Vol.3, No.10, 613-619 (2011)
doi:10.4236/health.2011.310103
C
opyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
Health
Effects of change in smoking habits on bladder cancer
incidence in Tunisia
Dhafer Mrizak1, Fatma B'chir1,2, Mehdi Jaida ne3, Maurice Jean Arnaud4, Saâd Sa guem1*
1Metabolic Biophysics and Applied Pharmacology Laboratory, Department of Biophysics, Medicine Faculty of Sousse, Sousse,
Tunisia; *Corresponding Author:
2Laboratory of Natural Substances, National Institute of Research and Physical Chemical Analysis, Technopole Sidi Thabet, Tunisia;
3Urolology Department of Central Hospital University (CHU), Sahloul-Sousse, T un isia;
4Nutrition & Biochemistry, Bourg Dessous 2A, La Tour de Peilz, Switzerland.
Received 22 January 2011; revised 19 February 2011; accepted 28, February 2011.
ABSTRACT
Bladder cancer is among the most frequently
diagnosed cancer. Tobacco smoking exposures
involving nitrosamines and aromatic amines
are the main cause of bladder cancer. Although
cigarette consumption has gradually decreased,
an increased incidence of bladder cancer was
registered among males Tunisian for the last tw o
decades. A similar increased incidence of lung
adenocarcinoma and bladder cancer was regis-
tered among Tunisian males. Epidemiological
results suggest that changes in cigarette smok-
ing may be the cause of the increased inciden-
ce of bladder cancer. The relationship between
CYP1A2 enzyme activities, a key enzyme for
activation of bladder carcinogens, and bladder
cancer risk was investigated. Variations in CYP1
A2 activities measured in patients with bladder
cancer and healthy smokers showed a signify-
cantly higher CYP1A2 metabolic activity in
patient group. Changing in cigarette smoking
habits associated to the variation in CYP1A2
activity seem to explain pa rtly the increase inci-
dence of bladder cancer observed in Tunisian
male population.
Keywords: Cigarette Filter; Smoking;
Bladder Canc er ; CYP1A2 Activity
1. INTRODUCTION
Bladder cancer is the most frequent urogenital cancer
in Tunisia and rank at the second position in the world
[1], among male populations.
Epidemiologic and biological studies have clearly es-
tablished that cigarette smoking is the main cause of
bladder cancer [2,3] and it was suggested that more than
60% of all bladder cancer cases may be attributed to
tobacco smoking [4,5].
Although stabilization and even decrease frequencies
of major cancers types which are associated to smoking
such as lung cancer and oral cavity cancer, a dramatic
increase in the incidence of bladder cancer was regis-
tered in Tunisian Cancer Centers for the last two decades.
This increasing prevalence has been also reported
worldwide [6,7] and may be the result of changes in
smoking behavior in particular when the cigarette with
filter had replaced those with no filter.
The concentrations of specific carcinogens such as toba-
cco-specific nitrosamines (TSNs) and Aromatic Amines
(AA) have been increased in filtered cigarette smoke [8].
It has been suggested that the consumption of those
cigarettes with filters may contribute to increase bladder
exposure to tobacco carcinogens thus leading to a higher
risk of cancer devel opment [8-10].
The aim of this study was to evaluate from epidemi-
ological and biological data whether the introduction of
cigarette with filter could explain the remarkable in-
crease of bladder cancer incidence observed in Tunisian
population.
2. MATERIALS & METHODS
2.1. Epidemiological Study
Data of epidemiological study were obtained from
CHU Central Tunisian hospital registers (Department of
Anatomy Pathology of CHU Farhat Hached of Sousse,
Tunisia) for the 1987-2005 time period and the evolu-
tion of bladder cancer incidence in Tunisian males was
evaluated.
2.2. Biological Study
This study was carried out at the Faculty of Medicine
of Sousse by the Biophysics laboratory where the as-
sessment of CYP1A2 enzyme activity was measured by

F. B'chir et al. / Health 3 (2011) 613-619
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
614
caffeine test among bladder cancer and healthy smoker ’s
subjects.
CYP1A2 activity was estimated in vivo using caffeine
as a probe drug and reversed–phase HPLC method was
developed to measure plasma caffeine and paraxanthine
concentrations to determine the CYP1A2 activity from
the ratio paraxanthine/caffeine.
