ROLE OF THE CYP1A2 GENE POLYMORPHISM ON
EARLY AGEING FROM OCCUPATIONAL EXPOSURE
Eshkoor SA1,2,*, Ismail P1, Rahman SA2, Moin S2, Adon MY3
*Corresponding Author: Dr. Sima A. Eshkoor, Institute of Gerontology, Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia (UPM), 43400, Malaysia; Tel.: +60-129-797-812; Fax: +60-389-472-744;
E-mail: simaataolahi@yahoo.com
page: 45
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MATERIALS AND METHODS
Permission and approval for the study were obtained
from the ethical committee of the Medical and
Health Sciences Faculty, University Putra Malaysia
(UPM), Ser-dang, Selangor, Malaysia [Reference
Number: UPM/ FPSK/PADS/T7-MJKEtikaPer/F01
(JSB-Aug (08)05]. The samples were epithelial cells
of buccal mucosa. For this project 120 mechanical
workshop workers were selected. The exposed group
included males aged 18 years and above. The considered
duration time of working in the workshops was
at least 1 year or more. Furthermore, 120 people who
were not exposed to petrochemical products such as
fruit sellers, textile shop keepers, sellers in mobile
phone shops, restaurant workers, sundry shops workers,
bank staff, photography shop workers, supermarket
staff, workers in computer centers, electronic
centers and optical examination centers, were selected
as a control group. Subjects were interviewed about
their health status, educational level, smoking habits,
alcohol consumption, work history, duration of working
at one occupation and other aspects relevant to
the study. In addition, workers were divided into two
groups, those with 5 or more years in one group and
those with less than 5 years in another group.
Respondents were asked to rinse their mouth
with water before collection of the buccal samples.
The cells were collected by scraping the inner part of
the cheeks both sides with a cytology brush. Then,
the cells were gently mixed with 0.9% sodium chloride
and phosphate buffered saline (PBS) in separate
micro-centrifuge tubes and brought to the laboratory.
The cells were treated for micronuclei (MN) test,
comet assay, real-time polymerase chain reaction (RTPCR)
and restriction fragment length polymorphism
(RFLP). The effect of polymorphism on the samples
was assessed by MN formation, comet tail length and
telomere length shortening as the biological parameters.
The methods of the MN test and comet assay
were performed according to a pattern described in
[9]. In the current study, genomic DNA was extracted
from the cells using QIAamp DNA blood MiniKit
(Qiagen, Courtaboeuf, France) and then was quantified
by Nanodrop™ 1000 spectrophotometer (Thermo
Scientific, Wilmington, DE, USA). Meanwhile, the
extracted DNA was run on a 0.7-1.0% agarose gel.
Genomic DNA was used to run RT-PCR and RFLP.
Real-Time Polymerase Chain Reaction. In RTPCR,
the reaction was performed to optimize primers
and determine a suitable annealing temperature. The
primers for telomere and 36B4 were those described in
a prior study [10]. The appropriate annealing temperature
for both primers was 56°C. The primer sequences
were: tel1 (5’-GGT TTT TGA GGG TGA GGG TGA
GGG TGA GGG TGA GGG T-3’); tel2 (5’-TCC CGA
CTA TCC CTA TCC CTA TCC CTA TCC CTA TCC CTA-3’); 36B4u (5’-CAG CAA GTG GGA AGG
TGT AAT CC-3’); 36B4d (5’-CCC ATT CTA TCA
TCA ACG GGT ACA A-3’). Real-time PCR improved
the assessment of relative telomere length
measurement. The telomere repeat copy number to
single gene copy number (T:S) ratio was detected by
using Corbett Rotor-Gene 6000 (Corbett Life Science,
Sydney, New South Wales, Australia) in a 36-well format.
The extracted DNA from buccal cells was used
in the procedure. During the reaction, each sample
was evaluated concurrently for both telomere and the
housekeeping gene 36B4. The primers were obtained
from Bioline (London, UK). For each PCR reaction,
a 25 mL volume of solution was prepared in the PCR
tube. The solution for the PCR reaction included 0.6
mL of each primer, 1 mL of Eva green (Biotium, Hayward,
CA, USA), 1 mL of DNA, 5 mL of master mix
Immomix (Bioline), and 16.8 mL of pure water. The
reaction proceeded one cycle of denaturation at 94°C
for 5 min., followed by 40 cycles at 94°C for 30 seconds,
56°C for 30 seconds, and 72°C for 50 seconds.
