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DRUG METABOLISM AND DISPOSITION
Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
DMD 34:647–652, 2006
Vol. 34, No. 4
8029/3099216
Printed in U.S.A.
INDUCTION AND RECOVERY TIME COURSE OF RAT BRAIN CYP2E1 AFTER
NICOTINE TREATMENT
Meenal Joshi and Rachel F. Tyndale
Centre for Addiction and Mental Health and Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada
Received October 28, 2005; accepted January 13, 2006
ABSTRACT:
hippocampus (1.8-fold, p < 0.01) and cerebellum (1.4-fold, p <
0.05), returning to basal levels by 24 h. In contrast, acute nicotine
treatment did not induce CYP2E1 in frontal cortex and hippocampus but increased CYP2E1 in cerebellum 8 h after treatment (1.6fold, p < 0.01). Brain CYP2E1 mRNA levels did not increase after
chronic nicotine treatment, suggesting nontranscriptional regulation. Thus, humans exposed to nicotine may have altered CYP2E1mediated metabolism of centrally acting drugs and toxins as well
as altered toxicity because of oxidative stress caused by CYP2E1.
Those affected may include current and passive smokers and
people that may be treated with nicotine such as smokers and,
potentially, patients with Alzheimer’s, Parkinson’s disease, or ulcerative colitis.
Cytochrome P450 2E1 (CYP2E1) is expressed in rat liver as well as
in various rat brain regions (Upadhya et al., 2000; Howard et al.,
2003). Besides a role in the inactivation of alcohol, CYP2E1 can
metabolize drugs such as acetaminophen, chlorzoxazone, and halothane and endogenous substrates such as arachidonic acid, gluconeogenic precursors, and estrogenic metabolites (Lieber, 1999; Ohe et al.,
2000). CYP2E1 bioactivates several procarcinogens (e.g., tobaccoderived nitrosamines and benzene) and cytotoxins (e.g., carbon tetrachloride) to their reactive intermediates (Lieber, 1999). CYP2E1 can
also generate reactive oxygen species and substrate-derived radicals,
especially when induced; these can mediate lipid peroxidation, protein
inactivation, and DNA damage, leading to cellular injury (Cederbaum
et al., 2001). Elevated levels of CYP2E1 in the central nervous system
could thus lead to increased risk of neurotoxicity and influence the
cerebral disposition and pharmacodynamic effects of centrally acting
CYP2E1 substrate drugs.
Cigarette smoking accelerates chlorzoxazone metabolism in humans, most likely by induction of hepatic CYP2E1 activity (Benowitz
et al., 2003). Nicotine, present in tobacco smoke, is widely used in
replacement therapy for smoking cessation (Henningfield et al., 2005)
and may be useful in the treatment of Parkinson’s and Alzheimer’s
disease (Kelton et al., 2000; Newhouse et al., 2001). Nicotine may
contribute to the enhanced levels of CYP2E1 found in smokers’ brain
(Howard et al., 2003), because we have shown that chronic low doses
of nicotine induce CYP2E1 in rat liver and brain (Howard et al., 2001,
2003) as well as in monkey brain (Joshi et al., 2005).
Tissue-specific and inducer-specific regulation of P450s has been
reported previously (Lieber, 1997; Miksys and Tyndale, 2004). The
mechanism of CYP2E1 induction is complex and may depend on the
substrate, species, tissue, or cell type (Novak and Woodcroft, 2000;
Kessova and Cederbaum, 2003). For example, acetone increases rat
liver CYP2E1 activity and CYP2E1 protein levels in rat liver, kidney,
and nasal mucosa but has no effect on CYP2E1 in rat lung (Longo and
Ingelman-Sundberg, 1993; Lee et al., 1998). However, unlike in liver
and kidney, increases in nasal CYP2E1 are accompanied by a corresponding increase in CYP2E1 mRNA (Longo and Ingelman-Sundberg, 1993). Tissue-specific expression of CYP2E1 may be influenced
by the presence and regulation of transcriptional factors HNF-1, Sp1,
and nuclear factor-1-like binding proteins (Peng and Coon, 2000). The
upstream region of CYP2E1 gene contains an HNF-1 binding site, and
in vitro evidence suggests that the rat CYP2E1 gene is positively
regulated by HNF-1 or a similar protein (Liu and Gonzalez, 1995). Pig
CYP2E1 promoter is also activated by HNF-1 (Tambyrajah et al.,
2004), which is predominantly present in liver and kidney but not in
lung and brain (Xanthopoulos et al., 1991).
Chronic nicotine treatment increases rat liver CYP2E1 activity and
protein levels (Howard et al., 2001), with maximal increases occurring
4 h after nicotine treatment (Micu et al., 2003). Induction of hepatic
CYP2E1 by nicotine is not accompanied by a change in mRNA and
is short-lived; protein levels return to basal levels by 24 h (Howard et
This study was supported by the Canadian Institutes of Health Research (MOP
14173), Centre for Addiction and Mental Health, Canada Research Chair (R.F.T.)
and Canadian Society for Clinical Pharmacology and Canadian Institutes of
Health Research Strategic Training program in Tobacco Use in Special Populations (M.J.).