2.3. Subjects
14 patients with bladder cancer and 14 healthy subje-
cts were recruited for this study.
All subjects are current smokers. They gave their info-
rmed consent and the study protocol was approved by
the local Ethical Committee.
All subject abstained to drink any beverages contain-
ing caffeine for more than 24 hours before and during
the study.
2.4. Methods
The subjects were asked to fill a questionnaire where the
total consumption of coffee and other caffeine-containing
beverages as well as the number daily cigarettes smoked
and the period of smoking were recorded (Table 1).
The subjects were coming at the hospital in the morn-
ing at 8 am and ingested 140 mg of caffeine disso lved in
150 ml warm water. A single 5 ml blood sample was co-
llected directly in EDTA tube four hours after caffeine
ingestion.
Blood samples were immediately centrifuged at 3000
rpm for 15 minutes and plasma samples collected were
stored at –20˚C until analyzed.
2.5. HPLC Plasma Analysis and Caffeine
Concentrations
To 0.5 ml defrost plasma was added 300 mg of ammo-
nium sulfate and after 1 minute vigorous shaking (Vor-
tex), 3 ml of ethyl acetate and isopropyl alcohol (8/2, v/v)
were added. After shaking for 2 minutes, the samples
were centrifuged at 4000 rpm for 25 minutes and 2.5 ml
of the organic phases were collected, dried at 56˚C under
a flow of nitrogen gas and the residue obtained was dis-
solved in 100 µl KH2PO4 buffer solution.
Table 1. Averages daily cigarettes smoked and smoking period
in bladder cancer patients and healthy controls.
Groups Bladder cancer
patients Healthy control
Daily cigarettes smoked 25 20
Smoking period (years) 43.5* 26.78
*P < 0.05
HPLC (Agilent 1200) separation was performed using
two reversed phase columns in series (Hypersil ODS 4 ×
250 mm, 5 µm) and (Eclipse XD B-C18 4.6 × 150 mm, 5
µm) maintained at 33˚C with a direct injection of 5 µl of
the extract. An U.V diode array was used for the analytic
detection. Elution was performed by gradient mode us-
ing two mobiles phases A and B. Phase A solution was
composed of water, tetrahydrofurane (THF), acetic acid
and acetonitrile (986/3/1/10 v/v respectively). The phase
B solution was acetonitrile. The gradient program was
chosen as follows: B was set at 0% for the 20 first min-
utes, increased linearly to 4% at 35minutes, reached to
8% from 45 to 50 minutes and return to 0% at 55 min-
utes.
Plasma caffeine and the concentrations of its metabo-
lites were calculated by CHEMSTATION software using
calibrations curves already estab lished.
2.6. Measurement of Caffeine Metabolic
Ratio
Caffeine is mainly metabolized by the hepatic CYP1A2
enzyme and is currently used as a probe for the measure-
ment of CYP1A2 activity. In the metabolism of caffeine,
the first demethylation steps gave paraxanthine (PX, 17X),
theobromine (TB, 37X) and theophylline (TP, 13X).
About 80% of the met abolism of caffeine is going through
the paraxanthine pathway mediated by CYP1A2 [11].
The measurement of the concentration ratio PX to
caffeine (PX/CA) was used as a metabolic marker to
evaluate the CYP1A2 activity.
2.7. Statistical Analysis
Mann–Whitney U test was used to examine differen-
ces in distributions of smoking period and caffeine meta-
bolic ratio between cases and controls.
3. RESULTS
3.1. Epidemiological Study
3.1.1. Ev olution of the Incidence o f Tobacco
Related Cancers in Tunisia
This study recorded the most frequent cancer associ-
ated to tobacco use in the Tunisian male population: lar-
ynx, oral cavity, bladder and lung.
The frequencies of these four cancers obtained from
CHU Cancer register of Sousse are calculated for two
periods 1987-1990 (PI) and 2002-2005 (PII). In Figure
1 is shown the evolution of the incidence curves of lung,
bladder, oral cavity and larynx cancers for the time pe-
riod 1987-2005.
