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TOXICOLOGICAL SCIENCES 87(1), 285–295 (2005)
doi:10.1093/toxsci/kfi238
Advance Access publication June 23, 2005
The Diverse Actions of Nicotine and Different Extracted Fractions
from Tobacco Smoke against Hapten-Induced Colitis in Rats
Joshua K. S. Ko*,1 and Chi-Hin Cho†
*School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China; †Department of Pharmacology,
Faculty of Medicine, The University of Hong Kong, Hong Kong, China
Received March 24, 2005; accepted June 7, 2005
The etiology of ulcerative colitis (UC) remains unknown,
although the risk of developing UC is apparently higher in nonsmokers and ex-smokers. We have demonstrated in a colitis
animal model that exposure to tobacco smoke could attenuate
UC pathogenesis. The present study aimed to investigate and
compare between the modes of action of nicotine and different
fractions of tobacco smoke extract in the development of
experimental colitis. The hapten 2,4-dinitrobenzene sulfonic acid
(DNBS) was used to induce colitis in Sprague-Dawley rats.
Results indicated that both tobacco smoke exposure and subcutaneous nicotine differentially reduced colonic lesion size,
myeloperoxidase (MPO) activity, luminol-amplified free radical
generation, and leukotriene B4 formation in the inflamed colon of
colitis animals. These phenomena were accompanied by the
downregulation of colonic interleukin (IL)-1b and monocyte
chemoattractant protein (MCP)-1 protein expression. By treating
the colitis animals with various tobacco extracts, we further
discovered that ethanol extract from filtered tobacco smoke could
attenuate DNBS-evoked colonic damage and the elevated MPO
activity, while at the same time it downregulated colonic IL-1b
and MCP-1 protein expression. In contrast, the highest dose of the
chloroform extract from the cigarette filter caused aggravating
effects and overexpression of the pro-inflammatory cytokines and
chemokines. These data suggest that effective attenuation of
DNBS-induced colitis by tobacco smoke could be due to its
nicotine content and possibly other flavonoid components found in
the ethanol smoke extract.
Key Words: nicotine; tobacco smoke; neutrophils; chemotactic
factors; colitis.
INTRODUCTION
Ulcerative colitis (UC) and Crohn’s disease (CD) are
dissimilar forms of inflammatory bowel disease (IBD) that
have differential etiologies and unknown pathogenesis. One of
the interesting but unexplained differences between UC and
1
To whom correspondence should be addressed at School of Chinese
Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon
Tong, Hong Kong, China. Fax: 852-3411 –2461. E-mail: [email protected].
CD patients is the correlation with the incidence of cigarette
smoking. The risk of developing UC was found to be greater in
both ex-smokers and non-smokers, and the risk of developing
CD was greater in active smokers (Harries et al., 1982;
Koutroubakis et al., 1996). We also demonstrated the same
phenomenon by using two distinct animal models that resemble human UC and CD, respectively (Guo et al., 1999; Ko
et al., 2001). Nicotine, one of the major components of tobacco
smoke, has been used as an alternative therapeutic agent for
treating UC in some clinical trials (Guslandi and Tittobello,
1998; Sandborn et al., 1997). However, its low efficacy and the
associated systemic side effects remain controversial
(Kennedy, 1996). In fact, there are constituents in tobacco
smoke other than nicotine that have been reported to possess
anti-oxidative properties (Chen and Loo, 1995; Kamisaki et al.,
1997; Lapenna et al., 1995). These include flavonoids, which
are antioxidants and potent inhibitors of low-density lipoprotein oxidation (Bravo, 1998).
One of the pathogenic characteristics of IBD is the infiltration of neutrophils into intestinal tissues. When tissue
influx of polymorphic nuclear cells (PMN) and macrophages
occurs, a marked increase in the production of reactive oxygen
metabolites (ROM) and leukotrienes (LT) will result as the
secondary amplification of the inflammatory responses
(Lauritsen et al., 1989). For this reason, new therapeutic agents
derived for the treatment of IBD include inhibitors of
neutrophil formation and activation. For instance, corticosteroids are capable of decreasing the margination of neutrophils, inhibiting neutrophil aggregation and interfering with
neutrophil eicosanoid metabolism (Claman, 1984). This could
explain why neutrophilic chemotactic activity was found to be
higher in UC mucosa than in normal mucosa. 5-Lipoxygenase
(5-LOX), primarily localized in leukocytes, is responsible for
the synthesis of LT and other products that have played
important roles in tissue inflammation. As a result, most of
the chemotactic activities were blocked by anti-LTB4 antibody
as demonstrated in some in vitro studies, suggesting that LTB4
could be one of the major chemotactic factors in UC (Lobos
et al., 1987). Moreover, there have been several reports
showing that LTB4 is responsible for the pathophysiology in
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both human and rat colitis models (Nakamaru et al., 1994;
Schmidt et al., 1995; Zhou and Mineshita, 1999). In fact, an
increased colonic level of both LTB4 and luminol-amplified
ROM was documented in patients with active IBD (Mahida
et al., 1989; Suematsu et al., 1987). In a clinical study, dietary
supplementation of fish oil in patients with IBD resulted in the
reduction of rectal dialysate LTB4 level and improvement in
UC disease markers (Stenson et al., 1992).
Studies have consistently revealed that chemokines are
upregulated in the colonic tissues of IBD patients (Banks
et al., 2003). One of the specific examples is that the percentage
of cells expressing monocyte chemoattractant protein (MCP)-1
was found to be significantly enhanced in all colonic biopsies
from active UC patients, when compared with the data from
healthy individuals (Uguccioni et al., 1999). In turn, the
upregulated chemokine expression appeared to correlate with
increased disease activities (Banks et al., 2003), which could be
related to the spontaneous release of pro-inflammatory cytokines from the inflamed intestinal mucosa (Reimund et al.,
1996). Among these, tumor necrosis factor (TNF)-a was
significantly increased only in the colonic biopsies from CD
patients, but not in UC patients (McCormack et al., 2001). It is
therefore of great interest to determine the involvement of other
major pro-inflammatory cytokines such as interleukin (IL)-1b
and the associated chemokine upregulation during UC development and after chemotherapy.
