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Anwar Anwar Mohamed


1-   ABSTRACT: Anwar Gamal Mohamed Louisville


Anwar Gamal Mohamed realized that We have previously shown that the p38 MAPK inhibitor SB203580 (SB) significantly
Cyp1a1 gene expression at the mRNA and activity levels, whereas it dramatically inhibited
the induction of Cyp1a1 by TCDD in murine hepatoma Hepa 1c1c7 cells. However, the molecular
mechanisms involved were not investigated yet. Therefore, the current study aims to examine the
capacity of SB to induce the constitutive
CYP1A1 gene expression in Hepa 1c1c7 and HepG2 cells
and to explore the mechanisms involved. Our results showed that SB induced the Cyp1a1 mRNA,
protein, and activity levels in a concentration-dependent manner in Hepa 1c1c7 cells. The increase
in Cyp1a1 mRNA by SB was completely blocked by the transcriptional inhibitor, actinomycin D, implying that SB increased
de novo RNA synthesis. In addition, the lack of Cyp1a1 induction by SB in mutant aryl hydrocarbon receptor (AhR)-deficient C12 cells and with cotreatment with the AhR antagonist, R-naphthoflavone, clearly suggests an AhR-dependent induction. This was further supported by the ability of SB to induce Cyp1a1 independent from its effect on MAPKs, and to bind to and activate AhR transformation and its subsequent binding to the xenobiotic responsive element (XRE). This is the first demonstration that the p38 MAPK inhibitor, SB can directly bind to and activate AhR-induced Cyp1a1 gene expression in an AhR-dependent manner and represents a novel mechanism by which SB induces this enzyme.



2-                   INTRODUCTION

Anwar Anwar Mohamed research shows that  The cytochrome P450 1A1 (CYP1A1) is a monooxygenase
enzyme that is involved in a number of cellular functions such as
the metabolism of xenobiotics.
1 CYP1A1 has been shown to be
responsible for the bioactivation of a variety of environmental
carcinogens such as polycyclic aromatic hydrocarbons (PHAs) to
epoxide and diol-epoxide intermediates.
2 The biochemical and
carcinogenic e
ffects of PAHs are primarily initiated by binding to
and activation of a cytosolic ligand-activated transcription factor,
the aryl hydrocarbon receptor (AhR). Mechanistically, upon binding with its ligands, such as 2,3,7,8-tetrachlorodibenzo-
(TCDD), AhR dissociates from its inhibitory proteins
3,4 allowing
it to translocate to the nucleus, where it heterodimerizes with a
nuclear transcription factor protein called the AhR nuclear translocator (ARNT).
5 The heterodimeric AhR-ARNT complex then binds to specific DNA recognition sequences, GCGTG, within
the xenobiotic responsive element (XRE) located in the promoter
region of all AhR-dependent genes, including
CYP1A1.68 Although the classical AhR ligands and CYP1A1 inducers such as PAHs are structurally similar and share several physiochemical properties, recent findings have demonstrated the structural diversity of CYP1A1 inducers.9 Consequently, activation of AhR is not
just restricted to these compounds, in that a large number of
newly identi
fied AhR ligands whose structures and physiochemical properties significantly differ from those of PAHs have been previously reported.10,11 Although the majority of these nonclassical
AhR ligands are weak CYP1A1 inducers and possess a low probability of human exposure, this list has expanded to include a number of widely prescribed drugs such as omeprazole,
12 primaquine,13 and
The AhR has been identified as a target of several signaling
pathways that cross-talk with its own regulatory pathway, such as
proteasomal degradation,
15 redox-sensitive transcription factors,16
and the mitogen-activated protein kinases (MAPKs).17 Among
those MAPKs, p38 MAPKs are important enzymes involved in
cellular signaling, apoptosis, carcinogenesis, and in the pathogenesis of variety of diseases.
17 The pyridinyl imidazole SB203580 (SB) (Figure 1) has been reported to be a potent and selective
inhibitor of p38 MAPK and hence has become the pharmacological inhibitor of choice for assessing the role of p38 MAPKs in mediating biological processes, including the AhR pathway.
In this regard, several previous studies have investigated the effect
of SB on the AhR-CYP1A1 pathway. In particular, it has been
reported that SB signi
ficantly suppressed CYP1A1 gene induction by TCDD through the p38 MAPK-independent pathway in different mammalian cell lines, such as murine hepatoma Hepa
18,20 human hepatoma HepG2,18 and monkey fibroblast
kidney COS-7
19 cells. Unfortunately, none of these previous studies