Although stabilization and even a small declin e in the

F. B'chir et al. / Health 3 (2011) 613-619
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/HEALTH/
615615
Figure 1. Incidence evolution of four tobacco-related cancers
in Tunisia between the time periods PI (1987-1990) and PII
(2002-2005).
incidence was observed for lung, larynx and oral cavity
cancers, an increase of the incidence of bladder cancer
was observed for the 2002-2005 period when compared
to the 1987-1990 period. Bladder cancer frequencies
exhibited increase from 7.69% in 1990 to 13.03% in
2005 and thus doub le in 15 years.
3.1.2. Evolution of Lung Cancer and Its
Histological Forms Incidence in Tunisian
Population
According to our previous study [12], the distribution
of lung cancer incidence compared to its major histo-
logical types the adenocarcinoma (AD) and the squam-
ous cell carcinoma (SCC) was not similar for the last
two decades in Tu nisi an p op u lat i on.
As shown in Figure 2 a slow decline in the incidence
for both total lung cancer and SCC was observed from
1990 up to 2005 per iod, while a con tinuous incr ease was
registered for AD incidence.
3.1.3. Comparative Analysis of the Bladder
Cancer and AD Frequenci e s Evolution
In Figure 3 is shown the evolution of the incid ence of
AD and bladder cancer for the 1987-2005 period. The
evolution of both frequencies of bladder cancer and AD
were similar during the same period of time. Bladder
cancer and AD have gradually and simultaneous in-
creased for these last two decades in Tunisian males.
3.2. Biological Study
3.2.1. Separation of Caffeine and Its Metabolites
The Figure 4 illustrates the chromatographic profiles
of caffeine and its metabolites measured at 280nm in
plasmatic sample of one patient and one healthy subject.
The chromatogram shows a complete separation of caf-
feine and its metabolites, in particular for TP, TB and PX.
This good separation allows an accurate quantification
of caffeine metabolites concentrations and consequently
Figure 2. Incidence evolution of AD, SCC and lung cancer in
Tunisia between the time periods PI (1987-1990) and PII
(2002-2005).
Figure 3. Evolution profile of AD and bladder cancer for the
1987-2005 period.
37X: 3,7 dimethylxanthine (theobromine); 17X: 1,7-dimethylxanthine
(paraxanthine); 13X: 1,3-diméthylxanthine (theophylline), SI: Inter-
nal Standard; CA: 1,3,7-trimethylxanthine (caffeine).
Figure 4. Chromatograms of plasma samples of caffeine and
its metabolites among one patient (blue) and one control sub-
ject (red).
the determination of the PX/CA metabolic ratios. The
chromatographic profiles of plasma show that there was
no interference with endogenous compounds.
3.2.2. CYP1A2 Activity Evaluation
Variations in PX/CA ratio were observed in Figure 5
between patients and healthy subjects. These variations
suggest wide interindividual differences in CYP1A2 ac-
tivities. The average PX/CA ratio was 0.42 for the bladder

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616
Patients
Controlgroup
0,1
0,2
0,3
0,4
0,5
0,6
0,7
0,8
Plasmatic ratio PX/CA
Figure 5. Distribution of PX/CA plasmatic ratio values among
patients and control group.
cancer group and 0.3 for healthy group of subjects. The
CYP1A2 activity was thus significant higher in the
bladder cancer patient group compared to healthy group
(P < 0.05).
4. DISCUSSION
In the present study, biological and epidemiological
investigations show that the rapid increase in the inci-
dence of bladder cancer observed for the last two dec-
ades in Tunisian population may be related to the change
in smoking behavior when the cigarettes with filter had
substituted progressively those with no filter.
More than 50 years ago, tobacco use was suggested as
a major etiologic risk factor for bladder cancer [8,13]
and 60% was attributed to cigarette smoking [4]. Many
case-control studies reported that smokers have at least a
two-fold higher risk of developing bladder cancer com-
pared to non smokers [8,14]. Bladder cancer is estimated
as the second cancer localization attributed to smoking
after the lung [15].
The epidemiological results of this study show an op-
posite evolution of bladder and total lung cancer fre-
quencies for the time period 1987-2005. Despite the sta-
bility of lung cancer incidence in Tunisian males for the
1987-2005 period (Figure 3), a two-fold increased
bladder cancer frequency was registered from 7.69% in
1990 to 13.06% in 2005. This dramatic increase of
bladder cancer incidence registered in Tunisia was also
reported all over the world during the same time period.