In the present study, the effects of unfiltered or filtered
tobacco smoke, nicotine, and various extracted tobacco smoke
components in experimental UC development were explored in
a rat colitis model. Moreover, the involvement of various
chemotactic factors in these processes was also investigated.
MATERIALS AND METHODS
Animals. Male Sprague-Dawley rats (180–200 g) were kept in a room with
controlled temperature (22° ± 1°C), humidity (55–60%) and 12-h light–dark
cycles, and were acclimatized for at least 2 weeks prior to experimentation. The
animals had free access to standard laboratory chow (Ralston Purina, USA) and
tap water ad libitum. The experimental procedures were approved by our
institutional animal research ethics committee with reference to the European
Community Guidelines for the Use of Experimental Animals.
Induction of colitis and tissue biopsy. 2,4-Dinitrobenzene sulfonic acid
(DNBS), a hapten molecule, was used to induce experimental colitis in rats (Ko
et al., 2001). Histopathological observation had proven that DNBS-induced
damage resembles human UC colitis (Hawkins et al., 1997). Nevertheless, in
studying chronic UC, other hapten models such as the dextran sulfate sodium
(DSS) method could be employed instead (Elson et al., 1995). Intracolonic
administration of DNBS (30 mg in 250 ll of 50% ethanol) was accomplished
by inserting a siliconized rubber cannula intrarectally into a ketamine/xylazineanesthetized rat such that the tip was 8 cm proximal to the anus. All
experimental animals were sacrificed 4 days after colitis induction. We had
previously demonstrated that colonic inflammation began to emerge 1 h after
intracolonic DNBS administration, and that hemorrhagic lesions and ulcers
were visualized in the distal colon by 6 h. The choice of 4 days after colitis
induction as the experimental endpoint designates the beginning of active
wound healing, which is susceptible to drug intervention. After macroscopic
lesion areas were assessed, biopsies were taken from the distal colonic region at
2–8 cm proximal to the anus and stored at 85°C for various assays and
Western analyses.
Treatment with tobacco smoke and nicotine. The passive smoking
method employed in this study was modified from the original design by
Chow and Cho (1996) and was further discussed in Guo et al. (1999). Rats were
exposed to a fixed concentration (4% v/v) of smoke from commercial cigarettes
(Kings, UK), with a nicotine content of 1.1 mg/cigarette and a tar content of
15 mg/cigarette, in a ventilated smoking chamber (39 3 23.5 3 21 cm3). A
lighted cigarette with the mouthpiece filter removed (by cutting the wrapping
paper circumferentially at the point where the glass-fiber filter meets the
tobacco leaves) was plugged into a home-made glass mouthpiece attached to a
3-way stopcock. To maintain a constant 4% v/v smoke/air concentration inside
the chamber, two peristaltic pumps (Masterflex, Cole-Parmer Instrument Co.,
Vernon Hills, IL) were used to deliver fresh tobacco smoke (at 40 ml/min) and
fresh air (at 960 ml/min) simultaneously. Control rats were subject to the same
procedures, except that they received only fresh air from the two pumps. The
duration of smoke exposure (or pure air in the control group) was 1 h daily for
3 consecutive days (days 2–4), commencing 24 h after enema DNBS administration on day 1. The experiment was then repeated using cigarettes with
intact mouthpiece attached (in the filtered smoke group). To ensure a consistent
supply of tobacco smoke to the chamber during both smoking sessions with
non-filtered or filtered smoke, the cigarette would be replaced by a new one
when there was a 5-mm unburned length (with tobacco leaves) left in the glass
mouthpiece. In addition, the whole set-up for the smoking experiments was
placed inside a chemical fume hood. This smoking method was proved not to
affect the blood pH and O2/CO2 balance in the animals based on blood gas data
(pH 7.43, 32.14–32.88 mm Hg pCO2 and 91.21–91.28 mm Hg pO2). The mean
serum concentration of nicotine in rats’ blood collected 45 min after a 1-h
exposure to 4% v/v tobacco smoke was measured at 0.136 ng/ll with nonfiltered smoke and 0.128 ng/ll with filtered smoke.
Alternatively, nicotine (Sigma, St. Louis, MO) dissolved in normal saline
was injected subcutaneously into rats after enema DNBS administration by
three regimens, as follows: a single dose of 4 mg/kg on day 2; a single dose of
8 mg/kg on day 2; multiple doses of 4 mg/kg on three consecutive days from
day 2 to day 4.
Extraction of filtered tobacco smoke and cigarette filter. Extracts of
either the filtered smoke or the used cigarette filter were obtained according to
methods described previously (Chow et al., 1997; Ma et al., 2000). The
constituents in tobacco smoke were extracted by a perfusion system including
four bottles of 96% ethanol and two bottles of chloroform with the flow rate of
700 ml/min using a peristaltic pump. The extraction process and different
extracts obtained are summarized in Figure 1. First, puffs of smoke from
cigarettes with filters attached were allowed to sieve through the filter and pass
into ethanol and chloroform for extraction, resulting in the collection of the
ethanol extract of filtered smoke (FSEE) and the chloroform extract of filtered
smoke (FSCE), respectively. The used filters of the cigarettes were then soaked
in 5% hydrochloric acid for 1 h with sonication. The resulting solution was
mixed with chloroform, followed by separation from the acidic aqueous layer
(forming the first chloroform extract of filter, FCE1). Sodium hydroxide was
added to the aqueous layer to a pH of 10. Chloroform extraction was repeated
once again to obtain the second chloroform extract of the filter (FCE2). All the
extracted products were concentrated to form different extracts by using
a rotary evaporator connected to a cooling system (Buchi, Germany), followed
by lyophilization (Labconco, Kansas City, MO).