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.105.008029.
ABBREVIATIONS: P450, cytochrome P450; HNF-1, hepatic nuclear factor 1; ANOVA, analysis of variance.
647
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CYP2E1, the primary ethanol-metabolizing cytochrome P450, metabolizes endogenous substrates (e.g., arachidonic acid) and
drugs (e.g., acetaminophen, chlorzoxazone) and bioactivates procarcinogens (e.g., tobacco-specific nitrosamines) and toxins (e.g.,
carbon tetrachloride). Nicotine from tobacco smoke may contribute to the enhanced hepatic CYP2E1 activity in smokers. We have
previously shown that chronic nicotine treatment can increase
CYP2E1 in rat liver and brain. In this study, induction of brain
CYP2E1 was assessed after a single acute or a 7-day chronic
treatment with saline or nicotine (1 mg/kg s.c.), with sacrifice
performed at various times after the last injection. Chronic 7-day
nicotine treatment showed the highest levels of CYP2E1 12 h after
the last injection in frontal cortex (1.4-fold, p < 0.05) versus 8 h in
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JOSHI AND TYNDALE
al., 2001; Micu et al., 2003). We have shown that cDNA-expressed
CYP2E1 is not stabilized by nicotine (Micu et al., 2003). In rat brain,
CYP2E1 expression is induced after chronic nicotine treatment in a
regiospecific manner; CYP2E1 was elevated in the frontal cortex and
cerebellum but not in the hippocampus (Howard et al., 2003). However, the duration of induction, time of maximal induction, and
mechanism(s) are not known. We examined CYP2E1 induction in rat
brain after chronic and acute nicotine treatment and assessed the
duration of the induction over 24 h after the last drug treatment to
determine how long the inductive effect persists, when it peaks, and
whether it also induces mRNA levels.
Materials and Methods
Results
Detection of CYP2E1 in Rat Brain Regions. A quantitative
Western blot assay was developed to measure CYP2E1 in rat brain,
where basal expression is low. Detection of CYP2E1 in serially
diluted brain membrane protein from saline-treated animals was linear, and 30 ␮g of protein was used for all subsequent experiments
(Fig. 1). Brain CYP2E1 protein migrated slightly faster at higher
protein concentrations. cDNA-expressed CYP2E1 or rat liver microsomes was therefore added to confirm comigration and detection of
brain CYP2E1 and to ascertain the linearity of the assay at potentially
higher levels of CYP2E1 in nicotine-treated rat brain (Fig. 1).
Induction of CYP2E1 in Rat Brain Regions by Chronic Nicotine Treatment. To determine whether the induction of CYP2E1 in
rat brain regions was seen at a later time point than previously
investigated (Howard et al., 2003), we examined CYP2E1 levels 8 h
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Materials. Nicotine bitartrate and cotinine were purchased from SigmaAldrich Canada Ltd. (Oakville, ON, Canada). The protein assay dye reagent
concentrate, Bio-dot microfiltration apparatus, and nylon membrane were
obtained from Bio-Rad (Hercules, CA). TRIzol was from Invitrogen Canada
Inc. (Burlington, ON, Canada), and the RNeasy kit was from Qiagen Inc.
(Mississauga, ON, Canada). Prestained molecular weight protein markers were
purchased from MBI Fermentas (Flamborough, ON, Canada). Lymphoblastoid
cDNA-expressed rat CYP2E1 and polyclonal NADPH-P450 reductase antibody were obtained from BD Biosciences (Mississauga, ON, Canada). The
polyclonal rabbit anti-rat CYP2E1 antibody was generously provided by Dr.
Magnus Ingelman-Sundberg (Department of Physiological Chemistry, Karolinska Institute, Stockholm, Sweden). The monoclonal Na,K-ATPase, ␤1 subunit antibody was from Upstate Biotechnology (Lake Placid, NY). Horseradish
peroxidase-conjugated goat anti-rabbit antibody was from Chemicon International Inc. (Temecula, CA). SuperSignal West Pico and Femto chemiluminescence substrate and peroxidase-conjugated goat anti-mouse antibody were
from Pierce Chemical (Rockford, IL). Peroxidase-conjugated anti-goat antibody was from Roche Diagnostics (Laval, QC, Canada). BioTrace NT nitrocellulose membrane was from Pall Corporation (Pensacola, FL), and autoradiography film was purchased from Ultident Scientific (St. Laurent, PQ,
Canada). All other reagents were obtained from standard commercial sources.
Animals. Adult male Wistar rats (250 –300 g; Charles River, St-Constant,
PQ, Canada) were housed two per cage and allowed to adapt to the novel
controlled environment with a 12-h artificial light/dark cycle (light on at 6:00
AM) for 1 week. 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 University of
Toronto.
