicotine at 0.1, 0.3, and
1.0 mg/kg for seven consecutive days resulted in increased
CYP2E1 protein (1.4-, 1.8-, and 1.5-fold, respectively)
(Howard et al., 2001). Moreover, we observed increased
CYP2E1 activity (increased Vmax, no change in Km) in the
same animals, of similar magnitude to the observed increase
in CYP2E1 protein, as assessed by chlorzoxazone metabolism
(Howard et al., 2001). We confirmed that the enhancement of
chlorzoxazone hydroxylation was mediated by CYP2E1 by
chemical inhibitory studies with specific CYP2E1 inhibitors.
The apparent saturation of CYP2E1 protein induction
prompted us to examine whether nicotine can increase
CYP2E1 at doses lower than 0.1 mg/kg. In the present study,
Western blot analyses were carried out to investigate the
dose-response relationship of nicotine on hepatic CYP2E1
protein levels. Chronic (7-day) treatment of rats with nicotine
at the doses of 0, 0.001, 0.003, 0.005, 0.01, 0.02, 0.03, 0.1, and
1.0 mg/kg/day resulted in 1.02-, 1.05-, 1.17-, 1.40- (p ? 0.005),
1.50- (p ? 0.05), 1.57- (p ? 0.01), 1.55- (p ? 0.001), and
1.62-fold (p ? 0.01) increases in CYP2E1 protein levels com-
pared with saline controls (Fig. 3). The ED50value for the
observed increase in CYP2E1 protein level was approxi-
mately 0.01 mg/kg. Results for treatment with 0.1 and 1.0
mg/kg nicotine were consistent with our previous findings
(Howard et al., 2001).
Plasma nicotine and cotinine levels were also measured in
this study. Due to the low doses used in this study, and due
to the sacrifice time (4 h post-treatment), plasma nicotine
levels were undetectable in rats treated with 0.001 to 0.03
mg/kg s.c. nicotine (Table 1). Even at the highest doses tested
(0.1 and 1.0 mg/kg), the plasma nicotine levels were very low
(1.6 and 19.7 ng/ml, respectively). Cotinine, which has a
much longer elimination half-life compared with nicotine,
was detectable for doses ranging between 0.005 and 1.0
mg/kg (4.0–272.0 ng/ml, respectively) (Table 1). Nicotine and
cotinine levels measured in plasma were reflective of the
administered nicotine dose, which demonstrates that our
treatment was effective.
Nicotine Does Not Induce CYP2E1 by Protein Stabi-
lization. The mechanism by which the majority of CYP2E1
inducers, including ethanol and low-molecular weight li-
gands, increase CYP2E1 is by protein stabilization, particu-
larly at low doses. Previous studies indicated that substrates
or ligands for CYP2E1 can stabilize the protein and inhibit
its degradation, resulting in an increase in the steady-state
level of the enzyme. In the present study, there was no
change in chlorzoxazone (CYP2E1 substrate) metabolism, or
Fig. 2. Acute versus chronic effects of nicotine on CYP2E1 levels. A, no
significant difference was observed in rats treated with a single dose of
1.0 mg/kg nicotine compared with their respective saline controls,
whereas chronic treatment for 7 and 18 days results in a significant
increase in CYP2E1 levels. Significance from saline is indicated by ?, p ?
0.05 and ??, p ? 0.01. B, representative immunoblot of 1 day (acute)
versus 7 day (chronic) nicotine treatment and their representative saline
controls; three animals per treatment group.
Fig. 3. Dose-dependent induction of hepatic CYP2E1 by nicotine. A,
dose-response curve of CYP2E1 induction as a function of nicotine- (Nic)
relative to saline- (Sal) treated animals. Each point represents mean
Nic/Sal of three animals/group ? S.D. Significant difference from saline
is indicated by ?, p ? 0.05. B, representative immunoblot of rats treated
with 0, 0.01, 0.1, and 1.0 mg/kg Nic; three animals per treatment group.
Micu et al.
at ASPET Journals on November 3, 2015
in the levels of immunodetectable CYP2E1 protein in nico-
tine-treated cells, suggesting that the addition of nicotine
failed to inhibit the degradation of CYP2E1 during the over-
night incubation in cells expressing either rabbit or rat
CYP2E1 (data not shown). Addition of the CYP2E1 ligand
4-methylpyrazole decreased the degradation of CYP2E1 as
reported previously (Huan and Koop, 1999), resulting in a
2.5-fold increase in immunodectable CYP2E1 protein. These
results suggest that nicotine does not inhibit the degradation
of CYP2E1 and that the induction of CYP2E1 by nicotine
does not occur via stabilization of the enzyme.
