According to the world health organization (WHO), lung
cancer has been the primary cause of cancer mortality all
over the world in recent years and it is considered as the
most prevalent fatal cancer. Annually lung cancer is the
cause of about 20 percent of all deaths caused by cancer.
Reports have demonstrated an excessive increase of lung
cancer and associated mortality in the Iranian community. Lung cancer has a weak prognostic; in spite of developments related to the treatment of this disease, 5-year survival rate of lung cancer patients was less than 15 percent
(1). Tobacco smoke is the most important factor in lung
cancer and 85 to 90 percent of lung cancer mortality is
due to tobacco smoking (2, 3). Exposure to polycyclic aromatic hydrocarbons (PAHs) which are primarily found in
tobacco smoke lead to lung cancer induction (4, 5). PAHs
are among various groups of primary carcinogens, which
exist in tobacco smoke and perform locally (6, 7). Higher
levels of PAHs and DNA-aromatic adducts exist in tobacco
smokers' blood and lung tissue in comparison with nontobacco smokers and lung cancer expansion probability
in tobacco smokers is 10-20 times more (8, 9). However,
according to the undeniable role of tobacco in lung cancer, only 10 percent of individuals who are exposed to tobacco smoke for a long time are afflted to lung cancer;
therefore, other factors such as lung disease, age, gender,
nutritional and genetic factors have a determining role
in the incidence of lung cancer (2). In fact, even after controlling tobacco smoking among relatives, lung cancer
risk is inflenced by genetic diffrences, especially polymorphisms in enzymes involved in carcinogen metabolism, thus, diffrent individuals with similar amounts
of received carcinogens contain various levels of DNA
damage (10, 11). One of the metabolizing enzyme groups
of xenobiotic compounds is cytochrome P450, which is
involved in various exogenous and endogenous chemical
compound metabolisms such as carcinogens and medicines.
CYP1B1 is one of the related enzymes of CYP450, which
consists of a genetic polymorphism in a specifid population; in addition, genetic susceptibility of malignancy
affction is inflenced by enzyme genotype (12-14). CYP1B1
has a prominent role in oxidation of various kinds of carcinogen compounds such as PAHs and arylamines. CYP1B1
gene is located on chromosome 2, in the 2p21-22 region.
Length of this gene is 5.1 Kb and encodes a protein with
543 amino acids (15). There is usually a polymorphism
in the CYP1B1 gene, which causes a C to G change in 4236
nucleotides leading to a change from Leucine to Valine
amino acid, in 432 codon (CYP1B1 L432V). In this polymorphism, C allele leads to an increase in enzyme activity,
thus increasing the risk of lung cancer (15, 16). Genotypes
related to CYP1B1 L432V polymorphism is represented in
table 1; C allele encodes Leucine amino acid and G allele
encodes Valine amino acid (17).
In the present study, the effct of the mentioned polymorphism on lung cancer susceptibility in the Iranian
population is surveyed by the RFLP-PCR method.
3. Materials and Methods
3.1. Studied Population
In the current case control study, 65 lung cancer patients who were fist diagnosed by standard histopathological procedures at the Chronic Respiratory Diseases,
Research Center, Masih Daneshvari Hospital, Tehran,
Iran were investigated. Blood collection was always performed under supervision of a medical oncology specialist to avoid possible error in choosing primary lung
cancer cases (patients sampling takes 6 months). 80
cancer free volunteers, unrelated to the patients, randomly selected among those who had referred to clinics
of Masih Daneshvari Hospital for regular health checks,
formed the control group. Before blood sample collection, a structured questionnaire was completed during a brief face to face interview to obtain demographic
characteristics, such as gender, age and smoking habits.
Data were collected from the subject’s closest relative or
from the medical fie, registered by physicians at admission time for patients who were unable to participate in
an interview. Blood sampling was done based on patient
satisfaction and an agreement was signed between the
University of Isfahan and Masih Daneshvari Hospital. The
blood samples were collected in tubes containing anticoagulant ethylene diamine tetraacetic acid (EDTA) and immediately stored at –20 ºC until DNA isolation.
3.2. DNA Genotyping
Using a salting method, genomic DNA was extracted
from peripheral white blood cells (18). To analyze CYP1B1
L432V polymorphism, a distinct region with 549 bp
length including the variation site was amplifid with
the PCR technique by using forward and reverses primers (Table 2).