Drug Treatment and Time-Course Study. Rats were injected subcutaneously with 1 mg of nicotine base per kilogram body weight, in the form of
nicotine bitartrate in sterile saline, pH 7.4 (Micu et al., 2003). Control rats were
injected with saline alone. For the chronic recovery time course, rats (n ⫽
3/group) were treated with saline or nicotine once a day for 7 days and
sacrificed at 0.5, 2, 4, 8, 12, 18, and 24 h after the last drug treatment. For the
acute study, rats (n ⫽ 4/group) received a single injection of saline or nicotine
and were sacrificed 4, 8, or 12 h after treatment. After sacrifice by decapitation,
brains were rapidly removed, dissected within 5 min, frozen immediately in
liquid nitrogen, and stored at ⫺80°C. There were no significant differences in
body weights of rats administered nicotine either acutely or chronically compared with rats in the respective saline control groups.
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 stored at ⫺20°C. Nicotine and cotinine plasma
concentrations were measured by standard high-performance liquid chromatography technique using 5-methylnicotine as the internal standard (Xu et al.,
2002).
Membrane Preparation for Western Blotting. Whole brain membranes
were prepared because it has been shown that brain P450s are present in
microsomal and mitochondrial membranes (Miksys et al., 2000). Briefly, brain
regions were homogenized in 100 mM Tris (pH 7.4) with 0.1 mM EDTA, 0.32
M sucrose, and 0.1 mM dithiothreitol on ice. Homogenates were centrifuged
twice at 3000g for 5 min to remove cellular and nuclear debris, and then the
supernatant was centrifuged at 110,000g at 4°C for 60 min. The resulting
membrane pellets were resuspended in 100 mM Tris (pH 7.4), 0.1 mM EDTA,
0.1 mM dithiothreitol, 1.15% (w/v) KCl, and 20% (v/v) glycerol and stored in
aliquots at ⫺80°C until used. Microsomes from rat liver were prepared as
described previously (Howard et al., 2001). The protein content of each sample
was assayed using a Bio-Rad protein assay kit.
Western Blotting. Membrane proteins were separated by SDS-polyacrylamide gel electrophoresis (4% stacking and 10% separating gels), transferred
overnight onto nitrocellulose membrane, blocked with 3% skim milk in Trisbuffered saline containing 0.1% BSA and 0.05% Triton X-100, and probed
with polyclonal rabbit anti-rat CYP2E1 antibody diluted 1:1500. Blots were
then incubated with peroxidase-conjugated anti-rabbit antibody (1:4000) and
developed using chemiluminescent detection and autoradiography film. The
antibody used is specific for CYP2E1, and cross-reactivity was not observed
with other cDNA-expressed rat P450s (Howard et al., 2001). Untreated rat
membranes from each brain region tested were serially diluted and used to
construct standard curves to determine the linear range of detection for the
immunoblotting assay. Microsomes from rat liver and cDNA-expressed rat
CYP2E1 were used as positive controls. Equal amounts of membrane protein
from saline- and nicotine-treated animals were loaded, and equivalence was
confirmed by Coomassie Blue gel staining, Ponceau S staining of the transferred protein on the membrane, and reprobing membranes with NADPH-P450
reductase antibody and Na,K-ATPase antibody. After confirmation that each
of these steps provided similar results, equal loading was used in subsequent
experiments. Digital images of immunoblots were analyzed using MCID Elite
software (Imaging Research Inc., St. Catherine’s, ON, Canada), and the
relative density of each band was expressed as arbitrary density units after
correcting for blot background.
RNA Isolation and Slot Blot Hybridization. Total RNA from rat brain
samples was isolated using RNeasy kit, and its quality assessed by electrophoresis in 1.2% agarose gel. Serially diluted CYP2E1 cDNA was used as a
positive control to detect linearity of the assay. Brain RNA (5 ␮g) was applied
directly to nylon membranes using a Bio-Dot microfiltration apparatus, and
hybridization was carried out according to manufacturer’s instructions (BioRad). Radiolabeled oligonucleotide probes specific for rat CYP2E1 and ␤-actin were made as described previously (Howard et al., 2001). Autoradiography
films were analyzed using MCID Elite software.
Data Analyses. CYP2E1 protein levels were expressed as density units
(mean ⫾ S.D.), average values obtained from n animals per treatment group,
from at least three separate experiments. Relative CYP2E1 levels were calculated as a ratio of CYP2E1 in nicotine-treated animals to levels in salinetreated animals. For the recovery time course, an average saline value from all
time points was used to determine the CYP2E1 ratio because no significant
differences between saline CYP2E1 levels were observed. For mRNA,
CYP2E1 levels were an average of two experiments and were expressed
relative to ␤-actin mRNA to control for loading efficiency and quality of the
RNA. The differences between brain regions, post-treatment time points, and
time of the day, within treatment groups, were tested by one-way analysis of
variance (ANOVA or Kruskal-Wallis) followed by a post hoc test (Duncan’s
new multiple range test). The differences between treatment groups were tested
using unpaired Student’s t tests.
