Comparative Medicine Vol 62, No 5

Copyright 2012 October 2012

by the American Association for Laboratory Animal Science Pages 395–399

Original Research

A Rat Model of Cigarette Smoke Abuse Liability

Petros Ypsilantis,1,* Maria Politou,1 Constantinos Anagnostopoulos,2 Alexandros Kortsaris,2 and Constantinos Simopoulos1

We sought to develop a rat model of cigarette smoke exposure (CSE) that created cotinine serum levels comparable to those
of smokers and induced conditioned place preference (CPP) suggestive of cigarette smoke abuse liability. Rats were exposed to
sidestream cigarette smoke delivered semicontinuously for 2 periods of 20 (group S20), 40 (group S40), or 60 (group S60) min daily
for 12 wk. Serum cotinine concentration in blood samples was determined at 1 and 20 h after CSE. A biased (black versus white
chamber) CPP paradigm was used. In the high CSE group (group S60), serum cotinine at 1 h (250 to 300 ng/mL) was comparable
to average cotinine levels reported for addicted smokers (around 300 ng/mL). Cotinine levels at 20 h after CSE were higher than
the smoker–nonsmoker cut-off value (greater than 14 ng/mL) in all smoking groups, with the S60 group having the highest levels.
All rats preferred the black chamber to the white chamber during the preexposure CPP test. The time spent in the white chamber
was increased compared with 0-wk values in group S40 at 8 wk, group S60 at 4 and 8 wk, and the control group at 4 and 8 wk but
not at 12 wk; however, the shift in CPP was significantly higher at 8 wk in group S60 compared with other groups. In conclusion,
interrupted 2-h daily CSE for 8 wk induced serum cotinine levels in rats comparable to those of smokers and induced CPP sugges-
tive of cigarette smoke abuse liability.

Abbreviations: CPP, conditioned place preference; CSE, cigarette smoke exposure.

The devastating consequences of smoking on health have been
studied extensively in numerous clinical and animal studies over
time. This chronic habit leads to dependence on tobacco smoke,
with nicotine, a main active ingredient of tobacco products, being
recognized as the basic addictive substance.32
The known health benefits of smoking cessation motivate
smokers to quit tobacco use. However, unaided efforts usually
are unsuccessful, resulting in smoking relapse. The fight against
nicotine addiction may be undermined by potential weight gain
after smoking cessation, potentially discouraging those attempt-
ing to quit smoking and contributing to relapse. During the past
few years, research has been focused on 2 main areas of inter-
est toward this direction: understanding the underlying biologic
mechanisms related to nicotine addiction to effectively design
therapeutic strategies to support those who wish to quit smok-
ing and investigating the hormonal and molecular mechanisms
responsible for weight gain after smoking cessation.
So far, animal models used to study the consequences of smok-
ing cessation involved the administration of nicotine as a sole
agent until addiction was achieved.23 However, nicotine-ad-
ministration models do not completely represent the toxic and
addictive effects of cigarette smoke, given that smoke contains
more than 4000 chemicals whose actions or coactions have not
been thoroughly evaluated yet.1 Cigarette smoke exposure (CSE)
animal models have been used in studies investigating the met-
abolic changes conferred by smoking10-12 but not in those after
Received: 20 Feb 2012. Revision requested: 03 Apr 2012. Accepted: 13 May 2012.
1
Laboratory of Experimental Surgery and Surgical Research and 2Laboratory of
Biochemistry, School of Medicine, Democritus University of Thrace, Alexandroupolis,
Greece.
*
Corresponding author. Email: pipsil@med.duth.gr
its cessation. In toxicity studies, animals are exposed to tobacco
smoke for various periods, which depend on the side effect un-
der investigation.18,25,27 Smoke exposure timetables usually do not
involve weekends for practical reasons, and addiction of animals
to tobacco smoke is not assessed in current models.
In our opinion, an ideal animal model of cigarette smoke abuse
liability suitable for the study of smoking cessation resembles
the clinical situation in terms of chronic daily inhalation of ciga-
rette smoke sufficient to attain blood nicotine levels compara-
ble to those of smokers and in cessation of the CSE period after
achieving tobacco smoke abuse liability. In the present project,
we sought to establish such a model in rats by defining the daily
timetable of CSE to induce serum levels of cotinine, nicotine’s
major proximate metabolite, comparable to those of smokers and
by determining the minimum total CSE period required to induce
abuse liability to cigarette smoke. We assessed the CSE period by
using a biased conditioned place preference (CPP) paradigm.8
Materials and Methods
Animals. Male Wistar rats (Rattus norvegicus; n = 42; age, 5 to 6
mo; weight, 380 to 420 g) provided by our laboratory’s inhouse
breeding colony were used. They were the 20th inbred generation
of Wistar rats obtained from the conventional breeding facility
of the Democritos National Center of Physics Research (Athens,
Greece). Colony health status was monitored semiannually, and
rats were found to be free of Mycoplasma spp., adventitious virus-
es, respiratory and enteric bacteria, and ecto- and endoparasites.
The rats were housed in polycarbonate cages, 3 rats per cage, at 20
to 22 °C, on a 14:10-h light:dark cycle and were given commercial
pelleted diet (4RF25, Mucedola, Milan, Italy) and tap water ad
libitum. The facilities were in accordance with Directive 86/609/

