ARTICLE
Auteur(s) : Slah
Ouerhani1, Raja Marrakchi3, Rym
Bouhaha3, Mohamed Riadh Ben Slama2, Mohamed
Sfaxi2, Mohsen Ayed2, Mohamed
Chebil2, Amel Ben Ammar El Gaaied3
1Laboratoire de génétique, immunologie et pathologies
humaines, Faculté des Sciences, El Mannar I, 2092 Tunis,
Tunisia
2Charles Nicole Hospital, Department of Urology, Tunis,
Tunisia
3Faculty of Sciences, Laboratory of Genetic, Immunology
and Human Pathology, Faculty of Sciences, Tunisia
It is well established that cigarette smoking and occupational
or environmental exposure to chemical carcinogens are the strongest
known risk factors in bladder cancer development [1, 2]. Once
introduced into the organism, these xenobiotic are bio-transformed
by several enzymes. The xenobiotic-metabolising machinery includes
oxidative enzymes (phase I), which may generally activate
compounds to become carcinogenic, and phase II conjugating
enzymes, considered mainly protective since they detoxify a number
of reactive chemical carcinogens [3]. The activation process is
mainly controlled by the large family of cytochrome P450 (CYP)
oxidative enzymes [4]. The superfamily of cytochrome P450 enzymes
catalyse oxidation of a large number of endogenous and exogenous
chemicals such as polycyclic aromatic hydrocarbons and aromatic
amines. Therefore, inheritance of polymorphic CYP metabolizing
enzymes is likely to be an important determinant of interindividual
variations in susceptibility to cancer [5]. Among phase I
enzymes; the CYP2D6 enzyme has focused most interest due to the
involvement of this protein in the metabolism of drugs such as
antiarhythmics, antihypertensives, 4-blockers, monoamine oxidase
inhibitors, morphine derivatives, antipsychotics and tricyclic
antidepressants [6] as well as nicotine [7]. Furthermore,
carcinogenic DNA adducts have been shown to be reduced in
individuals who do not express functional CYP2D6 protein [8]. The
CYP2D6 gene is located on the chromosome 22 (22q13.1) consisting of
9 exons [9] with more than 80 identified alleles [10]. The most
common polymorphism is the CYP2D6*4 variant affecting the site of
BstNI enzyme and was frequently encountered in all populations
studied [11, 12]. This mutation results in the decrease or the loss
of CYP2D6 activity. Allelic variations of CYP2D6 have been
investigated with respect to incidence of several cancers, such as
bladder cancer, breast cancer, head cancer and neck cancer [11-14].
The association between these alleles and cancer development is
rather complicated and a great heterogeneity of the results was
noticed [13, 15-18]. This heterogeneity is mainly due to ethnic
difference and to the techniques used for identification of CYP2D6.
In Tunisia, bladder cancer was the most prevalent cancer of the
urogenital tract and the second most frequent cancer affecting men
[19]. A recent study has shown that in Tunisian population, the
GSTM1*0 variant, a phase II enzyme, was implicated in bladder
cancer development [20]. However, in this population, there is no
study on the role of phase I enzymes in bladder cancer
development. Thus, in this study, we wanted to address the
relationship between CYP2D6*4, the most frequent variant of CYP2D6,
and bladder cancer susceptibility in a case-control study.
Materials and methods
The study was performed in patients from the urology department of
the Charles Nicolle hospital from Tunis, Tunisia. Eighty patients
with transitional cell cancer (TCC) of bladder cancer and
109 healthy volunteers, who served as controls for genetic
characterization, were included in the present study. In total,
91.96% of the patients were men. The mean age for patients at
diagnosis was 69.45 ± 7.67. Among the patients, 80% were smokers
and 23.75% of them (19/80) were exposed to known professional risk
factors. Among anatomopathologically confirmed cases (74/80),
24.32% (18/74) were with invasive tumour and 27.02% (20/74) were
with an advanced grade tumours (G3). The control group,
characterized by the absence of malignant disease, was similar to
the cases group according to the sex’s proportion, the age average
and the tobacco using. After giving informed consent, peripheral
blood samples were collected from all patients and healthy
volunteers into tubes with EDTA.