INDUCTION OF RAT BRAIN CYP2E1 BY NICOTINE
649
FIG. 1. Linear detection and specificity of rat brain CYP2E1 quantification by
Western blot. Dilution curve of cerebellum membrane protein (CB; mean of three
independent experiments ⫾ S.E.M.). Top inset shows a representative CB dilution
curve and rat liver microsomes (RL; 1 ␮g). Bottom inset shows comigration of CB
(30 ␮g) alone and combined with RL (0.4 ␮g).
FIG. 3. Recovery time course of CYP2E1 after chronic nicotine treatment. CYP2E1
levels (mean ⫹ S.D.) in nicotine-treated (Nic) group (n ⫽ 3) relative to the
saline-treated (Sal) group over 24 h after treatment in frontal cortex, hippocampus,
and cerebellum. CYP2E1 levels varied between times for frontal cortex (ANOVA,
p ⬍ 0.05) and hippocampus (ANOVA, p ⫽ 0.05).
after the last nicotine injection. The amount of CYP2E1 from salineand nicotine-treated animals varied among brain regions (ANOVA,
p ⬍ 0.01 for both treatments). Levels of CYP2E1 in frontal cortex,
hippocampus, and cerebellum after chronic nicotine treatment, compared with the corresponding saline controls, showed an increase in
the frontal cortex (1.4-fold, p ⬍ 0.01), hippocampus (1.5-fold, p ⬍
0.01), and cerebellum (1.4-fold, p ⬍ 0.05; Fig. 2). This prompted us
to examine a wider recovery time course (30 min–24 h) in each brain
region.
Recovery Time Course of CYP2E1 in Rat Brain. The recovery
time course of CYP2E1 showed distinct patterns among the brain
regions (Fig. 3), with highest levels of CYP2E1 occurring at 12 h after
the last drug treatment in frontal cortex and at 8 h post-treatment in
hippocampus and cerebellum relative to the saline-treated group. In
the frontal cortex, ANOVA was significant ( p ⬍ 0.05), and post hoc
analysis indicated that CYP2E1 levels at 12 h were significantly
different from those at 2, 4, 18, and 24 h. In addition, significant
increases in CYP2E1 levels of 1.4-fold were seen at 4 h ( p ⬍ 0.05),
8 h ( p ⬍ 0.01), and 12 h ( p ⫽ 0.01) post-treatment compared with
their respective saline controls. In the hippocampus ( p ⫽ 0.05), post
hoc analysis indicated that CYP2E1 levels at 8 h were different from
levels at 0.5, 2, and 24 h. Compared with its respective saline controls
at 8 h, CYP2E1 levels increased 1.8-fold ( p ⬍ 0.01) in nicotinetreated animals. In the cerebellum, CYP2E1 levels increased 1.4-fold
( p ⬍ 0.05) in nicotine-treated animals compared with its respective
saline controls at 8 h and trended toward an increase of 1.4-fold ( p ⫽
0.07) at 12 h. In all three regions, levels returned to those in salinetreated animals by 24 h.
CYP2E1 mRNA in Rat Brain after Chronic Nicotine Treatment. We examined whether levels of mRNA were increased in brain
tissues of chronically treated rats. Detection of CYP2E1 cDNA (Fig.
4A) was linear, as was detection of CYP2E1 and ␤-actin in a serial
dilution of rat liver RNA. A reliable CYP2E1 signal was detected
from 5 ␮g of brain RNA from chronic saline- and nicotine-treated
animals, which was within the linear range of the assay. The ␤-actin
RNA levels did not differ ( p ⫽ 0.4) between treatment groups within
each brain region. Levels of CYP2E1 mRNA, normalized to ␤-actin
mRNA, were not increased after chronic nicotine treatment at any
time tested and were significantly lower than saline levels in hippocampus (at 4 and 8 h, p ⬍ 0.01) and in cerebellum (at 4 h, p ⬍ 0.05;
Fig. 4B, Table 1).
CYP2E1 mRNA levels from saline-treated animals were different
at the post-treatment sacrifice time points in cerebellum (ANOVA,
p ⬍ 0.01) and hippocampus (ANOVA, p ⬍ 0.05). CYP2E1 levels
varied with the time of the day in frontal cortex (ANOVA, p ⬍ 0.05)
and cerebellum (ANOVA, p ⬍ 0.01), and higher levels were seen at
12 h post-treatment sacrifice time, corresponding to 6 PM in both
regions (Table 1). CYP2E1 mRNA levels did not correlate with
CYP2E1 protein levels in any brain region for either treatment group
(0.2 ⬍ p ⬍ 0.9).
Induction of CYP2E1 in Rat Brain by Acute Nicotine Treatment. To determine whether induction of brain CYP2E1 occurs after
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FIG. 2. Induction of CYP2E1 by chronic nicotine at 8 h post-treatment. CYP2E1
levels (n ⫽ 3/group, mean ⫺ S.D.) in frontal cortex, hippocampus, and cerebellum
after chronic treatment with nicotine or saline; inset shows a blot from cerebellum.
Significant differences from saline-treated animals are indicated by ⴱ, p ⬍ 0.05; ⴱⴱ,
p ⬍ 0.01. CYP2E1 levels varied between regions for saline- (ANOVA, p ⬍ 0.01)
and nicotine-treated (ANOVA, p ⬍ 0.01) groups.