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EEC, which refers to “the protection of animals used for experi-
mental and other scientific purposes.”13
Experimental design. We randomly assigned 24 rats to 3 smok-
ing (groups S20, S40, and S60) and a nonsmoking control group
(group C) that consisted of 6 rats each. Rats in the smoking groups
were exposed to interrupted sidestream cigarette smoke for vari-
ous periods on a daily basis for 12 wk. Three rats at a time were
confined in a white smoking chamber for 2 periods of 20 (group
S20), 40 (group S40), or 60 (group S60) min each, with a 60-min
interval between exposures. The color of the chamber was that
of the ‘nonpreferred’ side (white), as defined through a preex-
posure, black compared with white chamber, CPP test. Rats in
the control group were confined on a daily basis in an identical
chamber without smoke for 2 periods of 60 min, with a 60-min
interval, for 12 wk. Blood samples were collected around 20 h
after the first smoking session at 0, 5, and 9 wk during the CSE
period, as well as 2 d after the end of the CSE period, to determine
serum cotinine concentration. Every 4 wk, rats were subjected to
a biased CPP test to assess abuse liability to cigarette smoke. In
a separate set of smoking control experiments, 3 groups of 6 rats
each (groups SC20, SC40, SC60) were assigned to 1 wk of CSE
exposure as described earlier, and blood samples were collected
at 1 h after the last day’s smoking session to determine cotinine
levels. The experimental protocol was approved by the Animal
Care and Ethics Committee of the local Veterinary Service and
was in compliance with Directive 86/609/EEC.13
CSE system. The system used to expose rats to sidestream ciga-
rette smoke consisted of a ventilator, a smoke-generating cham-
ber, and a whole-body CSE chamber that were serially connected
via silicone tubes interlined by Heimlich valves (Becton Dickin-
son, Franklin Lakes, NJ) to avoid smoke regression (Figure 1). The
ventilator (Veterinary anesthesia ventilator model 2000, Hallowell
EMC, Pittsfield, MA) was set to provide 150 mL of air every 10 s.
The smoke-generating chamber consisted of an acrylic cylinder
(radius, 8 cm; height, 27 cm) corresponding to 5430 cm3 total vol-
ume into which one cigarette (tar, 8 mg; nicotine, 0.6 mg; carbon
monoxide, 9 mg) at a time was constantly kept lit. Smoke then
was delivered to an acrylic white chamber (length, 40 cm; width,
20 cm; height, 25 cm) of 20,000 cm3 total volume and exhausted
through a hole. The level of carbon monoxide inside the CSE
chamber was kept between 150 and 250 ppm by transient inter-
ruption, if necessary, of the diluted sidestream smoke delivery.
The carbon monoxide concentration in air samples collected from
the chamber interior at 15-min intervals was determined by using
a portable carbon monoxide analyzer (piCO Smokerlyzer Breath
CO monitor, Bedfont Scientific, Kent, UK). Rats were exposed to
cigarette smoke between 0900 and 1300.
CPP test. The CPP apparatus consisted of 2 acrylic chambers, a
black and a white, that were connected by a door (height, 10 cm;
width, 7 cm). The dimensions of each chamber were the same
with that of the CSE chamber (length, 40 cm; width, 20 cm; height,
25 cm; volume, 20,000 cm3). Each rat was placed individually in
the apparatus, at the level of the interconnecting door, and the
time the rat spent in each chamber was recorded for a period of
10 min (600 s). The chamber in which the rat spent less than 300 s
was designated as the nonpreferred side.
Before commencement of the CSE period, rats underwent a
preexposure CPP test to determine their place preference. There-
after, CSE (and control exposure) took place in acrylic chambers
identical in color and dimensions as those of the nonpreferred
396
cm12000025.indd 396
Figure 1. The smoke-exposure system.
side. CPP tests were considered to be biased and were conducted
in the same manner.
Determination of serum cotinine concentration. Blood samples
were collected from the tail artery under sevoflurane anesthesia.
Collection of blood samples for cotinine measurement in rats that
underwent CSE was performed at 1 h after CSE (that is, 1400).
Control samples were collected between 0800 and 0900, approxi-
mately 20 h after their last session. Serum was separated by cen-
trifugation at 3000 × g for 20 min and then stored at −70 °C until
later analyzed. The concentration of cotinine was determined by