Genomic DNA was extracted from leukocytes by a phenol–chloroform
procedure [21]. The quality of genomic DNA was controlled by
electrophoresis on 1% gel stained with ethidium bromide. Genomic
DNA (50 to 100 ng) was amplified in a final volume of 20μl,
containing 5 pmols of each primer, 4 μl of 5X PCR Buffer
(+ MgCl2), 0.2 mM of each dNTP and 1.5 U of Go TaqTM DNA
polymerase (Promega). The PCR conditions were 5 minutes
denaturation at 95 °C, followed by 30 cycles at
94 °C for 1 minute, 60 °C for 1 minute,
72 °C for 1 minute and a final extension at 72 °C
for 10 minutes. The PCR products were incubated with BstOI
restriction enzyme. The digestion products were separated
electrophoretically on 1.5 agarose gel and visualized by UV
radiation after ethidium bromide staining. The presence, or the
absence, of BstNI polymorphism site in CYP2D6 gene allows
distinguishing between the CYP2D6*4 allele and others variants. The
wild allele (CYP2D6*1) is assumed when CYP2D6*4 was not found. In
the CYP2D6*4/ CYP2D6*4 genotype, the 1934 G>A transition affects
a splice site sequence and a unique fragment of 334 bp is
observed. Heterozygous individuals display three bands
corresponding to the restricted normal allele and to the CYP2D6*4
variant.
Statistical analysis
The relative risk associated with certain alleles or genotypes was
estimated by calculating the odds ratio (OR) with CI95% at the 0.05
significance level [22]. All statistical analyses were performed
using Epi info (version 6.0).
Results
PCR amplification followed by BstOI digestion and electrophoresis
was performed. CYP2D6*4 allele frequency was respectively 8.25% in
control group and 10.62% in bladder cancer cases. The comparison of
CYP2D6*4 frequencies in all groups of patients and controls, did
not show a significant statistic difference (p = 0.43, OR = 0.76;
CI95% = 0.36-1.60) (table 1). With
considering the tobacco status of patients, the CYP2D6 polymorphism
did not appear to be a factor affecting bladder cancer
susceptibility (table 2). Indeed,
alleles frequencies between smokers and non smokers patients did
not differ significantly (p = 0.07, OR = 0.84; CI95% = 0.18-3.44).
To investigate the association between CYP2D6*4 variant and clinic
characteristics of bladder tumours, CYP2D6*4 was distributed and
analysed according to tumours stage and grade. The comparison of
CYP2D6*4 frequencies in two groups of patients with low and high
grade, did not show a statistically significant difference (data
not shown). In the subgroup of patients with superficial tumours, a
higher frequency of CYP2D6*4 variants compared to patients with
invasive tumours (p < 0.05) lead to a low value of odds ratio
(table 3). This results suggest a
protective role of CYP2D6*4 variant against bladder cancer
severity. As the consequence the heterozygous genotype was
overrepresented among patients with superficial tumours
comparatively to those with invasive tumours. However, the
corrected “p value” is higher than 0.05 which did not show a
statistically significant difference.
Table 1 Genotype and gene distribution for CYP2D6
polymorphisms in all cases and controls from Tunisia
|
Genotype
|
Controls (%)
|
Patients (%)
|
P
|
OR
|
CI95%
|
|
CYP2D6*1/CYP2D6*1
|
94 (86.23)
|
63 (78.75)
|
-
|
1*
|
-
|
|
CYP2D6*1/CYP2D6*4
|
12 (11)
|
17 (21.25)
|
0.06
|
2.11
|
0.88-5.10
|
|
CYP2D6*4/CYP2D6*4
|
3 (2.75)
|
0 (0)
|
0.19
|
0
|
0.0-4.13
|
|
CYP2D6*4
|
18 (8.25)
|
17 (10.62)
|
0.43
|
0.76
|
0.36-1.60
|
Table 2 Risk of bladder cancer from CYP2D6 genotypes by
smoking status
|
Genotype
|
Tobacco status
|
P
|
OR
|
CI 95%
|
|
No smokers
|
Smokers
|
|
CYP2D6*1/CYP2D6*1
|
13
|
50
|
-
|
1*
|
-
|
|
CYP2D6*1/CYP2D6*4
|
3
|
14
|
0.078
|
0.82
|
0.16-3.77
|
|
CYP2D6*1
|
29
|
114
|
-
|
1*
|
-
|
|
CYP2D6*4
|
3
|
14
|
0.07
|
0.84
|
0.18-3.44
|
Table 3 Distribution of the CYP2D6 genotypes in the
examined groups
|
Genotype
|
Superficial tumours (%) (N = 56)
|
Invasive tumours (%) (N = 18)
|
P
|
OR
|
CI 95%
|
|
CYP2D6*1/CYP26*1
|
71.42 (40)
|
94.45 (17)
|
-
|
1*
|
|
|
CYP2D6*1/CY2D6*4
|
28.58 (16)
|
5.55 (1)
|
0.043
|
0.15
|
0.01-1.22
|
|
CYP2D6*1
|
85.71 (96)
|
97.22 (35)
|
-
|
1*
|
|
|
CYP2D6*4
|
14.29 (16)
|
2.78 (1)
|
0.046
|
0.17
|
0.01-1.31
|
Discussion
The epidemiologic studies have shown the importance of the
environmental components (tobacco, professional exposure, urinary
infections....) on bladder cancer occurrence [1]. The elimination
of these products is carried out by enzymes metabolising the
xenobiotics. Genetic polymorphisms affecting these enzymes can
modify their activity with an effect in individual susceptibility
for cancers [23]. The CYP2D6*4 variant is frequently encountered in
all studied populations and is characterized by the decrease of
CYP2D6 enzyme activity. In this study the CYP2D6*4 allele was
analysed in 109 healthy controls and 80 bladder cancer
cases. In our cohort CYP2D6*4 allele was observed at only 8.25% of
the control population. This value deviates from that observed in
other populations estimated at 20.8% [14, 24]. The CYP2D6*4 variant
was present preferentially in the heterozygous state, indeed only
2.75% of control population carried the homozygous genotype
CYP2D6*4/CYP2D6*4. In the bladder cancer group, the CYP2D6*4 allele
is present at 10.62%. This frequency did not differ to that
reported in the control group. The same result was observed when we
stratified patients according to these genotypes. This finding is
similar to other studies which did not show any association between
CYP2D6 genotype and cancers occurrence [15, 17]. Conversely, Anwar
et al. [13] found an increased risk of bladder cancer in
individuals with wild homozygous genotype (CYP2D6*1/CYP2D6*1).