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JOSHI AND TYNDALE
TABLE 1
CYP2E1 mRNA levels in saline- and nicotine-treated animals
CYP2E1 RNA Levelsa
Time of Dayc
Sacrifice Timed
Saline
Nicotine
0.72 ⫾ 0.55
1.00 ⫾ 0.24
3.16 ⫾ 2.15
1.17 ⫾ 0.74
1.26 ⫾ 0.13
0.41 ⫾ 0.03
0.86 ⫾ 0.07
1.77 ⫾ 0.9
1.61 ⫾ 0.28
0.70 ⫾ 0.11
4.67 ⫾ 2.25
0.46 ⫾ 0.12
0.69 ⫾ 0.39
0.77 ⫾ 0.12
1.77 ⫾ 0.81
1.19 ⫾ 0.11
0.56 ⫾ 0.16**b
0.20 ⫾ 0.01**b
N.D.
0.52 ⫾ 0.14
1.00 ⫾ 0.17*b
0.71 ⫾ 0.36
2.12 ⫾ 0.06
N.D.
h
Frontal cortex
Hippocampus
Cerebellum
12:00 PM
3:30 PM
6:00 PM
3:30 PM
12:00 PM
3:30 PM
6:00 PM
3:30 PM
12:00 PM
3:30 PM
6:00 PM
3:30 PM
4
8
12
24
4
8
12
24
4
8
12
24
N.D., not detectable.
a
Values are mean ⫾ S.D. (n ⫽ 3/group).
b
Significant differences from saline-treated animals indicated by *, p ⬍ 0.05; and **, p ⬍ 0.01.
c
Variation in mRNA levels from saline-treated animals between times of day was significant
in frontal cortex (ANOVA, p ⬍ 0.05) and cerebellum (ANOVA, p ⬍ 0.01).
d
Variation in mRNA levels from saline-treated animals between the sacrifice times was
significant in cerebellum (ANOVA, p ⬍ 0.01) and hippocampus (ANOVA, p ⬍ 0.05).
acute nicotine treatment, we examined the effect of a single dose of
saline or nicotine on CYP2E1 levels in rat brain sacrificed 4, 8, or 12 h
after treatment. CYP2E1 was induced significantly in cerebellum at
8 h (1.6-fold, p ⬍ 0.01), and CYP2E1 levels were 1.2-fold ( p ⫽ 0.37)
and 1.4-fold ( p ⫽ 0.15) above saline levels at 4 and 12 h, respectively
(Fig. 5). In the hippocampus, CYP2E1 levels in nicotine-treated rats
were 1.2-fold ( p ⫽ 0.54) at 8 h. In addition, no significant induction
was observed in the frontal cortex. Plasma nicotine and cotinine levels
were 12 and 230 ng/ml at 4 h (Micu et al., 2003). Plasma nicotine
levels were undetectable at 8 and 12 h, and cotinine levels were 190 ⫾
40 and 95 ⫾ 15 ng/ml at 8 and 12 h, respectively.
Discussion
In the current study, we have characterized the recovery time course
of induction of brain CYP2E1 after chronic nicotine treatment, examined CYP2E1 mRNA levels, and investigated the effect of acute
nicotine treatment. We have shown that there was variation in the
amount of CYP2E1 among brain regions in both saline- and nicotinetreated rats 8 h after treatment. Chronic nicotine treatment produced a
1.4- to 1.5-fold increase in CYP2E1 protein levels within 12 h, with
a return to the control saline levels by 24 h in all three brain regions
studied. The results presented here support a short-lived induction of
CYP2E1 in the brain like that in the liver (Micu et al., 2003).
However, CYP2E1 levels in the brain peaked later than the 4-h peak
seen in the liver (Micu et al., 2003).
Region-specific and time-dependent changes in transcriptional response of various neural genes have been shown in rat brain after
nicotine exposure (Konu et al., 2001; Li et al., 2004). We have shown
that CYP2E1 mRNA levels were unchanged or decreased in frontal
cortex, hippocampus, and cerebellum of nicotine-treated rats compared with saline controls. In rat liver, CYP2E1 mRNA levels were
also not increased in response to nicotine treatment, whereas protein
levels were (Howard et al., 2001). Despite increases in CYP2E1
protein, decreases in hepatic CYP2E1 RNA levels have been shown in
mice exposed to chronic acetone treatment (Forkert et al., 1994), in
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FIG. 4. Rat brain CYP2E1 RNA detection by slot blot. A, linear detection of a serial
dilution of CYP2E1 cDNA (mean of two independent experiments ⫾ S.E.M.) was
linear; inset shows a representative dilution curve. B, CYP2E1 mRNA levels
relative to ␤-actin (n ⫽ 3 animals/group, mean ⫺ S.D.) from frontal cortex,
hippocampus, and cerebellum at 8 h after chronic treatment. Significant difference
from saline-treated animals is indicated by ⴱⴱ, p ⬍ 0.01. C, representative blot of
CYP2E1 and ␤-actin mRNA from frontal cortex, hippocampus, and cerebellum at
8 h after chronic treatment. S, saline; N, nicotine-treated animals.