Treatment with tobacco extracts. Various tobacco smoke or filter extracts
suspended in Tween 80 (Sigma, St. Louis, MO) were injected into different
groups rats intraperitoneally daily for 3 consecutive days after colitis induction,
with a similar schedule as that used for tobacco smoke exposure. The doses
used were 5, 10, or 20 mg/kg for FSEE, 2.5, 5, or 10 mg/kg for FCE1 and 0.68,
1.36, or 2.72 mg/kg for FCE2, respectively. The median doses of the extracts
used (10 mg/kg for FSEE, 5 mg/kg for FCE1, and 1.36 mg/kg for FCE2) were
calculated based on the experiment using 4% v/v of tobacco smoke (Chow
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ACTION OF TOBACCO COMPONENTS AGAINST COLITIS
Assessment of ROM in colonic tissue. Reactive oxygen metabolite
production in the excised colonic tissues can be measured by a luminolamplified chemiluminescence assay as described by Simmonds and co-workers
(1992). Luminol was dissolved in Dulbecco’s phosphate-buffered saline (PBS)
at a concentration of 300 lmol/l with added glucose (5 mmol/l) on the day of
experiment. Luminol can react with ROM to form 3-aminophthalate. The excited electrons in this compound reverted to their ground state with the emission
of energy as light (chemiluminescence), which was detected by the photomultiplier tubes of a scintillation counter (Beckman LS6500, Ramsey, MN).
The final value of each colonic sample was represented as count per minute.
FIG. 1. Flowchart illustrating how various fractions of tobacco smoke
components are obtained through ethanol and chloroform extractions of filtered
tobacco smoke or used cigarette filters. The collected fractions include the
ethanol (FSEE) and chloroform (FSCE) extracts obtained from filtered tobacco
smoke, as well as the first chloroform extract obtained from a used filter (FCE1)
and the second chloroform extract obtained from the resulting acidic aqueous
layer after the initial chloroform extraction (FCE2). Detailed extraction
procedures are described in Materials and Methods. The FSEE fraction
contains mainly alkaloids, including nicotine, that can pass through the
cigarette filter. The FCE1 fraction contains terpenoids and phenolic compounds, as well as tar phase substances such as the reactive oxygen species,
hydrocarbons, and phenols that are trapped in the cigarette filter. The FSCE
fraction contains a trace amount of the terpenoids and phenolics that can pass
through the cigarette filter, and the FCE2 fraction contains some alkaloids that
are retained in the filter.
et al., 1997). Control animals received intraperitoneal Tween 80 under the same
schedule as the other treatment groups. We had not performed a thorough
investigation of the effect of FSCE because this fraction was found to have no
significant pharmacological action in our preliminary study.
Assessment of colonic damage. Rats in control and treatment groups were
sacrificed by an overdose of ketamine (100 mg/kg, i.p.). The entire colonic
segment from rectum to colocecal junction was removed through a midline
laparotomy. The isolated colon was excised longitudinally, and pathological
conditions such as edema, inflammation, and shortening of the colon were
noted. The areas of hemorrhagic lesions and ulcers in the distal colon were
measured by tracing onto a transparency with 1-mm grids, with the total colonic
lesion area being expressed in mm2 (Ko et al., 2001).
Determination of neutrophil recruitment in colonic tissue. Myeloperoxidase (MPO) is localized inside the azurophil granules of neutrophils and plays
an important role in bactericidal action using halide ions as co-factor (Suzuki
et al., 1983). Hence, tissue MPO activity was regarded as an index of neutrophil
recruitment. Myeloperoxidase activity in the excised colonic tissues was
measured by a modified method as described previously (Ko et al., 2001), using
hydrogen peroxide and 3,3#,5,5#-tetramethylbenzidine. Horseradish peroxidase
was used as standard (ranging from 0 to 0.04 U/ml). One unit of horseradish
peroxidase (from colonic samples or standard solutions) will oxidize 1 lmole
of the reaction product 2,2#-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)
per min at 25°C under pH 5.0. The final values were represented as units per
gram of colonic wet tissue.
Measurement of colonic LTB4 concentration. Colonic tissue samples
were homogenized in buffer containing 50 mM Tris, 100 mM NaCl, 1 mM/l
CaCl2, 1 mg/ml D-glucose, and 28 lM/l indomethacin for 30 s. The resulting
homogenate was centrifuged at 15,000 3 g for 15 min at 4°C. Colonic LTB4
contents in the supernatant were measured by means of an enzyme
immunoassay system (Amersham Pharmacia Biotech, UK) with the range of
0.3 to 40 pg/well. Absorbance of each sample was read in a microplate reader
(MRX, DYNEX Technologies, Chantilly, VA) at 405 nm. The LTB4
concentration was expressed as picograms per milligram of colonic tissue.
Analysis of IL-1b and MCP-1 protein expressions in colonic tissue. To
further explore the contribution of other leukocytic chemotactic factors in the
pathogenesis of experimental colitis as well as in the actions of nicotine and
tobacco smoke components, protein expression of the pro-inflammatory
cytokine IL-1b and of the CC chemokine MCP-1 in the colonic tissues was
determined by Western blot analysis (Bless et al., 2000; Di Loreto et al., 2004).