Each 25 µl of PCR reaction mixture consisted of 2 µl of
10 mM of each forward and reverse primers, 2.5 µl of 10x
solution buffr, 0.5 µl of a 10 µM of the four mixed dNTPs,
0.75 µl of 50 mM of MgCl2, 0.25 µl of 5 u.µl -1 Taq DNA polymerase (Cinnagene, Co., Iran), 2 µl of genomic DNA (80
ng.µl-1) and 15 µl of sterile H2O. The amplifiation reaction
using a thermal cycler was carried out at the following
conditions: initial denaturation at 95 °C for 3 min, followed by 35 cycles with melting at 95 °C for 30 sec, annealing at 62°C for 35 sec, and primer extension at 72 °C for 40
sec. A fial extension completed the PCR reaction at 72 °C
for 10 min. The amplifid products were then separated
by electrophoresis through 1% gel agarose and visualized
with ethidium bromide staining.
PCR amplifid products were digested to determine
genotypes. EcoR571 restriction enzyme (New England
Biolabs) was used to distinguish the CYP1B1 L432V polymorphism, at 37 °C overnight. This enzyme only affcted
the C allele sequence and it did not have any inflence
on the G sequence. 549bp fragment cut in the polymorphism location with EcoR571 enzyme led to the creation
of fragments with 221 and 328 bp lengths; therefore, recognition and discrimination of two alleles in the fial
PCR products was conveniently possible; also, the created
restricted and non-restricted fragments were easily recognizable and discriminated by electrophoresis.
3.3. Statistical Analyses
To compare the distribution of diffrent genotypes and
allele frequencies for the polymorphism between patient
and control groups, the chi-square test was used. The
same test was also used to determine possible signifiant
associations between lung cancer risk and CYP1B1 L432V
variant alleles. The odd ratios (ORs) and their corresponding 95% confience intervals were calculated to estimate
relative association between each CYP1B1 L432V variant allele and lung cancer. Analysis of all data was down with
the SPSS software version 16. A P value less than 0.05 was
considered statistically signifiant.
The cases and controls were nearly similar regarding
gender distribution, since males and females had almost the same proportions between the two groups. The
mean age was computed as 53.39 ± 1.12 for the patients
and 51.71 ± 1.3 for the controls. Therefore, there were no
signifiant diffrences in the distribution of age between
cases and controls (for age and gender distribution, P
values > 0.05). However, according to smoking status a
signifiant diffrence was obtained between two groups,
as compared to the healthy control group; more tobacco
smokers were present among cases with lung cancer
(P value = 0.001) (Table 3). In addition, the average duration of smoking was much more in patients than controls, and cases smoked more intensively than healthy
controls (data not shown) .
Table 4 illustrates the distribution of diffrent lung
cancer histological types. Adenocarcinoma was more
frequent in patients, while cases with small cell lung carcinoma and squamous cell carcinoma were much more
likely to have a positive smoking history, although no noticeable diffrence was observed (P value > 0.05).
4.1. RFLP-PCR Method with EcoR751 Enzyme
Regional amplifiation of CYP1B1 gene, which consists
of L432V polymorphism, has created a band with 549 bp
length. Genotypes of controls and patients have been
determined by the EcoR571 restriction enzyme on the
PCR products. From each amplifid sample, two equal
volumes of individuals’ genomic DNA was taken and
digested in separated vials, where one received no enzyme and the enzyme inflenced the other one. The fist
sample was considered as control and without digestion.
Then, control and digested genomic DNA fragments were
respectively loaded in adjacent wells and the produced
bands were separated on 1% agarose gel. Electrophoresis
was performed with 65 mv of voltage for 90 minutes. Two
bands with 221 and 328 bp lengths were achieved for CC
homozygosis and one band with 549bp lengths for GG
homozygosis. For GC heterozygosis all three 221, 328 and
549bp bands were observed on the gel (Figure1).
4.2. RFLP Results Analysis Using Image J Software
For precise assessment of enzyme digestion, bands resulting from electrophoresis were evaluated using the
Image J software. The Image J software draws two charts
for each case. The area below the chart 1 shows the light
emitted from non-restricted band and the area under
chart 2 is the result of light from restricted bands of
equal samples under the effct of enzymatic digestion.
The evaluation of one of the samples obtained from the
electrophoresis is shown Figure 2.
Measurement of the exact area under the charts gives
two numbers in the results section, which show equal
area under the charts. Thus, light emitted from nonrestricted band and resultant emitted light of restricted
bands is exactly equal and enzyme digestion was performed correctly.
4.3. CYP1B1 L432V Polymorphism Investigation of
Lung Cancer Patients and Controls
In the present study, CC, CG and GG genotypes were
observed respectively in 36.25, 52.25 and 12.50 percent
of control samples; while these genotypes were instead
present 47.70, 46.15 and 6.15 percent of all lung cancer patients. CYP1B1 L432V polymorphism genotype distribution
for lung cancer patients and controls has been represented by table 4. The comparison of the genotype distributions of the two studied groups shows that CC genotype
frequency is about 1.7 times more than GG genotype in
lung cancer patients in comparison with controls; however, diffrences in the mentioned genotypes distributions of patients and controls were not signifiant and
those genotype distributions are according to HardyWeinberg equilibrium (P > 0.05). In addition, signifiant
diffrences have not been observed in patient and control groups (P > 0.05). Table 5 compares the allelic distribution between patient and control groups.