FIG. 5. Induction of CYP2E1 by acute nicotine treatment. CYP2E1 levels in
nicotine-treated (Nic) group (n ⫽ 4, mean ⫹ S.D.) relative to respective saline
controls (Sal) in frontal cortex, hippocampus, and cerebellum at 4, 8, and 12 h
post-treatment. Significant difference from saline is indicated by ⴱⴱ, p ⬍ 0.01.
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INDUCTION OF RAT BRAIN CYP2E1 BY NICOTINE
detected in the neurons of substantia nigra in rats (Watts et al., 1998).
We have not investigated whether nicotine induces CYP2E1 levels in
rat substantia nigra; however, we have seen an increase in CYP2E1
levels after nicotine treatment in substantia nigra in monkeys, as
detected by immunohistochemistry (Joshi and Tyndale, 2005). The
induction of CYP2E1 may have significant effects on local drug and
metabolite concentrations. The different areas of the brain investigated in this study are known to play diverse roles in behavior and
may thus be differently affected by increased CYP2E1 levels, metabolism of neuroactive substrates, and/or CYP2E1-mediated oxidative
stress.
In conclusion, we have shown that acute and chronic nicotine
treatment induced CYP2E1 expression in rat brain in a region-specific
manner. The induction of CYP2E1 after chronic treatment was not
regulated at the transcriptional level, and the recovery time course
varied among brain regions with CYP2E1 levels returning to basal
levels by 24 h after treatment. Induction of brain CYP2E1 may result
in altered neuroactive substrate metabolism, increased oxidative
stress, activation of procarcinogens and toxins, and increased risk of
cell and tissue damage. Nicotine may thus increase CYP2E1-related
toxicity in the central nervous system and may be of relevance in
current smokers, passive smokers, and people treated with nicotine
such as smokers and, potentially, patients with Alzheimer’s disease,
Parkinson’s disease, or ulcerative colitis.
Acknowledgments. We thank Dr. Sharon Miksys for expert technical guidance and review of the manuscript, Dr. Raj Sharma for
analyses of nicotine and cotinine, and Dr. Magnus Ingelman-Sundberg for the gift of CYP2E1 polyclonal antibody.
References
Badger TM, Huang J, Ronis M, and Lumpkin CK (1993) Induction of cytochrome P450 2E1
during chronic ethanol exposure occurs via transcription of the CYP 2E1 gene when blood
alcohol concentrations are high. Biochem Biophys Res Commun 190:780 –785.
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.
Cederbaum AI, Wu D, Mari M, and Bai J (2001) CYP2E1-dependent toxicity and oxidative
stress in HepG2 cells. Free Radic Biol Med 31:1539 –1543.
Forkert PG, Redza ZM, Mangos S, Park SS, and Tam SP (1994) Induction and regulation of
CYP2E1 in murine liver after acute and chronic acetone administration. Drug Metab Dispos
22:248 –253.
Henningfield JE, Fant RV, Buchhalter AR, and Stitzer ML (2005) Pharmacotherapy for nicotine
dependence. CA Cancer J Clin 55:281–299; quiz 322–283,325.
Howard LA, Micu AL, Sellers EM, and Tyndale RF (2001) Low doses of nicotine and ethanol
induce CYP2E1 and chlorzoxazone metabolism in rat liver. J Pharmacol Exp Ther 299:542–
550.
Howard LA, Miksys S, Hoffmann E, Mash D, and Tyndale RF (2003) Brain CYP2E1 is induced
by nicotine and ethanol in rat and is higher in smokers and alcoholics. Br J Pharmacol
138:1376 –1386.
Joshi M, Miksys S, and Tyndale RF (2005) Chronic nicotine increases CYP2E1, an enzyme
associated with alcohol and nicotine dependence, in monkey brain, in Proceedings of the
Society for Research in Nicotine and Tobacco, pp 80, Prague, Czech Republic.
Joshi M and Tyndale RF (2005) Regional and cellular distribution of CYP2E1 in monkey brain
and its induction by chronic nicotine. Neuropharmacology 20.
Kelton MC, Kahn HJ, Conrath CL, and Newhouse PA (2000) The effects of nicotine on
Parkinson’s disease. Brain Cogn 43:274 –282.
Kenny PJ, File SE, and Rattray M (2000) Acute nicotine decreases and chronic nicotine increases
the expression of brain-derived neurotrophic factor mRNA in rat hippocampus. Brain Res Mol
Brain Res 85:234 –238.
Kessova I and Cederbaum AI (2003) CYP2E1: biochemistry, toxicology, regulation and function
in ethanol-induced liver injury. Curr Mol Med 3:509 –518.
Kim SG and Novak RF (1990) Induction of rat hepatic P450IIE1 (CYP 2E1) by pyridine:
evidence for a role of protein synthesis in the absence of transcriptional activation. Biochem
Biophys Res Commun 166:1072–1079.