Colonic tissues were weighed and homogenized for 30 s at 4°C in lysis buffer
(50 mM Tris, 150 mM NaCl, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate
(SDS), 2 mM EDTA, 1% Triton X-100, and 10% glycerol) with 1 mM
phenylmethanesulfonyl fluoride, aprotinin (5 mg/ml), and pepstatin A (5 mg/ml)
added freshly. The colonic tissue samples were then centrifuged at 13,000 3 g
for 20 min and the resulting supernatant was collected. Sodium dodecyl sulfate
(SDS)-polyacrylamide gel electrophoresis was carried out on the protein
samples (100 lg). The separated proteins were transferred to a nitrocellulose
membrane, which was then blocked in TBST (0.2 M Tris, 1.37 M NaCl, 1N
HCl, and 0.1% Tween 20) with 5% non-fat dry milk for 1.5 h at room
temperature. The blotted membrane was then incubated with the respective
primary antibodies (anti-IL-1b or anti-MCP-1) or actin (as internal control)
overnight at 4°C, and subsequently incubated in secondary antibody for 1 h at
room temperature. Upon incubation in the ECL Western blotting detection
reagent (Amersham, USA) for 1 min, the IL-1b and MCP-1 bands were visualized by autoradiography on x-ray film (Fuji, Japan). Quantification of individual band density was carried out by a video densitometer (Scan Maker III,
Microtek, Taiwan, ROC) expressed as volume count 3 mm2, which was
normalized by using actin as internal control.
Statistical analysis. Results were expressed as mean ± SE. Differences
between two groups were examined using one-way analysis of variance
(ANOVA) with various post-hoc tests. Dunnett’s test was first performed to
compare between the effects of the individual treatment group (or the normal
group) with the respective control. Additional analysis was performed by
Duncan’s new multiple-range test, with which statistical differences can be
found with smaller mean differences (e.g., between the non-filtered smoking
group and the control). Moreover, the Newman-Keuls procedure was conducted
to compare between the magnitude of effects between different treatment
groups. Finally, the Pearson correlation coefficient test was conducted to
determine if there were any dose–response relations in the effects of various
extracts from tobacco smoke and filter. A p value of less than 0.05 was
considered significantly different.
RESULTS
Alleviation of DNBS-Induced Colitis by Tobacco
Smoke and Nicotine
Enema DNBS caused lesion formation and severe inflammation in the distal colon of control rats. The pathogenic
features were associated with tissue edema, represented by the
colonic edema index (expressed as the colonic weight per unit
body weight; data not shown), along with shortening of the
inflamed colon. Daily exposure of non-filtered tobacco smoke
288
KO AND CHO
Myeloperoxidase activity is a marker for neutrophil activation, which signifies the degree of local tissue inflammation. In
this study, colonic MPO activity was dramatically elevated by
more than threefolds in the inflamed colon of control rats, when
compared to the intact colonic tissue of non-diseased animals (Normal). Exposure to non-filtered smoke, filtered smoke,
and the two regimens of nicotine treatment (4 mg/kg 3 3 and
8 mg/kg 3 1) all significantly decreased the DNBS-activated
MPO activity (Fig. 3), a finding that was in line with the relative
degrees of amelioration in colonic lesion formation (Fig. 2).
Again, there was no significant difference between the effects
on MPO activity induced by various effective treatment groups
(Newman-Keuls analysis). Alternatively, the mucosal level of
LTB4, a neutrophil chemotactic factor, was also drastically
increased in the control colitic tissues when compared with its
basal level in normal colonic tissues (Fig. 4). Filtered tobacco
smoke exposure and nicotine regimens caused marked inhibition of such elevation in colonic LTB4 content. Nonetheless, although non-filtered tobacco smoke had a tendency to
produce similar inhibitory action on LTB4 production, the effect did not arrive at a statistically significant level after performance of two different post-hoc (Dunnett’s and Duncan’s) tests
(Fig. 4).
Luminol-amplified chemiluminescence assay can be used to
detect free radicals generated predominantly via a MPOcatalyzed reaction (Hawkins et al., 1997). Only a negligible
amount of luminol-amplified chemiluminescence was observed
in the excised colonic tissues of normal animals. Our findings
also showed that the vast amount of MPO-derived free radicals
found in the inflamed colonic tissue was significantly decreased
after treatment with either filtered tobacco smoke or nicotine
FIG. 2. Exposure to non-filtered or filtered tobacco smoke as well as
different regimens of nicotine reduces the area of colonic damage in colitis rats.
Enema DNBS was administered to rats to induce experimental colitis. In the
smoke-treatment groups, rats were exposed to 4% (v/v) non-filtered (S) or
filtered (FS) tobacco smoke in a ventilated smoking chamber 1 h daily for
3 consecutive days after colitis induction. In the nicotine treatment groups,
subcutaneous nicotine was given either with a single dose of 4 mg/kg or 8 mg/kg
on the day after colitis induction (Nic[4] and Nic[8]), or with multiple doses of
4 mg/kg for 3 consecutive days after colitis induction (Nic[4 3 3]). All animals
were sacrificed 4 days after colitis induction. The colonic lesion area was
measured in mm2. Data represent the means ± SE of seven animals. An asterisk
(*) indicates a significant difference between a treatment group and the DNBS
control (*p < 0.05, **p < 0.01).
FIG. 3. Exposure to non-filtered or filtered tobacco smoke as well as
different regimens of nicotine attenuate the activated colonic myeloperoxidase
(MPO) activity in colitis rats. Treatment regimens were the same as those stated
in Figure 2. Myeloperoxidase activity was determined by a modified tetramethylbenzidine method as described in Materials and Methods. The final values
of MPO activity were represented as units per gram of colonic tissue. Data
represent the means ± SE of seven animals. The pound sign (#) indicates
a significant difference between the group of normal animals without colitis and
the DNBS colitis control group (#p < 0.001). An asterisk (*) indicates
a significant difference between a treatment group and the DNBS control
group (*p < 0.05, **p < 0.01).
following colitis induction significantly reduced the area of inflammation and active ulceration in the colitis animals (Fig. 2),
as well as alleviating the associated pathogenic conditions.