Effects of Cotinine on CYP2E1 Protein Levels. Our
mechanistic studies suggest that nicotine does not increase
CYP2E1 protein by two common induction mechanisms, spe-
cifically transcriptional regulation (Howard et al., 2001) or
protein stabilization. Furthermore, in the time-course stud-
ies, we observed similar patterns of CYP2E1 induction and
plasma cotinine levels over time; nicotine plasma levels were
undetectable at 4 h post-treatment when we observed maxi-
mal CYP2E1 induction. These findings suggested the possi-
bility that the major nicotine metabolite cotinine may be
responsible for the observed CYP2E1 induction by nicotine.
Treatment of rats with 1.086 mg/kg s.c. cotinine (dosing for
equal molarity to 1.0 mg/kg nicotine) for 7 days resulted in
plasma cotinine levels of 837, 489, and 295 ng/ml at 0.5, 4,
and 8 h post-treatment (Fig. 4B). Despite the high plasma
cotinine levels achieved with this treatment, we observed no
significant increase in CYP2E1 protein levels (Fig. 4, A and
B). At 4 and 8 h post-treatment, plasma cotinine levels (Fig.
4B) were similar to those found at 4 h postnicotine (1.0
mg/kg) treatment (Fig. 1B) when maximal CYP2E1 induction
was observed in nicotine-treated rats (Fig. 1A). However,
CYP2E1 levels were not elevated in cotinine-treated rats at 4
or 8 h after the last injection (Fig. 4A). These results indicate
that cotinine does not induce CYP2E1.
Previous studies conducted in our laboratory demonstrated
that relatively low doses of nicotine induce CYP2E1 protein
and activity in the rat liver (Howard et al., 2001). The objec-
tive of the current study was to further characterize this
effect in terms of the dose-response relationship, the on and
off time course of induction, and to investigate mechanisms
for CYP2E1 regulation by nicotine.
Based on our findings, the induction of hepatic CYP2E1 by
nicotine requires multiple doses (Fig. 2). After 7 days of
treatment the induction was rapid, as evidenced by the sig-
nificant elevation in protein levels within 0.5 h of nicotine
treatment (Fig. 1A). This induction is also best characterized
as short lasting, because the elevated levels of CYP2E1 in
nicotine-treated rats returned to near control levels by 18 to
24 h after the last nicotine injection (Fig. 1A). We have also
established that CYP2E1 levels peak at 4 h after nicotine
treatment which suggests that the highest risk in terms of
CYP2E1-associated toxicity may be at approximately 4 h
after nicotine exposure at which time the production of
CYP2E1-mediated toxic metabolites is expected be at its
CYP2E1 inducers, including ethanol (Petersen et al.,
1982), acetone (Forkert et al., 1994), and pyridine (Kim and
Novak, 1990), are known to exert their stimulatory effects on
CYP2E1 in both an acute and a chronic setting. In contrast,
treatment of rats with a single dose of nicotine failed to alter
hepatic CYP2E1 protein levels (Fig. 2). This finding implies
that induction requires more than one exposure to nicotine,
suggesting a required priming effect and is consistent with
the idea that the induction of CYP2E1 by nicotine is not via
protein stabilization. These data also suggest that a single
exposure to nicotine may be insignificant in enhancing
CYP2E1-associated hepatotoxicity, at least at the dose and
paradigm tested here.
We have also shown that CYP2E1 induction is very sensi-
tive to chronic nicotine with an estimated ED50value of 0.01
mg/kg s.c. Plasma nicotine levels in rats treated with 0.01
Plasma nicotine and cotinine levels after s.c. injections of 0.001 to 1.0
Rats were sacrificed at 4 h after last nicotine treatment. Data are means ? S.D. of
three animals in each treatment group.