using a commercially available ELISA kit (Cotinine Direct ELISA,
BioQuant, San Diego, CA) according to the manufacturer’s in-
structions. The lower limit of detection was 1 ng/mL.
Statistical analysis. Data were analyzed by using SPSS 13.0 sta-
tistical software (SPSS, Chicago, IL). Data were expressed as mean
± SE and underwent repeated-measures ANOVA followed by
the Student t test for comparisons between 2 groups or the Least
Significant Difference test for multiple comparisons. A probabil-
ity of less than 5% (P < 0.05) was considered to be statistically
significant.
Results
All rats survived the 12-wk experimental period.
Serum cotinine levels. Serum cotinine levels at 1 h after CSE
ranged between 147 and 167 ng/mL (mean ± SE, 160.0 ± 4.1 ng/
mL) for group SC20, 171 and 221 ng/mL (195.8 ± 11.2 ng/mL) for
group SC40, and 248 and 305 ng/mL (276.5 ± 12.7 ng/mL) for
group SC60.
At 20 h after CSE, serum cotinine concentration in all smoke-
exposed rats was increased above 14 ng/mL, which is the cut-off
value for being defined as a smoker.20 Cotinine concentrations
were significantly (P < 0.001) higher in group S60 compared with
groups S20 and S40. Cotinine returned to preexposure levels 2
d after completion of the smoke-exposure period (Figure 2). No
changes were noted in the cotinine levels of control rats (data not
shown). Serum cotinine concentration did not differ among time
points within each smoke-exposed group, suggesting that the
CSE setup induced a reliable and reproducible increase in serum
cotinine.
CPP. During the preexposure CPP test, all rats preferred the
black over the white chamber (Figure 3 A). The time spent in the
white chamber was significantly increased in group S40 at 8 wk
(P < 0.05) and group S60 at 4 wk (P < 0.05) and 8 wk (P < 0.001)
but not at 12 wk of exposure. Similarly, the time control rats spent
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Figure 2. Mean serum cotinine concentration (n = 6) of rats at 20 h after
daily smoke exposure during the CSE period (0, 1, 5, 9 wk) and at 2 d
after cessation of the CSE period (C2d). Error bars represent standard
errors. Values of 1 ng/mL or less are denoted as 1 ng/mL (minimum
detection limit). The dotted line depicts the 14 ng/mL cut-off limit for
being considered a smoker. CSE, cigarette smoke exposure; *, significant
(P < 0.001) difference between S20 and S40.
in the white chamber was increased at 4 wk (P < 0.01) and 8 wk (P
< 0.05). The white chamber remained the nonpreferred side (time
spent, less than 300 s) in all groups studied (Figure 3 A).
To compare the shift in place preference among groups at each
time point, the relative increase (%) of the initial time spent in the
white chamber was calculated. This percentage was significantly
higher (P < 0.05) in group S60 compared with group S20 and con-
trol rats after 8 wk of exposure (Figure 3 B).
Discussion
In the present study, daily exposure of rats to freshly generated
sidestream cigarette smoke delivered semicontinuously for 120
min into a whole-body CSE chamber for an 8-wk period induced
serum cotinine levels comparable to those of addicted smokers
and that remained over the smoker–nonsmoker cut-off value
throughout the day. The model also led to the development of
CPP suggestive of tobacco smoke abuse liability.
In a cigarette smoke abuse liability animal model, tobacco
smoke inhalation needs to result in serum nicotine levels equiva-
lent to those of smokers. Cotinine has a longer half-life (18 to 20 h)
than that of nicotine (1 to 2 h)6 and therefore is considered a more
reliable biomarker for smoking status. In our study, the smok-
ing habit was simulated in terms of serum cotinine levels, which
were around 250 to 300 ng/mL in the high-CSE rats at 1 h after
exposure, because these levels are comparable to average blood
cotinine concentration reported for addicted smokers (around 300
ng/mL).5 Moreover, cotinine concentrations in our rats remained
above the accepted cut-off limit for being considered a smoker
(14 ng/mL)20 throughout the day, according to measurements in
blood samples taken 20 h after CSE; data at this time point also
ensured that rats began subsequent days at a nonzero cotinine
concentration. Recent studies in humans have lowered the smok-
er–nonsmoker cut-off point to 3 ng/mL to better discriminate ac-
tive smokers from nonsmokers exposed to secondhand smoke.4
Abuse liability is the potential that a drug has for addiction. A