Owing to the fact that some detrimental compounds such as
hydrocarbons and terpenoids could be trapped in the cigarette’s
filter, while potentially colitis-ameliorating compounds like
nicotine and other alkaloids could pass through (Chow et al.,
1997), we exposed the experimental animals to filtered tobacco
smoke so as to determine whether there would be a further
improvement in lesion and inflammation development. On the
one hand, results had shown that the differences in antiinflammatory effects and diminution of lesion size between
the non-filtered and filtered tobacco smoke groups were not
reaching a statistically significant level. On the other hand,
subcutaneous nicotine also demonstrated significant and similar
anti-inflammatory and lesion-alleviating effects with either
a single dose of 8 mg/kg or multiple doses of 4 mg/kg nicotine,
whereas a single dose of 4 mg/kg nicotine had no effect (Fig. 2).
There was no significant difference between the magnitudes
of anti-lesion actions in various effective treatment groups
(Newman-Keuls analysis).
Attenuation of the Exaggerated Neutrophil Recruitment
and Free Radical Formation
ACTION OF TOBACCO COMPONENTS AGAINST COLITIS
289
radical formation, as in the case of LTB4 level (Dunnett’s and
Duncan’s tests).
Modulation of the Protein Expression of IL-1b and MCP-1
in the Inflamed Colonic Tissue
FIG. 4. Exposure to filtered tobacco smoke as well as different regimens of
nicotine attenuate the elevated colonic LTB4 level in colitis rats. Treatment
regimens were the same as those stated in Figure 2. The LTB4 level was
determined by an enzyme immunoassay method as described in Materials and
Methods. The final values of LTB4 level were represented as picograms per
milligram of colonic tissue. Data represent the means ± SE of seven animals. A
pound sign (#) indicates a significant difference between the group of normal
animals without colitis and the DNBS colitis control group (#p < 0.01). An
asterisk (*) indicates a significant difference between a treatment group and the
DNBS control group (*p < 0.05).
regimens (Fig. 5), much like their respective inhibitory actions
on LTB4 production (Fig. 4). When compared with the
inhibitory effect on MPO activity, non-filtered tobacco smoke
exposure did not significantly alleviate MPO-derived free
FIG. 5. Exposure to filtered tobacco smoke as well as different regimens of
nicotine alleviate MPO-derived free radical formation in colitis rats. Treatment
regimens were the same as those stated in Figure 2. Myeloperoxidase-derived
free radical formation was determined by a luminol-amplified chemiluminescence assay as described in Materials and Methods. Reactive oxygen
metabolite was quantified by the photomultiplier tubes of a scintillation counter
and the final values were represented as count per minute (3 103 cpm). Data
represent the means ± SE of seven animals. A pound sign (#) indicates
a significant difference between the group of normal animals without colitis and
the DNBS colitis control group (#p < 0.001). An asterisk (*) indicates
a significant difference between a treatment group and the DNBS control
group (*p < 0.05, **p < 0.01).
Report from animal colitis model had demonstrated significant elevation in mRNA expression of the cytokines IL-1b and
IL-6, and of the chemokine macrophage inflammatory protein1a and MCP-1, which could persist for 2 weeks (Sun et al.,
2001). In fact, MCP-1 activates macrophages and increases the
migration of monocytes into tissue during inflammation,
whereas IL-1b upregulates constitutive MCP-1 mRNA level
in colonic cell culture (Reinecker et al., 1995). Figures 6 and 7
illustrate that colonic IL-1b and MCP-1 proteins were both
overexpressed in colitis tissues, and the overexpression was
concomitantly attenuated by filtered cigarette smoke exposure
and nicotine regimens. However, no significant difference was
found between the modulation of protein expression by various
treatment groups (Newman-Keuls analysis). Representative
bands of IL-1b, MCP-1, and actin for each experimental group
are shown in Figure 12A.
Effects of Ethanol Extract of Filtered Tobacco Smoke and
Filter Chloroform Extract 1 and 2 on DNBS-Induced
Colitis and Neutrophil Activation
Ethanol and chloroform extracts from tobacco smoke were
analyzed for their chemical types by thin-layer chromatography
FIG. 6. Exposure to filtered tobacco smoke as well as different regimens of
nicotine attenuate the upregulation of colonic IL-1b protein expression in
colitis rats. Treatment regimens were the same as those stated in Figure 2.
Western blot analysis with specific antibody against IL-1b and actin (internal
control) was performed. Colonic tissue extracts were obtained as described in
Materials and Methods. Quantification of individual band density was carried
out by a video densitometer and was normalized by the actin band. Data
indicate the means ± SE of the ratio of IL-1b to actin band density from the
blots of colonic tissue extract of seven animals. A pound sign (#) indicates
a significant difference between the group of normal animals without colitis and
the DNBS colitis control group (#p < 0.001). An asterisk (*) indicates
a significant difference between a treatment group and the DNBS control
group (*p < 0.01).
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KO AND CHO
FIG. 7. Exposure to filtered tobacco smoke as well as different regimens of
nicotine attenuate the upregulation of colonic MCP-1 protein expression in
colitis rats. Treatment regimens were the same as those stated in Figure 2.
Western blot analysis with specific antibody against MCP-1 and actin (internal
control) was performed. Colonic tissue extracts were obtained as described in
Materials and Methods. Quantification of individual band density was carried
out with a video densitometer and was normalized by the actin band. Data
indicate the means ± SE of the ratio of MCP-1 to actin band density from the
blots of colonic tissue extract of seven animals. A pound sign (#) indicates
a significant difference between the group of normal animals without colitis and
the DNBS colitis control group (#p < 0.001). An asterisk (*) indicates
a significant difference between a treatment group and the DNBS control
group (*p < 0.01, **p < 0.001).
and gas chromatography/mass spectrophotometry. The chemicals identified in FSEE were mainly alkaloids, including
nicotine, whereas terpenoids, phenolic compounds, hydrocarbons, organic acids, fatty acids, and flavonoids were found
in FCE1, with no alkaloid being observed (Chow et al., 1997).
Furthermore, the acidic aqueous layer obtained from FCE2
contained a trace amount of alkaloid, which is much lower than
those found in the FSEE fraction from filtered smoke.