3-                   MATERIALS AND METHODS

According to Anwar Gamal Mohamed Materials. 7-Ethoxyresorufin, Dulbeccos modified Eagles medium
(DMEM), antigoat IgG peroxidase secondary antibody, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1
H-imidazole (SB203580),
and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
were purchased from Sigma Chemical Co. (St. Louis, MO). 2,3,7,8-
p-dioxin, >99% pure,was purchased from Cambridge
Isotope Laboratories (Woburn, MA). 2,3,7,8-Tetrachlorodibenzofuran
(TCDF) and [
3H]-TCDD (13 Ci/mmole) were obtained from Dr. Safe
(Texas A&M University). Amphotericin B and resorufin were purchased
from ICN Biomedicals Canada (Montreal, QC). TRIzol reagent and
lipofectamine kits were purchased from Invitrogen Co. (Grand Island,
NY). The High Capacity cDNA Reverse Transcription kit and SYBR
Green PCR Master Mix were purchased from Applied Biosystems
(Foster city, CA). The nitrocellulose membrane was purchased from
Bio-Rad Laboratories (Hercules, CA). Cyp1a1 goat polyclonal primary
antibody and goat anti-ARNT antibody were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Chemiluminescence Western
blot detection kits were obtained from GE Healthcare Life Sciences
(Piscataway, NJ). Actinomycin D (Act-D) was purchased from Calbiochem (San Diego, CA). Poly(dI.dC) were purchased from Amersham
Canada (Oakville, ON). [
γ-32P]ATP was supplied by the DNA Core
Services Laboratory, University of Alberta (Edmonton, AB). All other
chemicals were purchased from Fisher Scientific Co. (Toronto, ON).
Biohazard Precaution. TCDD is toxic and a likely human carcinogen. All personnel were instructed as to safe handling procedures.
Lab coats, gloves, and masks were worn at all times, and contaminated
materials were collected separately for disposal by the Office of Environmental Health and Safety at the University of Alberta.
Animals and Ethics. All experimental procedures involving animals were approved by the University of Alberta Health Sciences Animal
Policy and Welfare Committee. Male Hartley guinea pigs weighing
250300 g were obtained from Charles River Canada (St. Constant,
QC, Canada). All animals were exposed to 12 h of light and 12 h of
darkness daily and given free access to food and water.
Cell Culture and Treatments. Murine hepatoma Hepa 1c1c7
wild-type (WT), AhR-deficient murine hepatoma (C12), and human
hepatocellular carcinoma HepG2 cells (American Type Cell Cutler,
Manassas, VA) were maintained in DMEM, without phenol red


4-                   RESULTS

Dr. Anwar Gamal Mohamed Archives said that Effect of SB on Hepa 1c1c7 Cell Viability. To determine the
maximum nontoxic concentrations of SB to be utilized in the
current study, Hepa 1c1c7 cells were exposed for 24 h to increasing
concentrations of SB (1, 5, 10, 20, and 40
μM). The MTT assay
showed that the concentrations ranging from 1 to 20
μM did not
affect cell viability. However, the concentration of 40
μM decreased
cell viability by 30% (Figure 2). On the basis of these findings, SB
concentrations 1, 5, 10, and 20
μM were utilized in all subsequent
Concentration-Dependent Induction of Cyp1a1 Gene expression by SB in Hepa 1c1c7 WT Cells. To determine the
capacity of SB to alter the expression of the
Cyp1a1 gene, we
examined the effect of SB on Cyp1a1 mRNA, protein, and activity
levels in Hepa 1c1c7 cells. For this purpose, Hepa 1c1c7 WT cells
were incubated for the indicated time points with increasing
concentrations of SB (1, 5, 10, and 20
μM) or TCDD (1 nM),
as a positive control. Figure 3A shows that SB induced Cyp1a1
mRNA in a concentration-dependent manner as determined
by RT-PCR. The submaximal induction was achieved at a concentration 10
μM (3-fold), whereas the highest concentrations tested, 20 μM, increased Cyp1a1 mRNA by approximately 6-fold,
which was approximately 50% of what was observed with TCDD
(12-fold). To further examine whether the induction of Cyp1a1 mRNA
in Hepa 1c1c7 WT cells in response to SB treatment is translated
into functional protein and catalytic activity, Hepa 1c1c7 cells
were treated for 24 h with increasing concentrations of SB or
TCDD; thereafter, Cyp1a1 protein and catalytic activities were
determined by Western blot analysis and EROD assay, respectively. Figure 3B and C shows that in a pattern similar to what was observed with mRNA, SB induced Cyp1a1 protein and catalytic
activity in a concentration-dependent manner. The maximal
inductions of Cyp1a1 protein and catalytic activity were observed
at 20
μM (Figure 3).
Induction of CYP1A1 Gene expression by SB in HepG2
In order to examine the relevance of SB-associated inductions
of murine
Cyp1a1 gene to humans, HepG2 cells were incubated
for the indicated time points with only a single concentration of
SB (10
μM) that showed submaximal induction in Hepa 1c1c7
cells. CYP1A1 mRNA and catalytic activity were then determined using RT-PCR and EROD assays, respectively.

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