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Anwar Gamal Mohamed Louisville


      ABSTRACT: Anwar GaMAL Mohamed 

          Anwar Gamal Mohamed research shows that The use of doxorubicin (DOX) is limited by significant cardiotoxic

ity, nephrotoxicity, and hepatotoxicity. We have previously shown
that DOX cardiotoxicity induces several cardiac cytochrome P450
(P450) enzymes with subsequent alteration in P450-mediated ara
chidonic acid metabolism. Therefore, in the current study, we
investigated the effect of acute DOX toxicity on P450 expression
and arachidonic acid metabolism in the kidney and liver of male
Sprague-Dawley rats. Acute DOX toxicity was induced by a single
intraperitoneal injection (15 mg/kg) of the drug. After 6 and 24 h,
the kidneys and livers were harvested, and several
P450 gene and
protein expressions were determined by real-time polymerase
chain reaction and Western blot analyses, respectively. Kidney and
liver microsomal protein from control or DOX-treated rats was
incubated with arachidonic acid, and its metabolites were deter
mined by liquid chromatography-electron spray ionization-mass
spectrometry. Our results showed that acute DOX toxicity caused
an induction of CYP1B1 and CYP4A enzymes and an inhibition of
CYP2B1 and CYP2C11 in both the kidney and liver. CYP2E1 was
induced and soluble epoxide hydrolase (sEH) was inhibited in the
kidney only. In addition, DOX toxicity caused a significant increase
in epoxyeicosatrienoic acids formation in the kidney and a signif
icant increase in 20-hydroxyeicosatetraenoic acid formation in
both the kidney and the liver. In conclusion, acute DOX toxicity
alters the expression of several P450 and sEH enzymes in an
organ-specific manner. These changes can be attributed to DOX
induced inflammation and resulted in altered P450-mediated ara
chidonic acid metabolism.


 According to Anwar Gamal Mohamed  ,  Doxorubicin (DOX) could be a potent anthracycline cancer drug

used to treat a large type of malignancies. However, the clinical

use of this agent is restricted by a big dose-dependent cardiotoxicity, which can accomplish end-stage failure (Outomuro et al., 2007). additionally to DOX-induced cardiotoxicity, it

also causes nephrotoxicity and hepatotoxicity (Injac et al., 2008;

Bulucu et al., 2009). DOX-induced nephrotoxicity causes increased  capillary porosity and capillary vessel atrophy (Injac et al.,

2008). though the precise mechanism of DOX-induced nephrotoxicity has not been totally elucidated, it's thought to be mediate

through DOX-induced aerophilous injury (Liu et al., 2007). Likewise, DOX-induced hepatotoxicity is usually mediate through

the generation of free radicals (Bulucu et al., 2009). additionally to

oxidative injury, DOX toxicity has been shown to induce inflammatory changes within the heart, kidney, and liver tissues of DOXadministered rats (Deepa and Varalakshmi, 2005).

We have shown antecedently that DOX-induced cardiotoxicity induces soluble epoxide hydrolase (sEH) and several other haemoprotein P450

(P450) enzymes within the heart of male Sprague-Dawley (SD) rats as

well as within the heart-derived H9c2 cells with succeeding alteration of

P450-mediated arachidonic acid metabolism (Zordoky and El-Kadi,

2008; Zordoky et al., 2010). additionally, it's been recently according

that DOX activates the aryl organic compound receptor (AhR) with succeeding induction of CYP1A1 (Volkova et al., 2011). On the opposite hand,

administration of DOX to rats and mice has been shown to decrease

the chemical process activity of many viscus P450 enzymes, though it

does not cause mechanism-based inactivation of those enzymes (Di

Re et al., 1999). Therefore, the impact of DOX on P450 enzymes appears

to be tissue- and enzyme-specific.

P450 enzymes play a vital role in arachidonic acid metabolism additionally to the Cox and also the lipoxygenase pathways

(Roman, 2002). though the role of P450-derived arachidonic acid

metabolites within the vessel physiology and pathophysiology






Dr. Anwar Gamal Mohamed Archives said that grabbed the most important scientific attention (Elbekai and El-Kadi, 2006),

their roles within the urinary organ and liver can not be unheeded. urinary organ and internal organ P450 epoxygenases metabolise arachidonic acid to totally different epoxyei

cosatrienoic acid (EET) regioisomers, whereas P450 hydroxylases

metabolize it to hydroxyeicosatetraenoic acids (HETEs) (Roman,

2002; Sacerdoti et al., 2003). moreover, sEH enzyme, which cat

alyzes the conversion of EETs to the less biologically active dihy

droxyeicosatrienoic acids (DHETs), is additionally extravagantly expressed in

the urinary organ and liver in each human and experimental animals (Enay

etallah et al., 2004; Zordoky et al., 2008).



Several investigators have addressed  the role of P450-derived ara

chidonic acid metabolites in urinary organ and internal organ perform (Maier and

Roman, 2001; Sacerdoti et al., 2003). In general, it's been shown

that EETs dilate the preglomerular arterioles, whereas 20-HETE elic

its a vasoconstricting impact in these blood vessels (Zhao and Imig,

2003). However, each EETs and 20-HETE have a diuretic drug impact

through inhibition of metal biological process within the proximal tube

(Moreno et al., 2001). still, very little info is understood regarding

the role of those eicosanoids within the liver (Sacerdoti et al., 2003). In

contrast to the urinary organ, 11,12-EET has been shown to possess a vaso

constrictive impact on the portosinusoidal circulation in rat, whereas

20-HETE showed a weaker vasoconstricting impact that was cycloox

ygenase-dependent (Sacerdoti et al., 2003). additionally, EETs were

shown to be concerned in vasopressin-induced glycogenolysis in rat

hepatocytes (Yoshida et al., 1990).