Intraperitoneal administration of the two higher doses of
FSEE (10 and 20 mg/kg) significantly reduced DNBS-induced
colonic lesion formation (Fig. 8, top), a finding that was in
concert with their ameliorating potential on the elevated MPO
activity (Fig. 9, top). In contrast, intraperitoneal administration
of the highest dose of FCE1 (10 mg/kg) significantly aggravated DNBS-induced colonic damage (Fig. 8, bottom) with
concurrent potentiation of the elevated colonic MPO activity
(Fig. 9, bottom). But there was no dose–response relation in the
effect of FSEE and FCE1 (Pearson correlation coefficient test).
Treatment with FCE2 caused no effect in either colitis
development or in modulating colonic MPO activity.
Effects of Ethanol Extract of Filtered Tobacco Smoke and
Filter Chloroform Extract 1 and 2 on Colonic Protein
Expression of IL-1b and MCP-1
We further delineated the effects of various tobacco extracts
on colonic protein expression of IL-1b and MCP-1. Our results
FIG. 8. Ethanol extract of filtered tobacco smoke (FSEE) reduces the area
of colonic damage, whereas chloroform extract 1 of the filter (FCE1)
aggravates colonic injury in colitis rats. Enema DNBS was administered to
rats to induce experimental colitis. (Top) FSEE (5, 10, or 20 mg/kg) and
(bottom) FCE1 (2.5, 5, or 10 mg/kg) were given intraperitoneally to the rats
daily for 3 consecutive days after colitis induction. All animals were sacrificed
4 days after colitis induction. Colonic lesion area was measured in mm2. Data
represent the means ± SE of seven animals. An asterisk (*) indicates
a significant difference between a treatment group and the DNBS control
(*p < 0.05, **p < 0.01).
had shown that treatment with all three doses of FSEE induced
a downregulation of both IL-1b and MCP-1 protein expression
in colonic tissues (Fig. 10, top; Fig. 11, top), a result that
was somewhat consistent with their anti-inflammatory actions
(Fig. 8, top; Fig. 9, top) and that had effects similar to those
demonstrated by filtered tobacco smoke and nicotine treatments (Fig. 6 and 7). In contrast, treatment with FCE1 (10 mg/kg)
caused an upregulation of colonic IL-1b and MCP-1 protein expression (Fig. 10, bottom; Fig. 11, bottom), which
possessed a direct correlation with their pro-inflammatory
actions in the colon (Fig. 8, bottom; Fig. 9, bottom). Again,
no dose–response relation was found in the effect of FSEE and
FCE1 (Pearson correlation coefficient test). Representative
bands of IL-1b, MCP-1, and actin for each experimental group
are shown in Figure 12B and 12C.
ACTION OF TOBACCO COMPONENTS AGAINST COLITIS
FIG. 9. An ethanol extract of filtered tobacco smoke (FSEE) attenuates the
activated colonic MPO activity in colitis rats, whereas chloroform extract 1 of
the filter (FCE1) intensifies the activated colonic MPO activity in colitis rats.
Treatment regimens were the same as those stated in Figure 8. Myeloperoxidase activity was determined by a modified tetramethylbenzidine method as
described in Materials and Methods. The final values of MPO activity were
represented as units per gram of colonic tissue. Data represent the means ± SE
of seven animals. A pound sign (#) indicates a significant difference between
the group of normal animals without colitis and the DNBS colitis control group
(#p < 0.01). An asterisk (*) indicates a significant difference between
a treatment group and the DNBS control group (*p < 0.05, **p< 0.01,
***p< 0.001).
DISCUSSION
The strong association between smoking and IBD development has been known for many years. We are the first group to
exemplify the anti-inflammatory and immunomodulating effects of tobacco smoke on experimental colitis (Ko et al.,
2001). In the present study, exposure to non-filtered and filtered
tobacco smoke reduced the severity of colonic inflammation
and lesion formation induced by DNBS. Because the antiinflammatory action of tobacco smoke involved reduction of
the elevated colonic MPO activity, prevention of neutrophil
activation and infiltration into the inflamed tissues could be
important. LTB4 is one of the most potent chemotactic and
chemokinetic metabolites of arachidonic acid, and it plays
a permissive role in the action of neutrophils during the
amplification of intestinal inflammatory reactions, including
colitis (Nielsen and Rask-Madsen, 1996). As a result, lowering
of the LTB4 level could attenuate neutrophil activation and
infiltration into the inflamed colonic tissue, and consequently
291
FIG. 10. Ethanol extract of filtered tobacco smoke (FSEE) attenuates while
the chloroform extract 1 of the filter (FCE1) intensifies the activated
upregulation of colonic IL-1b protein expression in colitis rats. Treatment
regimens were the same as those stated in Figure 8. Western blot analysis with
specific antibody against IL-1b and actin (internal control) was performed.
Colonic tissue extracts were obtained as described in Materials and Methods.
Quantification of individual band density was carried out by a video
densitometer and was normalized by the actin band. Data indicate the means
± SE of the ratio of IL-1b to actin band density from the blots of colonic tissue
extract of seven animals. A pound sign (#) indicates a significant difference
between the group of normal animals without colitis and the DNBS colitis
control group (#p < 0.01, ##p < 0.001). An asterisk (*) indicates a significant
difference between a treatment group and the DNBS control group (*p < 0.05,
**p < 0.01).
free radicals generated by activated neutrophils and other
granulocytes would also be markedly reduced. Our earlier
findings had indicated a significant decline in colonic LTB4
level and inhibition of ROM production by both nicotine and
filtered tobacco smoke treatments. Nicotine was previously
shown to decrease LTB4 production from rat alveolar macrophages (Sugiyama et al., 1989). Besides, it was also discovered
that nicotine could enhance neutrophil sequestration, possibly
by scavenging other oxidants in tobacco smoke (Aoshiba et al.,
1994). Other proposed protective mechanisms of nicotine on
UC include the boosting of mucin synthesis on colonic luminal
surface, release of nitric oxide that counterreacts with eicosanoid metabolism, and the increase in intestinal trefoil factors
292
KO AND CHO
FIG. 11. Ethanol extract of filtered tobacco smoke (FSEE) attenuates while
the chloroform extract 1 of the filter (FCE1) intensifies the activated
upregulation of colonic MCP-1 protein expression in colitis rats. Treatment
regimens were the same as those stated in Figure 8. Western blot analysis with
specific antibody against MCP-1 and actin (internal control) was performed.