1.6 ? 0.6
19.7 ? 1.3
4.0 ? 1.0
7.7 ? 1.53
10.0 ? 1.0
10.0 ? 2.65
24.3 ? 2.08
272.0 ? 28.2
Fig. 4. Effects of cotinine (Cot) treatment on CYP2E1 protein level. A, no
significant increase was observed in CYP2E1 levels at 0.5, 4, and 8 h
post-cotinine treatment relative to saline- (Sal) treated animals. B,
plasma cotinine levels over time. Each point represents the mean ? S.D.
of three animals per group.
Hepatic CYP2E1 Induction by Nicotine in the Rat
at ASPET Journals on November 3, 2015
mg/kg s.c. were undetectable at 4 h post-treatment, whereas
cotinine levels were approximately 7.7 ng/ml. Blood or
plasma concentrations of nicotine in smokers generally range
from 10 to 50 ng/ml (Benowitz, 1999a). Plasma cotinine levels
lower than 15 to 17.5 ng/ml are considered suggestive of
exposure to second hand-smoke or environmental tobacco
smoke (ETS). From our studies and previous investigations
(Pratt et al., 1983), subcutaneous nicotine administration to
rats leads to peak nicotine plasma levels after 30 to 60 min.
Although for the dose-response studies we have measured
nicotine and cotinine plasma levels at 4 h after the last
treatment (Table 1), plasma nicotine and cotinine levels were
measured at 30 min after a 7-day treatment with 1.0 mg/kg
s.c. nicotine in our recovery time-course studies (Fig. 3B)
where we found plasma nicotine of approximately 210 ng/ml
and cotinine of 120 ng/ml. Based on the information avail-
able, we can deduce that at the ED50value (0.01 mg/kg s.c.)
for effect on CYP2E1, peak plasma nicotine in rats at 30 min
would be approximately 2.1 ng/ml and peak cotinine plasma
levels at 4 h would be 2.4 to 7.7 ng/ml. Of note, plasma
cotinine in nonsmoking adults living with a smoking versus
a nonsmoking partner have been estimated at 2 and 0.31
ng/ml, respectively (Jarvis et al., 2001). Similar plasma coti-
nine levels have also been observed in young children living
with at least one smoking parent (2.9 ng/ml) versus children
with nonsmoking parents (0.26 ng/ml) (Tang et al., 1999).
Based on the similarities of plasma nicotine and cotinine
levels, we can postulate that nicotine doses of 0.01 mg/kg s.c.
(ED50), at which we observed induction of CYP2E1, would be
comparable with levels of ETS exposure in humans.
Therefore, in addition to smokers and people on nicotine
replacement therapies, individuals exposed to ETS may also
have increased CYP2E1 levels and increased risk of CYP2E1-
related toxicity and cancer development via amplified acti-
vation of tobacco smoke and other procarcinogens. Neverthe-
less, although smokers have higher levels of CYP2E1 in liver
(Benowitz et al., 1999b) and brain (Howard et al., 2003), the
induction by nicotine needs to be explicitly tested in humans,
and the consequences of this induction with clinical and
toxicological outcomes in passive and active smokers inves-
We have previously demonstrated that nicotine does not
regulate CYP2E1 expression by transcriptional mechanisms
(Howard et al., 2001); no change in mRNA levels was ob-
served after CYP2E1 induction by nicotine. An alternative
mechanism of CYP2E1 induction is protein stabilization.
Many CYP2E1 inducers, including ethanol (at low doses),
acetone, and pyrazole, interact with CYP2E1’s active site and
increase CYP2E1 levels by slowing its high NADPH oxidase
activity and the subsequent generation of reactive oxygen
species (Zhukov and Ingelman-Sundberg, 1999). These reac-
tive metabolites are thought to oxidize and modify the en-
zyme labeling CYP2E1 for autodegradation by cellular pro-
teolytic systems (Zhukov and Ingelman-Sundberg, 1999).
