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Anwar Gamal Mohamed


Abstract : Anwar Mohamed Louisville

Anwar Gamal Mohamed realized that Abstract We recently demonstrated that V5+ down
regulates 2,3,7,8-tetrachlorodibenzo-
p-dioxin (TCDD)-
mediated induction of Cyp1a1 mRNA, protein, and
catalytic activity levels in Hepa 1c1c7 cells through
transcriptional mechanism. Therefore, it is important to
investigate whether similar changes occur in humans.
For this purpose, we examined the effect of V
5+ (as
ammonium metavanadate, NH
4VO3) on the expression
of aryl hydrocarbon receptor (AhR)-regulated gene;
cytochrome P450 1A1 (CYP1A1) at each step of the
AhR signal transduction pathway in human hepatoma
HepG2 cells. Our results show a significant reduction
in TCDD-mediated induction of CYP1A1 mRNA,
protein, and activity levels after V
5+ treatment in a
dose-dependent manner. Investigating the effect of co
exposure to V
5+ and TCDD at transcriptional levels
revealed that V
5+ significantly inhibited TCDD
mediated induction of AhR-dependent luciferase
reporter gene expression. Looking at the posttranscrip
tional level, V
5+ did not affect CYP1A1 mRNA
stability, thus eliminating the possible role of V
5+ in
modifying CYP1A1 gene expression through this
mechanism. On the other hand, at the posttranslational
level, V
5+ was able to significantly decrease CYP1A1
protein half-life contributing to the inconsistency
between catalytic activity and transcriptional level.
Importantly, we showed that V
5+ did not significantly
alter the heme oxygenase-1 mRNA level, thus elimi
nating any possibility that V
5+ might have decreased
CYP1A1 activity through affecting its heme content.
This study demonstrates for the first time that V
downregulates the expression of CYP1A1 at the
transcriptional, posttranscriptional and posttranslational
mechanisms in the human hepatoma HepG2 cells.
Keywords Aryl hydrocarbon receptor . Cytochrome
P450 1A1 . Vanadium . Carcinogenesis
Electronic supplementary material The online version of this
article (doi:10.1007/s10565-010-9153-7) contains supplementary
material, which is available to authorized users.
G. Abdelhamid
: A. Anwar-Mohamed : A. O. El-Kadi (*)
Faculty of Pharmacy & Pharmaceutical Sciences,
3126 Dentistry/Pharmacy Centre, University of Alberta,
Edmonton, Alberta, Canada T6G 2N8
e-mail: aelkadi@pharmacy.ualberta.ca

Read more abour dr anwar Mohamed research 

1-    Introduction:

Anwar Gamal Mohamed research shows that  The aryl hydrocarbon receptor (AhR) is a ligand
activated cytoplasmic transcription factor that belongs
to the basic-helix
loop-helix protein family (Lubet et
1984). In the absence of a ligand, AhR is associated

with two 90-kDa heat shock protein-90, the 23-kDa
heat shock protein, and hepatitis B virus X-associated
protein 2. Following ligand binding, AhR translocates
to the nucleus, dissociates from the complex, and
forms a heterodimer with the AhR nuclear translocator
(Arnt) (Hankinson
1995). The whole complex then
acts as a transcription factor that binds to a specific
DNA recognition sequence, termed the xenobiotic
responsive element (XRE), located in the promoter
region of a number of AhR-regulated genes. Among
these genes, cytochrome P450 1A1 (CYP1A1) is the
most capable of bioactivating the toxic and environ
mental contaminants polycyclic aromatic hydrocarbons
(PAHs) and halogenated aromatic hydrocarbons
(HAHs) to carcinogenic metabolites (Denison and
2003). Of interest, it has been shown that the
toxicological effects of PAHs and the more toxic
HAHs, typified by 2,3,7,8-tetrachlorodibenzo-
(TCDD), are mainly mediated through the activation of
AhR and consequently CYP1A1. In fact, a well
established link between the induction of CYP1A1
and cancer has been previously reported (McLemore et

According to Anwar Gamal Mohamed  The toxicological effects of individual AhR ligands
have been extensively studied; yet, the combined
toxicological effects of these ligands with heavy metals
such as vanadium (V
5+) are currently at large. V5+
compounds exert protective effects against chemical
induced carcinogenesis in animals, by modifying
various xenobiotic enzymes, thus, inhibiting
carcinogen-derived active metabolites generation. The
anticarcinogenic effects of V
5+ combined with its low
toxicity have made V
5+ an attractive tool for the
treatment of different cancers. Moreover, recent studies
have suggested V
5+ as an effective non-platinum metal
antitumor agent (Kostova
2009). The major difference
between platinum anticancer agents and V
5+ is that the
former react with nitrogen atoms of DNA and
preferentially react with the N-7 atom of deoxygua
nylic acid (Knox et al.
1986). This type of DNA
damage produced by platinum anticancer agents is
thought to be the major reason for their effectiveness
(Rosenberg et al.
1969). On the contrary, V5+ is
believed to mediate its anticancer effect mainly through
three different mechanisms: firstly by inactivating the
carcinogens-generating metabolizing enzymes such as
CYP1A1, secondly through affecting cell proliferation,
and lastly, through inducing cellular oxidative stress