Konu O, Kane JK, Barrett T, Vawter MP, Chang R, Ma JZ, Donovan DM, Sharp B, Becker KG,
and Li MD (2001) Region-specific transcriptional response to chronic nicotine in rat brain.
Brain Res 909:194 –203.
Kubota S, Lasker JM, and Lieber CS (1988) Molecular regulation of ethanol-inducible cytochrome P450-IIEI in hamsters. Biochem Biophys Res Commun 150:304 –310.
Lavery DJ, Lopez-Molina L, Margueron R, Fleury-Olela F, Conquet F, Schibler U, and Bonfils
C (1999) Circadian expression of the steroid 15 ␣-hydroxylase (Cyp2a4) and coumarin
7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP. Mol Cell Biol 19:6488 – 6499.
Lee C, Watt KC, Chang AM, Plopper CG, Buckpitt AR, and Pinkerton KE (1998) Site-selective
differences in cytochrome P450 isoform activities. Comparison of expression in rat and rhesus
monkey lung and induction in rats. Drug Metab Dispos 26:396 – 400.
Li MD, Kane JK, Wang J, and Ma JZ (2004) Time-dependent changes in transcriptional profiles
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rats treated with a single dose of pyridine (Kim and Novak, 1990), and
in rabbits after imidazole and ethanol treatment (Porter et al., 1989).
However, increases in hepatic CYP2E1 mRNA have been reported in
hamsters after ethanol and pyrazole treatment and in rats after ethanol
treatment (Kubota et al., 1988; Badger et al., 1993). These data
indicate that CYP2E1 regulatory mechanisms may vary and can be
influenced by species, tissue, and inducing agent as well as its mode
of administration (Forkert et al., 1994). Changes were seen in
CYP2E1 mRNA levels from saline-treated rats dependent on the
post-treatment sacrifice time and time of the day when animals were
sacrificed, but the protein levels did not vary correspondingly. Higher
mRNA levels were seen at 6 PM, the beginning of the active/dark
cycle. Diurnal cycles have been previously reported in P450 expression, for example, CYP7 and CYP2A (Lavery et al., 1999). Mouse
hepatic CYP2A4 and CYP2A5 expression exhibits circadian rhythm,
with the highest expression levels occurring in the evening, and
mRNA levels showed more pronounced increases than protein levels
(Lavery et al., 1999; Su and Ding, 2004).
CYP2E1 inducers, including ethanol (Petersen et al., 1982), acetone
(Forkert et al., 1994), and pyridine (Kim and Novak, 1990), increase
hepatic CYP2E1 protein levels after acute and chronic treatment.
However, an acute dose of nicotine failed to alter rat hepatic CYP2E1
levels, whereas chronic nicotine treatment did increase hepatic
CYP2E1 (Micu et al., 2003). In rat brain, at 8 h after an acute dose of
nicotine, an increase in CYP2E1 protein was seen in cerebellum but
not in frontal cortex or in hippocampus. Chronic nicotine did induce
CYP2E1 levels in all three brain regions. Differences in acute and
chronic nicotine treatment have been previously observed; for example, levels of brain-derived neurotrophic factor mRNA in rat hippocampus decreased after acute treatment but increased after chronic
treatment (Kenny et al., 2000). Acute nicotine exposure in rats resulted in nicotine and cotinine plasma levels not different ( p ⬎ 0.1)
from those found after chronic nicotine treatment (Micu et al., 2003),
indicating that the treatment regime did not alter nicotine bioavailability in rats. One possible explanation for acute induction in cerebellum but not in the liver may be related to the subcutaneous route of
nicotine administration, whereby the brain may be exposed to higher
nicotine concentrations than the liver.
Several mechanisms have been described for the regulation of
CYP2E1 expression. Protein stabilization and increased translational
efficiency have been implicated in the regulation of CYP2E1 protein
expression by xenobiotics such as ethanol, acetone, and pyridine
(Novak and Woodcroft, 2000). We previously reported that nicotine
did not alter levels or activity of cDNA-expressed CYP2E1, suggesting that nicotine does not inhibit the degradation of CYP2E1 and that
the induction of CYP2E1 by nicotine is unlikely to occur via stabilization of the enzyme (Micu et al., 2003). We found no increase in
mRNA levels in the rat liver (Howard et al., 2001) or brain after
nicotine treatment, suggesting nontranscriptional regulation. Increased translation efficiency may be involved in CYP2E1 induction
by nicotine. Induction of CYP2E1 by pyridine involving increased
protein synthesis in the absence of transcriptional activation has been
reported (Kim and Novak, 1990).
Expression of brain P450s is highly localized in specific cells, and
induction of these P450s may alter local substrate and metabolite
concentrations of drugs (Miksys and Tyndale, 2004). Therefore, although the increase in CYP2E1 levels detected by Western blot was
modest, region- and cell-specific CYP2E1 expression and induction
(Howard et al., 2003) may lead to a substantial increase in CYP2E1
levels in the microenvironment. CYP2E1 has been suggested to play
a role in dopamine regulation (Nissbrandt et al., 2001) and/or neuronal
detoxification mechanisms (Vaglini et al., 2001, 2004) and has been
652
JOSHI AND TYNDALE
Porter TD, Khani SC, and Coon MJ (1989) Induction and tissue-specific expression of rabbit
cytochrome P450IIE1 and IIE2 genes. Mol Pharmacol 36:61– 65.