Colonic tissue extracts were obtained as described in Materials and Methods.
Quantification of individual band density was carried out with a video densitometer and was normalized by the actin band. Data indicate the means ±
SE of the ratio of MCP-1 to actin band density from the blots of colonic tissue
extract of seven animals. A pound sign (#) indicates a significant difference
between the group of normal animals without colitis and the DNBS colitis
control group (#p < 0.01, ##p < 0.001). An asterisk (*) indicates a significant
difference between a treatment group and the DNBS control group (*p < 0.05).
that eventually improve mucosal restitution (Wu and Cho,
2004). Although many clinical trials have suggested that
nicotine may not be effective in mono-therapy, evidence from
a 12-month study indicated that nicotine-induced remission of
UC could last longer than that obtained by conventional
therapeutic agents such as oral corticosteroids (Guslandi,
1999). In addition, this report had also shown that 6 mg of
both oral and transdermal nicotine could be well tolerated in
UC patients, and this was thus thought to be the highest
therapeutic dose with a low risk of adverse effects in the human
body (Green et al., 1999; Ingram et al., 2004).
Tobacco smoke could result in a comparable serum concentration of nicotine to the level in animals treated with non-
FIG. 12. Representative bands of IL-1b, MCP-1, and actin after different
treatments. (A) N ¼ normal, C ¼ control, FS ¼ multiple (33) treatments of 4%
v/v filtered tobacco smoke, Nic(4 3 3) ¼ multiple (33) treatments of 4 mg/kg
nicotine, Nic(8) ¼ single treatment of 8 mg/kg nicotine. (B) N ¼ normal, C ¼
control, FSEE(5) ¼ multiple (33) treatments of 5 mg/kg FSEE, FSEE(10) ¼
multiple (33) treatments of 10 mg/kg FSEE, FSEE(20) ¼ multiple (33)
treatments of 20 mg/kg FSEE. (C) N ¼ normal, C ¼ control, FCE1(2.5) ¼
multiple (33) treatments of 2.5 mg/kg FCE1, FCE1(5) ¼ multiple (33)
treatments of 5 mg/kg FCE1, FSEE(10) ¼ multiple (33) treatments of
10 mg/kg FCE1.
filtered smoke (Chow et al., 1997), a level much lower than that
achieved after nicotine treatments. Under such circumstances,
because tobacco smoke was capable of producing similar
protection against colitis development, as in the nicotinetreated animals, there must be some unknown substance(s) in
the tobacco smoke that could play a permissive role in the
amelioration of colitis. However, this hypothesis can only be
clarified by examining individual components of the tobacco
smoke. It should be noted that the cigarette filter can separate
the tar and gas phases of tobacco smoke. Most of the particles
in the tar phase, including ROS, hydrocarbons, and phenols, are
retained in the filter (Pryor and Stone, 1993). Our previous
studies also revealed that extracted compounds from the tar
phase of tobacco smoke could repress mucus synthesis in vivo
and in vitro by inhibiting polyamine synthesis (Ma et al., 2000)
and thus potentiate ethanol-induced gastric mucosal injury
(Chow et al., 1997). In accordance with this finding, the
ACTION OF TOBACCO COMPONENTS AGAINST COLITIS
detrimental compounds may aggravate colitis formation and
also antagonize the effects of other protective agents. Although
the gas phase of tobacco smoke also contains radicals such as
nitric oxide and reactive olefins, these organic radicals usually
have a shorter half-life (typically less than 1 s) than those
contained in tar phase smoke (Pryor and Stone, 1993). Hence,
most of the alkaloids in tobacco smoke, including nicotine, that
pass through the filter could produce prominent anti-oxidative
effects that contribute to colonic protection in a similar manner
to the action of systemic nicotine treatment alone.
To clarify the differential effects of filtered and non-filtered
smoke, we performed ethanol and chloroform extractions to
collect the chemical components of gas phase smoke and tar
phase smoke, respectively. The ethanol extract of filtered
smoke (FSEE) was shown to be protective against colitis
formation, whereas the administration of FCE1 at its highest
dose (10 mg/kg) was detrimental to colitis development. Ma
et al. (2000) reported that the chloroform extract of tobacco
smoke contains terpenoids and phenolic compounds. These
results actually supported our previous suggestion that the
beneficial effects of filtered smoke and nicotine were more
prominent than those of non-filtered smoke because of the
presence of damaging factor(s) in the latter. We also examined
the effect of FCE2 using the same colitis model, but there was
no significant effect observed (data not shown). These findings
indicate that the quantity of detrimental substances contained
in filtered smoke was not high enough to worsen colitis development, while the content of potential protective mediators
trapped in the filter was also too minimal to cause any effect.
Polymorphonuclear leukocytes migrate in response to chemotactic factors, which can be activated to produce certain
chemokines. Neutrophils and macrophages in inflamed intestine of IBD patients synthesize and secrete large amounts of
chemokines (MacDermott, 1999). Nonetheless, epithelial chemokine production could be an important target in the therapy
of IBD (van Deventer, 1997). Our findings actually demonstrated that there was concomitant inhibition of colonic MPO
activity and downregulation of MCP-1 protein expression by
filtered cigarette smoke, FSEE extract, and nicotine. The
expression of the MCP-1 gene in vessel-associated cells may
indicate its involvement in regulating the adhesion of blood
monocytes to endothelial cells (Mazzucchelli et al., 1996).