drug meets criteria for abuse liability if it produces effects medi-
ated by the brain sufficient to lead a substantial proportion of
cm12000025.indd 397
Cigarette smoke abuse liability model
Figure 3. (A) Time spent in the white chamber (P values indicate dif-
ferences compared with value at 0 wk) and (B) relative shift (%) of the
initial time spent in the white chamber after white compared with black
chamber-biased CPP test of rats exposed to cigarette smoke. Data are
presented as the mean of 6 rats; error bars represent standard error. The
dotted line depicts the time limit that characterized place preference.
Total test time, 600 s.
people or animals to repeated self-administration of the drug.
The likelihood that the self-administration of a drug will result
in persistent use or abuse is associated with its psychoactive or
CNS effects, which can have both positive and negative subjec-
tive effects; its reinforcing effects; and its ability to induce toler-
ance, craving, and withdrawal-associated effects after repeated
use of the drug.32 A broad range of tests can provide scientifically
validated basis for predicting the likelihood that a drug will be
abused or cause addiction. In the experimental setting, these tests
include assessment of intravenous self-administration, condi-
tioned place preference, and drug discrimination and the mea-
surement of withdrawal disturbances.22
Abuse liability of animals to cigarette smoke is essential to be
established when addiction-related side effects are to be studied.
Nicotine is a highly addictive psychoactive substance and can
induce enhanced reinforcement, as shown by behavioral tests
in animals, after only 3 d of intraperitoneal,7 7 d of continuous
subcutaneous,23 or 8 d of intravenous infusion in adult rodents33
and after a single subcutaneous injection in early adolescent ro-
dents.3 Although inhalation of tobacco smoke rapidly elevates
blood nicotine levels,26 abuse liability of rats to cigarette smoke,
as suggested by CPP, took considerably more time to be estab-
lished in the current study compared with the time needed for
nicotine addiction after its infusion as a sole agent, as reported in
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previous studies.7,23,33 In the current study, the diluted sidestream
smoke delivery of nicotine and other smoke constituents were
sufficient to support the development of CPP in our rat model af-
ter 8 wk of CSE. In humans, addiction to nicotine due to cigarette
smoking is a progressive procedure, starting with the use of 1 to 2
cigarettes daily and followed by a gradual escalation of smoking
frequency. In addition, nicotine overload is known to produce
conditioned place or taste aversion.22 Because the rats in the cur-
rent study were exposed to cigarette smoke for a standard exten-
sive daily period from the beginning of the study, they may have
experienced nicotine overload that was sufficient to have delayed
CPP development. Repeated CSE, and therefore nicotine intake,
for 8 wk may then have led to CPP development suggestive of
abuse liability. In addition, cigarette smoke contains numerous
other compounds, in addition to the well-known addictive agent
nicotine,9 whose potential addictive or aversive actions have not
yet been investigated thoroughly.
The biased CPP paradigm we used here evaluates the reward-
ing effect of an agent or procedure in terms of an increase in the
time spent in the initially preferred or nonpreferred environment
(for example, black compared with white chamber) after repeated
pairing with the agent or procedure under investigation. This test
has been previously used to evaluate rewarding effects of nicotine
and other abused agents.7,8,14,21,24 However, the magnitude of the
effect of nicotine is generally small and affected by environmen-
tal stimuli or previous handling history, suggesting its weaker
reinforcing effect compared with that of other agents.22 Accord-
ing to previous experimental setups, chambers used to evaluate
nicotine-induced CPP differed in both color and floor texture.8,34
In our study, rats showed a definite preference against the white
chamber (only a 50- to 150-s stay in the white chamber during a
600-s total test period) during the preexposure CPP test, even in
the absence of a difference in floor texture. Therefore, we presume
that sufficient time was available for the manifestation of a CPP