Su T and Ding X (2004) Regulation of the cytochrome P450 2A genes. Toxicol Appl Pharmacol
199:285–294.
Tambyrajah WS, Doran E, Wood JD, and McGivan JD (2004) The pig CYP2E1 promoter is
activated by COUP-TF1 and HNF-1 and is inhibited by androstenone. Arch Biochem Biophys
431:252–260.
Upadhya SC, Tirumalai PS, Boyd MR, Mori T, and Ravindranath V (2000) Cytochrome P4502E
(CYP2E) in brain: constitutive expression, induction by ethanol and localization by fluorescence in situ hybridization. Arch Biochem Biophys 373:23–34.
Vaglini F, Pardini C, Viaggi C, Bartoli C, Dinucci D, and Corsini GU (2004) Involvement of
cytochrome P450 2E1 in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced mouse
model of Parkinson’s disease. J Neurochem 91:285–298.
Vaglini F, Pardini C, Viaggi C, and Corsini GU (2001) Cytochrome p450 and parkinsonism:
protective role of CYP2E1. Funct Neurol 16:107–112.
Watts PM, Riedl AG, Douek DC, Edwards RJ, Boobis AR, Jenner P, and Marsden CD (1998)
Co-localization of P450 enzymes in the rat substantia nigra with tyrosine hydroxylase.
Neuroscience 86:511–519.
Xanthopoulos KG, Prezioso VR, Chen WS, Sladek FM, Cortese R, and Darnell JE Jr (1991) The
different tissue transcription patterns of genes for HNF-1, C/EBP, HNF-3, and HNF-4, protein
factors that govern liver-specific transcription. Proc Natl Acad Sci USA 88:3807–3811.
Xu C, Rao YS, Xu B, Hoffmann E, Jones J, Sellers EM, and Tyndale RF (2002) An in vivo pilot
study characterizing the new CYP2A6*7, *8 and *10 alleles. Biochem Biophys Res Commun
290:318 –324.
Address correspondence to: Rachel F. Tyndale, Department of Pharmacology, 1 King’s College Circle, Toronto, ON, Canada M5S 1A8. E-mail:
[email protected]
Downloaded from dmd.aspetjournals.org at ASPET Journals on April 29, 2017
within five rat brain regions in response to nicotine treatment. Brain Res Mol Brain Res
132:168 –180.
Lieber CS (1997) Cytochrome P-4502E1: its physiological and pathological role. Physiol Rev
77:517–544.
Lieber CS (1999) Microsomal ethanol-oxidizing system (MEOS): the first 30 years (1968 –
1998)—a review. Alcohol Clin Exp Res 23:991–1007.
Liu SY and Gonzalez FJ (1995) Role of the liver-enriched transcription factor HNF-1 alpha in
expression of the CYP2E1 gene. DNA Cell Biol 14:285–293.
Longo V and Ingelman-Sundberg M (1993) Acetone-dependent regulation of cytochromes
P4502E1 and P4502B1 in rat nasal mucosa. Biochem Pharmacol 46:1945–1951.
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.
Miksys S, Hoffmann E, and Tyndale RF (2000) Regional and cellular induction of nicotinemetabolizing CYP2B1 in rat brain by chronic nicotine treatment. Biochem Pharmacol 59:
1501–1511.
Miksys S and Tyndale RF (2004) The unique regulation of brain cytochrome P450 2 (CYP2)
family enzymes by drugs and genetics. Drug Metab Rev 36:313–333.
Newhouse PA, Potter A, Kelton M, and Corwin J (2001) Nicotinic treatment of Alzheimer’s
disease. Biol Psychiatry 49:268 –278.
Nissbrandt H, Bergquist F, Jonason J, and Engberg G (2001) Inhibition of cytochrome P450 2E1
induces an increase in extracellular dopamine in rat substantia nigra: a new metabolic
pathway? Synapse 40:294 –301.
Novak RF and Woodcroft KJ (2000) The alcohol-inducible form of cytochrome P450 (CYP
2E1): role in toxicology and regulation of expression. Arch Pharm Res (NY) 23:267–282.
Ohe T, Hirobe M, and Mashino T (2000) Novel metabolic pathway of estrone and 17betaestradiol catalyzed by cytochrome P-450. Drug Metab Dispos 28:110 –112.
Peng HM and Coon MJ (2000) Promoter function and the role of cytokines in the transcriptional
regulation of rabbit CYP2E1 and CYP2E2. Arch Biochem Biophys 382:129 –137.
Petersen DR, Atkinson N, and Hjelle JJ (1982) Increase in hepatic microsomal ethanol oxidation
by a single dose of ethanol. J Pharmacol Exp Ther 221:275–281.