2-    Materials and methods

DR Anwar Mohamed Louisville said Ammonium metavanadate (NH4VO3), 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT), cycloheximide (CHX), 7-ethoxyresorufin,
and protease inhibitor cocktail were purchased
from Sigma
Aldrich (St. Louis, MO). 2,3,7,8-
p-dioxin, >99% pure, was purchased from Cambridge Isotope Laboratories (Woburn,
MA). TRIzol reagent and Lipofectamine 2000 reagents
were purchased from Invitrogen (San Diego, CA). The
High-Capacity cDNA Reverse Transcription Kit and
SYBR Green PCR Master Mix were purchased from
  Applied Biosystems (Foster City, CA). Actinomycin-D
(Act-D) was purchased from Calbiochem (San Diego,
CA). Chemiluminescence Western blotting detection
reagents were from GE Healthcare Life Sciences
(Piscataway, NJ). Nitrocellulose membrane was purchased from Bio-Rad (Hercules, CA). CYP1A1 D-15
mouse anti-rat polyclonal primary antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA), anti-mouse IgG peroxidase secondary
antibody was purchased from R&D Systems (Minneapolis, MN, USA). pRL-CMV plasmid and luciferase
assay reagents were obtained from Promega (Madison,
WI). All other chemicals were purchased from Thermo
Fisher Scientific (Toronto, ON, Canada). Primers were
purchased from Integrated DNA Technologies, Inc.
(Coralville, IA) and are listed in Table

Cell culture Human hepatoma HepG2 cell line, ATCC number
HB-8065 (Manassas, VA), passages 4
15, was maintained in Dulbeccos modified Eagles medium
supplemented with 10% heat-inactivated fetal bovine
serum and 1% penicillin
streptomycin. Cells were
grown in 75-cm
2 cell culture flasks at 37°C in a 5%
2 humidified incubator.

A- Chemical treatments

 DR Anwar Mohamed Louisville  Cells were treated in serum-free medium with various
concentrations of V
5+ (251,000 µM) in the absence
and presence of 1 nM TCDD as described in figure
legends. TCDD was dissolved in dimethyl sulfoxide
(DMSO) and maintained in DMSO at
-20°C until
use. V
5+ was prepared freshly in double-deionized
water (10 mM stock). In all treatments, the DMSO
concentration did not exceed 0.05% (
Effect of V
5+ on cell viability
The effect of V
5+ on cell viability was determined
using the MTT assay as described previously (AnwarMohamed and El-Kadi
2009b). MTT assay measures
the conversion of MTT to formazan in living cells via
mitochondrial enzymes of viable cells. In brief,
HepG2 cells were seeded onto 96-well microtiter cell
culture plates and incubated for 24 h at 37°C in a 5%
2 humidified incubator. Cells were treated with
various concentrations of V
5+ (251,000 μM) in the
absence and presence of 1 nM TCDD. After 24 h
incubation, the medium was removed and replaced
with cell culture medium containing 1.2 mM MTT
dissolved in phosphate-buffered saline (PBS, pH 7.4).
After 2 h of incubation, the formed crystals were
dissolved in isopropanol. The intensity of the color in
each well was measured at a wavelength of 550 nm
using the Bio-Tek EL 312e microplate reader (BioTek Instruments, Winooski, VT).
RNA extraction and cDNA synthesis
Total RNA was isolated using TRIzol reagent
(Invitrogen) according to the manufacturer
s instructions and quantified by measuring the absorbance at
260 nm. Thereafter, first-strand cDNA synthesis was
performed by using the high-capacity cDNA reverse
transcription kit (Applied Biosystems) according to the
s instructions. Briefly, 1.5 μg of total
RNA from each sample was added to a mix of 2.0
10× RT buffer, 0.8
μl 25× dNTP mix (100 mM), 2.0 μl
10× RT random primers, 1.0
μl MultiScribeTM reverse
transcriptase, and 3.2
μl nuclease-free water. The final
reaction mix was kept at 25°C for 10 min, heated to
37°C for 120 min, heated for 85°C for 5 s, and finally
cooled to 4°C.

3-    Results

Dr. Anwar Anwar Mohamed showed that  Effect of co-exposure to V5+ and TCDD on cell viability
To determine the maximum nontoxic concentrations
of V
5+ to be utilized in the current study, HepG2 cells
were treated for 24 h with increasing concentrations
of V
5+ (251,000 µM) in the absence and presence of
1 nM TCDD. Thereafter, cytotoxicity was measured
using MTT assay. Figure
1 shows that V5+ alone at
the concentrations of 25 to 250 µM did not affect cell
viability. However, the highest concentration tested,
1,000 µM, significantly decreased cell viability to
approximately 57%. Similarly, co-exposure to V
and TCDD caused a significant decrease in cell
viability, only at the highest concentration tested
(1,000 µM), to approximately 52% (Fig.
Time-dependent effect of co-exposure to V
and TCDD on CYP1A1 mRNA

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