The melting temperature was arranged between 70°C
and 95°C. Finally, the products were resolved in 2.0%
gel to ensure amplification of the specific products.
All samples were run in triplicate for both genes, and
the threshold value was assessed. In addition, a serial
dilution of genomic DNA derived from one sample
was run to ensure having a good view of the efficiency
of the PCR reaction standard curve. The obtained data
from the samples were interpreted throughout the data
set to assess the threshold cycle (Ct) values. The T:S
ratio was calculated based on the fractional number
between the average 36B4 Ct value and the average
telomere Ct value for each sample. One sample was
used as reference in each run in triplicate for comparing
the results. The formula 2–DDwas used to calculate
the T/S ratio. For calculating, DCT = Ct (telomere) – Ct
(36B4) formula was applied. Final calculation was
based on the results of DCt(target) – DCt (reference)
formula to compare the measurements and take proper
T:S ratio. Furthermore, the RFLP method was applied
to identify the CYP1A2 gene polymorphism.
Polymerase Chain Reaction and Restriction
Fragment Length Polymorphism CYP1A2. Polymerase
chain reaction was performed to optimize
primers and obtain proper annealing temperature by
gradient PCR analysis. Primer sequences for CYP1A2
were: sense (5’-GCT ACA CAT GAT CGA GCT ATA
C-3’) and anti-sense (5’-CAG TCT CTT CAC TGT
AAA GTT A-3’). The forward and reverse primers
were selected from the published article [11]. The
solution volume in the PCR tube was 25 mL. Immomix
master mix (Bioline) containing dNTPs, Taq
polymerase, MgCl2 and a buffer were used for PCR
reactions. To prepare the product, each tube received
5 mL of immomix master mix, 0.6 mL of primer, 2 to
6 mL of genomic DNA and 12.8 to 16.8 mL of pure
water. The tubes were placed in a G-Storm Thermal
Cycler (Gene Technology Ltd, Braintree, Essex, UK)
for PCR reaction. The PCR was performed in 35 cycles.
The first cycle initiated by an incubation time at
94°C for 5 min. The reaction was then followed by a
denaturation step at 94°C for 30 seconds, annealing
at 62°C for 30 seconds, extension phase at 72°C for
45 seconds and a final extension at 72°C for 10 min.
after the last cycle. After completing amplification,
the samples were stored at 4°C until used. A negative
control without a DNA template was carried out in
every run. The specific PCR product was identified
by running 1.8 to 2.0% agarose gel electrophoresis,
and the gel was then viewed with an AlphaImager
analysis system (Alpha Innotech, San Leandro, CA,
USA). The product size was 596 bp.
Fifteen microlitres of PCR reaction product was
put aside for RFLP. First, 7.3 mL of double-distilled
water (ddH2O) was added to the PCR tube, then 1.5
mL of restriction enzyme (RE) buffer, and then 6.0
mL of amplified PCR product. Finally, 0.2 mL of each
RE including Fast Digest BslI and DdeI 10 U/mL was
added to the tube. The mixture was mixed well by gently
pipetting. The reaction mixture for each enzyme
was prepared in the discrete tubes and then incubated
on a heating block at 37°C for 16 hours. After incubation,
DdeI enzyme was inactivated by incubating
at 65°C for 20 min. on a heating block. Fast Digest
BslI enzyme inactivation could be done by phenol or
chloroform extraction. The size of the products was
then identified using agarose gel electrophoresis. The
gels used for PCR and RFLP products were 2.0 and
4.0%, respectively. Meanwhile, DNA ladders of 50
bp and 100 bp (Bioline) were used to identify the size
of the products. Finally, the gel was viewed under
UV light using the AlphaImager™ 2200 (Alpha Innotech)
system.
Statistical Analyses. All data were analyzed using
the Statistical Package for the Social Sciences
(SPSS, Chicago, IL, USA) software version 16.0.
The statistical tests were independent t-test and the Mann-Whitney U-test. Micronuclei frequency and
DNA damage tail length were tested using the non
parametric Mann-Whitney U-test. The analysis method
of the relative telomere length was the independent
t-test. The Hardy-Weinberg equilibrium was evaluated
by using the c2 to test the rightness of the fit between
adjusted samples. The critical level for rejection of
the null hypothesis (two-tailed test) was the p value
of 5.0% (p = 0.05).
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