Cancer registers show a larger increase in Denmark, in
USA and in France among men and a somewhat smaller
increase among women [16,17]. This incidence is still
increasing overtime. It has been suggested that heavy
cigarette smoking plays a major role in increasing blad-
der cancer incidence [18]. This conclusion was also con-
firmed in our work. Patients with bladder cancer are
heavier smokers that subjects in the control group (Table
1).
How can be explained the apparent disagreement be-
tween the increase incidence of bladder cancer and the
decline of the lung cancer frequency with a change in
smoking behavior. We suggested that the changes in
smoking behavior associated to the increased use of ciga-
rettes with filter may explain the changes observed [12].
In this study was reported a stabilization of the total inci-
dence of lung cancer while a continuous and significant
increase of AD over SCC forms was observed among
Tunisian population for the tim e-period 1987-2005.
Figure 6 shows that the 10.5% incidence of SCC is
unchanged while that of AD exhibits an increase from
3.1% in 1990 to 7.2% in 2005. Th is increase of AD was
related to the introduction of cigarette with filter [12].
This new smoking habit appeared to be more carcino-
genic through the increasing incidence of AD, the most
aggressive and metastasis cell types of lung carcinoma
[19]. The smoker of cigarette with filter is more exposed
to tobacco nitrosamines (TSNs) in particular the 4-me-
thylnitrosamino-1-3-pyridyl-1-butanone (NNK); a potent
carcinogen which induce lung tumors in the peripheral
lung regions, the areas where the AD occurs [12,20].
The significant increase of bladder cancer observed
during the 1987-2005 time-period parallels the evolu-
tion of AD incidence in Tunisian population suggesting
that both pathologies may have the same origin. The
most prominent change in smoking habit reported during
this period was the increased use of cigarettes with filter.
Filter cigarette deliver at least 60% less nicotine and tar
than plain cigarette [5,8].While this change leads to a
lower exposure of polycyclic aromatic hydrocarbons
(PAHs), the concentrations of aromatic amine increased
by 59% from 1968 to 1985 [8] and between 1978 and
1995, the concentrations of tobacco-specific nitrosmines
in filtered cigarettes increased 17% for N’-nitrosonorni-
cotine and 44% for 4-(methylnitrosamino)-1-(3-pyridyl)-
1-butanone [8,21]. Some N-nitroso-compounds such N NK
have been suggested to be involved as causative agents
in human lung carcinogenesis leading to the AD type
[20,22].
Figure 6. Trends of the evolution of AD and SCC in Tunisia.

F. B'chir et al. / Health 3 (2011) 613-619
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617617
Moreover, the smoker’s dependence on nicotine may
lead to smoking more cigarettes with filter because they
contain lower amounts of nicotine. To achieve the de-
sired physiological response to nicotine, smokers of low-
tar filter cigarettes tend to adapt their smoking patterns
by taking larger puffs, increasing puff frequency and
inhaling more deeply. Such changing smoking patterns
showed that although the smoker-weighted carbon mo-
noxide-yield of ventilated filter cigarettes was 21% less
than that of plain cigarettes, blood carboxyhemoglobin
level in ventilated filter cigarettes was 7% higher than
those found in men who smoked plain cigarettes [23].
These data are consistent with the conclusion that smok-
ers who are addicted to low-nicotine cigarettes may take
more intense smoking and deeper inhalation of the smo-
ke leading to a higher amount of carcinogenic tar parti-
cles, in the bronchi and bladder of the smoker [24].
Although the increasing incidences of AD and bladder
cancer may have the same origin with the use of ciga-
rette filter, they have different carcinogenic inducers.
PAHs such as benzo(a) pyrene (BaP) and TSNs like
NNK are the most known tobacco smoke potent car-
cinogens as shown on animal model [25,26]. PAHs and
NNK cause mainly lung cancers, PAHs induce the SCC
tumours occurring in bronchi and bronchioles of the
lungs and NNK induce AD predominately in the deeper
region of the lung. In animals exposed to the mainstream
cigarettes smoke develop bladder cancer while animals
treated with BaP or NNK develop cancers on other loca-
lizations than the bladder [27]. Thus, the induction of
bladder tumours by tobacco substances inhaled is not
dependent upon PAHs and TSNs, but may dependent on
other tobacco chemicals [18,28]. It was reported that
aromatic amines are potent bladder carcinogens [29].