Therefore, the binding of such substrates/inducers prolongs
CYP2E1 half-life by inhibiting the fast-phase component as-
sociated with this enzyme’s normal degradation. Because
nicotine does not seem to regulate CYP2E1 by transcrip-
tional mechanisms, we have conducted studies to examine
whether nicotine can induce CYP2E1 by protein stabilization
(Barmada et al., 1995; Huan and Koop, 1999). Using CHO
cells that constitutively express rabbit CYP2E1 and human
fibroblast cells that express rat CYP2E1, no change in chlor-
zoxazone metabolism and no elevation in CYP2E1 protein
levels after nicotine exposure was detected. These findings
suggest that nicotine does not inhibit the degradation of
CYP2E1 by interacting with its active site and that in vivo,
the induction of CYP2E1 by nicotine is unlikely to occur via
stabilization of the enzyme by nicotine. However, the molec-
ular triggers that target CYP2E1 for degradation are still
unclear and require further investigation. Thus, it is possible
that in vivo, nicotine may slow down CYP2E1 degradation by
altering some transduction pathways or targeting events
that are not present, or are different, in the CHO and in the
human fibroblast cell lines. This would, however, distinguish
the induction mechanism of nicotine from that of ethanol,
which has been shown to stabilize CYP2E1 in this experi-
mental model (McGehee et al., 1994). In addition, we have
demonstrated that nicotine does not inhibit CYP2E1 hy-
droxylation of chlorzoxazone in rat liver microsomes (Howard
et al., 2001). We interpret this as evidence that nicotine does
not interact with CYP2E1’s catalytic site and does not induce
CYP2E1 by protein stabilization directly.
Alternatively, nicotine could indirectly cause CYP2E1 pro-
tein stabilization in vivo, via its conversion to metabolites
such as cotinine, which would not occur in the in vitro system
in which we tested the stabilization of CYP2E1. Nicotine is
readily metabolized to cotinine in humans, primarily by he-
patic CYP2A6, whereas in rats hepatic CYP2B1 is the main
catalyst in the conversion of nicotine to cotinine (Nakayama
et al., 1993). Interestingly, in our recovery time-course study
we observed that plasma cotinine levels and CYP2E1 protein
induction displayed similar patterns over 24 h after nicotine
treatment. Moreover, nicotine plasma levels were undetect-
able at 4 h post-treatment when we detected maximal
CYP2E1 induction. Based on these finding, we hypothesized
that cotinine could be responsible for the observed CYP2E1
induction in nicotine-treated rats. However, cotinine treat-
ment did not result in a significant increase in hepatic
CYP2E1 levels (Fig. 4A); the role of intermediate metabolites
such as the nicotine-?1?(5?)iminium ion remains to be inves-
tigated. Although in rats the metabolism of nicotine to coti-
nine is less extensive, and other nicotine metabolites are
more predominant than in humans, the induction of this
enzyme by smoking or nicotine has been observed in humans
(Benowitz et al., 1999b) and in nonhuman primates (A. Lee,
S. Miksys, and R. F. Tyndale unpublished observations),
suggesting that a similar mechanism or compound is respon-
sible. In addition, the acute nicotine dose is unable to induce
CYP2E1, which suggests that the mechanism does not in-
volve protein stabilization by any of the rat metabolites.
Other CYP2E1 inducers, such as pyridine, increase CYP2E1
protein by enhancing CYP2E1 mRNA translational efficiency
(Kim et al., 1990; Lieber, 1999). Because nicotine bears struc-
tural similarity to pyridine, translational activation of
CYP2E1 may be the mechanism underlying nicotine’s induc-
tion of this enzyme. Further investigations are necessary to
elucidate the mechanism involved in CYP2E1 induction by
In conclusion, we have demonstrated that very low doses of
nicotine that are comparable with ETS exposure in humans
can induce hepatic CYP2E1 in the rat liver. Induction of
CYP2E1 results in increased oxidative stress, activation of
tobacco smoke, other procarcinogens, and hepatototoxicants,
Micu et al.
at ASPET Journals on November 3, 2015
which may increase the risk for organ damage and cancer Download full-text
development. Therefore, our data suggest that nicotine may
increase CYP2E1 related toxicity in smokers, passive smok-
ers, and people treated with nicotine (e.g., smokers, patient’s
with Alzheimer’s disease, ulcerative colitis, and neuropsychi-
atric motor disorders).
We thank Helma Nolte, Hernease Davis, and members of Dennis
Koop’s research team for the excellent technical assistance, and
Magnus Ingelman-Sundberg (Department of Physiological Chemis-
try, Karolinska Institute, Stockholm, Sweden) for providing the rab-
bit anti-rat CYP2E1 polyclonal antibody.
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Address correspondence to: Dr. Rachel F. Tyndale, Department of Phar-
macology, 1 King’s College Circle, University of Toronto, Canada M5S 1A8.
Hepatic CYP2E1 Induction by Nicotine in the Rat
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