4.4. Investigation of Tobacco Smoking Relationship with CYP1B1 L432V Polymorphism Genotypes
in Lung Cancer Patients
Patients were divided into two tobacco smoker and
non-tobacco smoker groups; then, CYP1B1 L432V polymorphism genotype distribution was compared between
these two groups (Table 6). The results showed that there
is a signifiant association between CC genotype and
lung cancer risk in tobacco smokers; in other words, CC
tobacco smokers are exposed to higher lung cancer risk
in comparison with GG genotype tobacco smokers (P >
0.029). There is no signifiant association between tobacco smoking and lung cancer risk in CG genotype (P
> 0.169). The investigation of tobacco association with
polymorphism genotype distribution showed that no tobacco smoker with GG genotype was found; therefore, OR
was not calculated (Table 7).
Lung cancer has been identifid as one of the most
prevalent and dangerous cancers. In 2005, more than
163000 individuals died as a result of lung cancer in the
U.S.A; thus, this kind of cancer has been considered as
the greatest cause of cancerous mortality (19). In Iran,
lung cancer is the fith factor leading mortality, but
lung cancer incidence has been improving in men and
women (1). Researches and studies on the risks of lung
cancer are very important due to the lack of suffient
knowledge, high mortality rate as a result of late identifiation (usually after metastasis) and expensive treatment costs. CYP1B1 enzyme has an important role in metabolism and activation of environmental carcinogens;
also, it has a dominant role in increasing the risk of lung
cancer (20, 21). On the other hand, metabolic capacity
has been affcted intensively by individual diffrences
such as genetic polymorphism of metabolized enzymes.
In recent decades, researches on various populations
represent the association between CYP1B1 polymorphism and lung cancer risk; in addition, contradictory
results have been achieved in several studies. Epidemiological studies which represent CYP1B1 L432V polymorphism role in lung cancer are restricted. Contradictory
results have been reported about this polymorphism;
for instance, leucine allele and valine allele have been
associated with an increase in the risk of lung cancer,
in some studies. Investigation of this polymorphism in
two non-tobacco smoker populations of Caucasian and
African-American races showed that there was a signifcant association between lung cancer susceptibility and
this polymorphism (CI 95% = 1.63-5.07; OR = 2.87 for at
least one valine allele) (22). In one research in Germany,
no association between CYP1B1 L432V polymorphism and
lung cancer was found; however, a signifiant increase
in primary lung cancer risk for minor allele carrier
women (G) was observed (P < 0.0001, OR = 1.97, CI 95%
= 1.32-2.94). The effct of this allele on the risk of lung
cancer is related to tobacco smoking; in other words,
women that are G allele carriers, who smoke more than
20 packs per year show a signifiant increase in primary
lung cancer risk (P < 0.0001, OR = 2.69, CI 95% = 1.49-4.84)
(14). In this study, in spite of the high frequency of CC
genotype in the patient population, there were no signifiant diffrences in two control and patient groups
regarding CYP1B1 L432V genotype distribution; therefore,
the two populations were almost the same. Generally,
the most control and patient groups have contained
heterozygous genotype and in spite of C allele higher
frequency in patients, there was no signifiant diffrences in C and G alleles distribution between the two
groups (P > 0.05); however, as it was pointed in previous
studies, C allele existence leads to the lung cancer risk
increase; in this case, CC homozygosis is more exposed
to lung cancer risk in comparison with heterozygosis.
The investigation of the relationship between genes and
tobacco in patients represents signifiant diffrences in
genotype distribution according to tobacco smoking
rate; in addition, tobacco smokers with two lue432 alleles (CC genotypes) have been shown to be at a greater
risk of lung cancer in comparison with tobacco smokers
with two val432 alleles (GG genotypes). Thus, tobacco
increases lung cancer risk in patients carrying the CC
genotypes. In summary, CYP1B1 L432V polymorphism has
an important role in lung cancer risk. Therefore, further
studies are recommended for investigation of other
related CYP1B1 gene polymorphisms, their association
with affctive genes and regulatory factors in the Iranian population.
We thank the graduate offi of University of Isfahan.
We would also like to acknowledge all the physicians and
nurses of the Masih Daneshvari Research Center.
All authors worked equally in the present study.
There is no fiancial disclosure.
This work was founded by the graduate offi and re
search department of university of Isfahan.