Bladder tumours induced in animals with tobacco aro-
matic amines carcinogens [30] showed that these com-
pounds do not elicit directly bladder tumours but the
effects were due to some of their metabolites. Among
these tobacco amines carcinogens, 2-naphthylamine and
4-aminobiphenyl were found to be the most potent
bladder carcinogens in man [31]. The most direct-acting
carcinogens that are reactive electrophilic agents are
produced through in vivo activation involving an initial
oxidation catalyzed primarily by hepatic cytochromes
P450 [31], the first critical step in this bio activatio n [32,
33]. 2-Naphthylamine and the 4-aminobiphenyl are me-
tabolized mainly in liver to N-hydroxy-4-naphthylamine
(N-OH-naphthylamine) and N-OH-aminobiphenyl re-
spectively. These reactive metabolites produce human
epithelium DNA adducts in liver and bladder. The enzy-
me primarily responsible for the N-hydroxylation of 2-
naphthylamine and the 4-aminobiphenyl, the initial step
for carcinogenic process, is the CYP1A2 [31]. This key
enzyme in the activation of bladder cancer carcinogens
exhibits wide variations in activity explaining individual
susceptibility to chemical carcinogenesis and thus to
bladder cancer development.
In smokers, the results of this study show a significant
higher CYP1A2 activity in patients with bladder cancer
when compared with healthy subjects. High CYP1A2
activity is therefore suggested as a susceptibility factor
of bladder cancer. The polymorphic CYP1A2 activity
may be related to environment and genetic factors. Ex-
posure to tobacco smoke is known as a potent inducer of
CYP1A2 activity [34,35] but there are other occupa-
tional and dietary inducers. Variation in CYP1A2 activ-
ity was also related to polymorphisms in the CYP1A2
genes and consequently the effect of cigarette filter on
the risk of bladder cancer development is dependent on
this polymorphism. Further investigations on the role of
CYP1A2 polymorphisms on the risk of bladder cancer
performed on larger population of smokers are needed.
5. CONCLUSIONS
This epidemiological study suggests that the increased
incidence of bladder cancer may be attributed t o change i n
smoking habit associated to the substitution of plain ciga-
rette to cigarette with filter while the effect of smoking
duration cannot be excluded. As bladder carcinogens,
such as aromatic amines, are mainly metabolised by
CYP1A2 enzyme, a signif icantly higher CYP1A2 activity
observed in bladder cancers patients when compared to
control subjects suggests that high CYP1A2 activity
could be a susceptible factor for bladder cancer in sm-
okers. Changing in cigarette smoking habit associated to
higher enzyme activity may be considered as a potential
biomarker in the aetiology of bladder cancer and must be
confirmed on a larger study.
Although filter is effective in reducing the amounts of
PAHs and nicotine, it is less effective to retain volatile
carcinogenic compounds such as TSNs and aromatic
amines so that smokers are more exposed to these potent
carcinogens. The users of cigarette with filter increase
the smoking frequency and inhales more deeply, retain-
ing smoke longer. This lead as a consequence to a higher
deposition of carcinogens smokes in all vascular body
regions. These carcinogens undergo biotransformation to
give active carcinogens which are able to form ADN
adducts initiating the first steps of cancer process. In-
creased use of cigarette with filter associated to the po-
lymorphic CYP1A2 enzyme activity could therefore be a
main cause in the bladder cancer increase.
It appears that cigarette with filter is not less danger-
ous than the plain cigarette as expected. The new smok-
ing behaviour is involved in the increase incidence of

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618
many aggressive cancer forms such as AD of the lung
and bladder cancer. In addition, the introduction of ciga-
rette with filter lead to an increase over time in the
number of cancers localization attributed to smoking [15,
36].
Finally, cigarette with filter and more generally smok-
ing is today a major public health problem and smoking
cessation remains the primary preventive way fo r to ba c co
related-cancers and pathologies.
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