shift after CSE, especially after having incorporated a biased CPP
procedure by pairing CSE with the less-preferred enclosure. The
time spent in the nonpreferred white chamber increased after
8 wk of pairing it daily with 2-h interrupted CSE. According to
our results, the magnitude of the increase proved to be depen-
dent on the CSE period and subsequently on the dose of addictive
agents taken in, when compared with shorter exposure periods.
Accordingly, 4-h daily exposure of rats to tobacco smoke has been
reported to induce nicotine dependence after 4 wk.28 Several stud-
ies have found a dose-dependent CPP to nicotine-paired environ-
ments.8 In addition to the rewarding effect of nicotine in tobacco
smoke, the anxiolytic effect of nicotine may have contributed to
the development of CPP in our rats. Nicotine alleviates stress,
as shown by a reduction in aversion toward a previously non-
preferred environment in the CPP procedure31 and according to
results of the social interaction test,16 a fear-conditioning model,29
and the elevated plus-maze test.15
We collected blood samples from rats after they had been anes-
thetized briefly with sevoflurane. The remote possibility that
sevoflurane anesthesia, even of a short duration (1 to 2 min) and
at such a sparse interval (4 wk), promoted place conditioning
due to potential residual effects was counterbalanced by simi-
larly anesthetizing control animals before collecting their blood
samples.
Time spent in the nonpreferred chamber was reduced at the
12th week of CSE. Nicotine has both reinforcing and aversive
398
cm12000025.indd 398
effects at high or sometimes the same dose.22 The reinforcing ef-
fect derived from cigarette smoke inhalation is a complex proce-
dure, during which the rewarding properties of cigarette smoke
overcome the aversive nature of smoke constituents. A number of
compounds, including acetaldehyde, act in concert with nicotine
to potentiate its brain-rewarding function,28,30 whereas others,
including ammonia, acrolein, and acetone, act as irritant toxins.19
In addition, combustion products are aversive because of eye or
airway irritation, and repeated nicotine intake may induce toler-
ance to its anxiolytic-like effects29 or place aversion due to poten-
tial residual anxiogenic effects.8 Furthermore, although repeated
CPP testing enable us to identify the time point with maximal
CPP shift, repetition of place conditioning might have affected
behavioral outcomes.
During the first 8 wk, we also noted an increase in the time that
control rats spent in the white chamber, which were confined for
2 h daily to that chamber. This finding suggests an acquaintance
of the animals to the nonpreferred environment after chronic
daily confinement, and this possibility should be considered
when evaluating addiction by using a biased CPP paradigm. In
longitudinal studies involving long-term pairing of a test sub-
stance or procedure with a certain type of environment, one can-
not rule out the possibility that repeated exposure of animals
may gradually decrease aversion to, for example, an enclosure
of a specific color. In addition, diverse ‘natural’ stimuli, includ-
ing food, water, and wheel-running, can condition an increase in
place preference.2
The present experimental protocol involved daily exposure
of rats to cigarette smoke, whereas this procedure in other rel-
evant models was interrupted during the weekends for practical
reasons.10,11 Undetectable serum cotinine levels at 2 d after ter-
mination of the CSE period confirmed the cessation of CSE and
emphasized the importance of the uninterrupted CSE procedure.
Although demanding, our time schedule closely resembled the
human smoking habit and excluded the possibility of inducing
short-term behavioral or organic effects due to transient cessation
of nicotine intake. Evidence indicates that nicotine withdrawal
is followed by behavioral abstinence signs, which appear within
the first 24 h,23 whereas the hypothalamic expression of orexigenic
neuropeptides increases after only 3 d.17
In conclusion, we here describe a rat model of chronic CSE that
induced serum cotinine levels comparable to those of smokers
and led to the development of CPP suggestive of cigarette smoke
abuse liability. This model may be useful in the study of the mech-
anism and side effects related to tobacco smoke abuse liability
and smoking cessation.
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