Anwar Gamal Mohamed Archives said that Several studies have rumored the modulation of urinary organ and he

patic P450-mediated arachidonic acid metabolism in many patho

physiological and experimental conditions, most notably fast,

increased dietary -linolenic acid, and inflammation (Qu et al.,

1998; Yu et al., 2006; Anwar-Mohamed et al., 2010; Theken et al.,

2011). However, there's very little info regarding the impact of DOX

toxicity on the expression of P450 enzymes within the urinary organ and liver.

Therefore, within the gift study, we have a tendency to investigated the impact of acute

DOX toxicity on the expression of urinary organ and internal organ P450 and sEH

enzymes. additionally, we have a tendency to tried to explore the mechanism by

which acute DOX toxicity might alter P450 expression. Finally, we

evaluated the impact of DOX toxicity on P450-mediated arachi

donic acid metabolism (Santa Cruz, CA). alternative chemicals were purchased from Thermo Fisher

Scientific (Waltham, MA).


Animals. All experimental procedures involving animals were approved by

the University of Canadian province Health Sciences Animal Policy and Welfare Com

mittee. Male Mount Rushmore State rats consideration 250 to three hundred g were obtained from Charles

Canada (St. Constant, QC, Canada). Animals were treated intraperitoneally

with one fifteen mg/kg dose of DOX (n  12). Weight-matched controls

received constant volume of traditional saline (n  12). Animals were euthanized

under inhalation anesthetic physiological state at half-dozen and twenty four h once the treatment. All animals were allowed free access to food and water throughout the treatment amount. The amount of food consumed by every animal was recorded, and therefore the animals were weighed before and twenty four h once DOX administration. The kidneys and livers were excised, straight off frozen in N, and keep at 80°C till



RNA Extraction and desoxyribonucleic acid Synthesis. Total RNA from the frozen tissues

was isolated victimisation TRIzol chemical agent (Invitrogen, Carlsbad, CA) consistent with themanufacturer’s directions and quantified by measure the absorbance at 260

nm. RNA quality resolve by measure the 260:280 magnitude relation. Thereafter,

first-strand desoxyribonucleic acid synthesis was performed by victimisation the High-Capacity desoxyribonucleic acid

Reverse Transcription Kit consistent with the manufacturer’s directions. In

brief, 1.5 g of total RNA from every sample was superimposed to a mixture of two.0 l of

10 polymerase buffer, 0.8 l of twenty five dNTP combine (100 mM), 2.0 l

of ten polymerase random primers, 1.0 l of MultiScribe reverse

transcriptase, and 3.2 l of nuclease-free water. the ultimate reaction combine was

kept at 25°C for ten min, heated to 37°C for a hundred and twenty min, heated for 85°C for five s,

and finally cooled to 4°C.



Quantification by period PCR. chemical analysis of specific

mRNA expression was performed with period PCR, by subjecting the

resulting desoxyribonucleic acid to PCR amplification victimisation 96-well optical reaction plates in

the ABI Prism 7500 System (Applied Biosystems). The 25-l reaction combine

contained zero.1 l of one0 M forward primer and zero.1 l of one0 M reverse primer,

12.5 l of SYBR inexperienced Universal MasterMix, 11.05 l of nuclease-free water,

and 1.25 l of desoxyribonucleic acid sample. The primers employed in this study were

chosen from antecedently revealed studies (Anwar-Mohamed et al., 2010) and

are listed in Table one. No-template controls were incorporated onto constant

plate to check for the contamination of any assay reagents. associate degree optical adhesive

cover was wont to seal the plate; thenceforth, thermocycling conditions were

initiated at 95°C for ten min, followed by forty PCR cycles of denaturation at

95°C for fifteen s and annealing/extension at 60°C for one min. Dissociation curves

were performed by the top of every cycle to verify the specificity of the

primers and therefore the purity of the ultimate PCR product.



Materials and Methods


 Anwar Gamal Mohamed Louisville said that  Materials. The High-Capacity cDNA Reverse Transcription Kit, SYBR

Green SuperMix, and 96-well optical reaction plates with optical adhesive
films were purchased from Applied Biosystems (Foster City, CA). Real-time
PCR primers were synthesized by Integrated DNA Technologies Inc. (Coralville, IA) according to previously published sequences. Arachidonic acid,
4-hydroxybenzophenone, and DOX were purchased from Sigma-Aldrich (St.
Louis, MO). Arachidonic acid metabolite standards 5,6-EET, 8,9-EET, 11,12-
EET, 14,15-EET, 5,6-DHET, 8,9-DHET, 11,12-DHET, 14,15-DHET, and
20-HETE were obtained from Cayman Chemical (Ann Arbor, MI). Reagents
used for liquid chromatographic-electron spray ionization-mass spectrometry
(LC-ESI-MS) were HPLC-grade. Acetonitrile and water (HPLC-grade) were
purchased from EM Scientific (Gibbstown, NJ). Acrylamide, N,N-bis-methylene-acrylamide, ammonium persulfate, -mercaptoethanol, glycine, nitrocellulose membrane (0.45 m), and N,N,N,N-tetramethylethylenediamine were
purchased from Bio-Rad Laboratories (Hercules, CA). Chemiluminescence
Western blotting detection reagents were purchased from GE Healthcare
(Little Chalfont, Buckinghamshire, UK). CYP1B1 rabbit polyclonal primary
antibody was purchased from BD Gentest (Woburn, MA). CYP2J and sEH
primary antibodies were obtained as generous gifts from Dr. Darryl Zeldin
(National Institute of Environmental Health Sciences, National Institutes of
Health, Research Triangle Park, NC) and Dr. Bruce Hammock (Department of
Entomology, University of California, Davis, CA), respectively. Other primary
and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc






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