Although monocyte recruitment is the primary biological role
of MCP-1, it was found that MCP-1 is also capable of attracting
neutrophils via the production of LTB4 (Matsukawa et al.,
1999). In contrast, LTB4 could reversibly induce MCP-1
production during active inflammation. In addition, findings
from other studies have suggested that MCP-1 depletion
inhibits neutrophil influx by downregulating LTB4 production
in intestinal tissues (Crooks and Stockley, 1998), which
resembles the situation in colitis animals treated with filtered
cigarette smoke, FSEE extract, or nicotine.
Along with chemokine activation, the expression of most
pro-inflammatory cytokines, notably IL-1, IL-6, and TNF-a, is
293
markedly enhanced in the intestinal mucosa of IBD patients
(Kmiec, 1998). Some protective agents could act by reducing
both colonic LTB4 and IL-1b levels, which would consequently
improve the state of oxidative stress in the colon (Galvez et al.,
2001). Our findings on the effects of filtered cigarette smoke,
FSEE extract, and nicotine on colonic IL-1b expression in fact
reflected the phenomenon of a clinical study on smokers with
UC, which had shown a significant reduction in IL-1b level in
the patients’ colonic mucosa (Sher et al., 1999). In spite of the
report that the Th-1 cytokine IL-1b could modulate LTB4
production from monocytes (Montero et al., 2000), other
groups have stated that synthesis of such cytokines is in turn
determined by LTB4 level in the inflammatory tissues (He
et al., 2002; Kuwabara et al., 2000). Moreover, the inhibition of
IL-1b production also seemed to be associated with the
downregulation of MCP-1 protein synthesis in the colonic
mucosa. Nonetheless, a similar correlation between MCP-1
and another pro-inflammatory cytokine, TNF-a, seems to occur
in CD patients only (McCormack et al., 2001). According to
the increased colonic protein expression of both MCP-1 and
IL-1b induced by administering the highest dose of FCE1
extract, it could be owing to the presence of detrimental
compounds like terpenoids, as stated earlier. We once reported
that chloroform extract from the tar phase of tobacco extract decreased colonic cell proliferation, which in turn modulates the
turnover of epithelium at the base of colonic crypt cells (Shin
et al., 2003). This could at least partially explain the deteriorating effect on colitis development by the FCE1 extract.
Although we have suggested the potential use of nicotine or
other protective alkaloids as found in the ethanol extract of
tobacco smoke, it is also crucial to stress the general
detrimental effects of smoking and the pathophysiological
modulation of many dreadful diseases by various components
in the smoke. There are 5 3 109 particles contained in 1 cc of
mainstream smoke. In gas phase smoke, 99.9% of which is able
to sieve through the cigarette filter, there are various gaseous
compounds such as HCN, NO, and acetone (amounting to
0.5 mg/cigarette). In the cigarette we used in the present study,
other than the 1.1 mg of nicotine, there are 15 mg of tar, and
12 mg equivalent of CO will be generated by the smoke. In
addition, although tar and the total particulate matter (in the
particulate phase) will be partially retained by the cigarette
filter, the remaining amount to be inhaled or released to the
environment still contains many detrimental compounds such
as glycols, terpenoids, carboxylic acids, paraffin wax, and worst
of all, the carcinogenic nitrosamines (amounting to 1.5–2 lg/
cigarette) and aromatic hydrocarbons (amounting to 3.6 lg/
cigarette, including 0.02 lg of benzo(a)pyrene). We had
demonstrated in a chronic colitis-induced adenoma study that
long-term treatment of 4% v/v tobacco smoke to DSS-treated
mice increased angiogenesis and reduced the apoptosis/
proliferation ratio, which resulted in a significantly increased
incidence of inflammation-associated adenoma/adenocarcinoma
formation (Liu et al., 2003). We further examined the
294
KO AND CHO
carcinogenic effects of the tobacco extract, and found that
both ethanol and chloroform extracts from tobacco smoke
would lead to elevation in the expression of the protooncogene c-myc in a gastric adenocarcinoma cell line,
although such expression was found to be reduced by the
same treatments in a well-differentiated colon adenocarcinoma cell line (Shin et al., 2003). To our surprise, we also
discovered that nicotine, the ‘‘protective’’ component in the
tobacco smoke component as shown in the present study,
would lead to increase in proliferation in colon adenocarcinoma cells, with concurrent overexpression of the 5-lipoxygenase enzyme and an increase in epidermal growth
factor receptor and c-Src phosphorylation levels, which
eventually contributed to the promotion of tumor growth in
nicotine-treated nude mice (Ye et al., 2004). These points
together raise an important question about how to balance the
‘‘beneficial’’ and harmful effects of tobacco smoke components, including the seemingly ‘‘protective’’ nicotine. The
implications being brought forth from findings of the present
investigation actually target the discovery of potential novel
anti-inflammatory agents and the underlying mechanisms that
could be used in the treatment of colitis diseases.
In conclusion, tobacco smoke contains various active
chemical components that exert differential effects in DNBSinduced colitis. When some of the damaging factors are
removed from the smoke after it is passed through the filter,
the net effect could be colonic protection. As a result, both
filtered tobacco smoke and nicotine have been demonstrated to
accelerate healing during active colitis, which also involves
inhibition of neutrophil-derived free radicals and downregulation of the pro-inflammatory chemokine and cytokine protein
synthesis in the colonic tissue. Nonetheless, it is remarkable
that the vast number of detrimental substances present in
tobacco smoke, including terpenoids, polyphenols, nitrosamines, and polycyclic hydrocarbons, plus the multiple systemic
side effects of nicotine have to be considered in the search of an
effective chemotherapeutic regimen for IBD. Perhaps the
verification of the precise protective alkaloids contained in
tobacco smoke could be of even a greater importance.
SUPPLEMENTARY DATA
Supplementary data are available online at www.toxsci.
oxfordjournals.org.
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