Moreover, the study of kaisary et al. [25] has shown an association
between rapid debrisoquine metabolism and aggressive form of
bladder cancer. The mechanism by which this enzyme induces
carcinogenesis is essentially by forming active compound which
directly interact with DNA and induce carcinogenesis. Indeed, the
study of Romekes et al. [26] indicates that activation of
procarcinogens by the wild CYP2D6 enzyme can be associated with
Retinoblastoma mutations. The distribution of the CYP2D6*4 variant
in bladder cancer group, with considering the tobacco status, did
not show an interaction between this polymorphism and tobacco use.
This result is similar to other findings which didn’t report any
interaction between tobacco use and CYP2D6 polymorphism [27]. We
can explain this result by the fact that only the nicotine is
metabolized by the CYP2D6 enzyme [7] however the other major
tobacco procarcinogens, such as PAH and aromatic amine, are mostly
activated by the CYP1A enzyme [28]. Conversely the recent study of
Sobti et al. [29] indicates a significant association between
CYP2D6 genotype and bladder cancer occurrence in heavy smokers with
an OR of 2.13. At the same way, the study of Saarikoski et al. [30]
indicates that the ultarapid metabolizer genotype of CYP2D6 can
increase the risk of bladder cancer in smokers. The genotype
distribution according to the tumour stage reveals an eventual
protective effect of the heterozygous genotype-results which must
be confirmed by elevating the number of cases-which was most
present in patients with superficial tumours. However patients
without CYP2D6*4 variants could had an elevated risk of bladder
cancer invasion. This suggests that subjects who are homozygous for
the wild-type CYP2D6 gene (CYP2D6*1/CYP2D6*1) may have higher
enzyme activities leading to higher body burden of reactive
metabolites and to have higher cancer risk than those with
heterozygous genotypes. This observation is consistent with reports
indicating that subjects who were heterozygous had reduced (about
half) oxidative capacity compared to homozygous wild genotype. So
the xenobiotic metabolism, via wild enzyme, increases the quantity
of electrophilic active products. Once activated, these products
have the capacity to adduct to the DNA molecules. This process
induces local somatic mutations in oncogenes and/or anti-oncogene,
initiating tumoral progression. Indeed the study of Romkes et al.
[26], has shown that an environmental procrcinogen fails to be
detoxified by CYP3A enzyme may preferentially induce p53 mutations.
P53 alterations are mostly associated with invasive forms of
bladder cancer, and more than 50% of these tumours were p53 mutated
[31] which increase the DNA instability in advanced tumours.
Patients with superficial tumours and homozygous wild genotype had
more risk to progress to invasive form. So to evaluate the effect
of CYP2D6 polymorphism in bladder cancer severity, we think that
the study of p53 mutations would very important. In this study only
one polymorphism were analysed. In spite of this limit we were able
to find an eventual protective role of CYP26*4. But we think that
is very important to remember that the xenobiotic metabolizing
process implies, in addition to the CYP2D6 enzyme (phase I),
other CYPs and also the phase II enzymes [32]. Indeed an
enzymatic defect, especially concerning GSTM1 and NAT2 genes, was
associated with an increased risk of bladder cancer initiation and
progression [20, 29]. With respect to this, the conclusion
concerning the polymorphism studied should be taken cautiously and
would be studied in association to others polymorphisms.
Conclusion
This study does not show an association between the CYP2D6*4
variant and bladder occurrence. However the correlation with the
tumour stage reveals an eventual positive association between the
wild variant and invasive tumours. This preliminary result could be
confirmed by increasing the number of cases and by analysing other
polymorphism in CYP2D6 gene and in other phase I and phase II
genes.
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