1: Clin Exp Pharmacol Physiol. 2007 May-Jun;34(5-6):399-405.\

Thymoquinone supplementation prevents the development of
gentamicin-induced acute renal toxicity in rats.
Sayed-Ahmed MM, Nagi MN.
Department of Pharmacology, College of Pharmacy, King Saud
University, Kingdom of Saudi Arabia. [email protected]
1. The present study investigated the possible protective effects of
thymoquinone (TQ), a compound derived from Nigella sativa with
strong anti-oxidant properties, against gentamicin (GM)-induced
nephrotoxicity. 2. A total of 40 adult male Wistar albino rats was
divided into four groups. Rats in the first group were injected daily
with normal saline (2.5 mL/kg, i.p.) for 8 consecutive days, whereas
rats in the second group received TQ (50 mg/L in drinking water) for 8
consecutive days. Animals in the third group were injected daily with
GM (80 mg/kg, i.p.) for 8 consecutive days, whereas animals in the
fourth group received a combination of GM (80 mg/kg, i.p.) and TQ
(50 mg/L in drinking water) for 8 consecutive days. 3. Gentamicin
resulted in a significant increase in serum creatinine, blood urea
nitrogen (BUN), thiobarbituric acid-reactive substances (TBARS) and
total nitrate/nitrite (NOx) and a significant decrease in reduced
glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) and
ATP levels in kidney tissues. 4. Interestingly, TQ supplementation
resulted in a complete reversal of the GM-induced increase in BUN,
creatinine, TBARS and NOx and decrease in GSH, GPx, CAT and ATP to
control values. Moreover, histopathological examination of kidney
tissues confirmed the biochemical data, wherein TQ supplementation
prevents GM-induced degenerative changes in kidney tissues. 5. Data
from the present study suggest that TQ supplementation prevents the
development of GM-induced acute renal failure by a mechanism
related, at least in part, to its ability to decrease oxidative stress and
to preserve the activity of the anti-oxidant enzymes, as well as it
ability to prevent the energy decline in kidney tissues.
PMID: 17439407 [PubMed - indexed for MEDLINE]
2: Phytother Res. 2007 May;21(5):410-4.

Thymoquinone supplementation attenuates hypertension and
renal damage in nitric oxide deficient hypertensive rats.
Khattab MM, Nagi MN.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, Kingdom of Saudi Arabia.
The present study was undertaken to evaluate the protective effect of
thymoquinone (TQ), the main constituent of the volatile oil from
Nigella sativa seeds, in rats after chronic inhibition of nitric oxide
synthesis with N(omega)-nitro-l-arginine methyl esters (l-NAME). Rats
were divided randomly into different treatment groups: control, lNAME, TQ and l-NAME + TQ. Hypertension was induced by 4 weeks
administration of l-NAME (50 mg/kg/day p.o.). TQ was administered
alone or in combination with l-NAME and continued for 4 weeks. The
animals were killed, and the serum and kidney tissues were isolated
for the determination of creatinine and glutathione (GSH),
respectively. Rats receiving l-NAME showed a progressive increase in
systolic blood pressure compared with control rats. Concomitant
treatment with TQ (0.5 and 1 mg/kg/day p.o.) reduced the increase in
systolic blood pressure induced by l-NAME in a dose dependent
manner. Kidney injury was demonstrated by a significant increase in
serum creatinine and a decrease in GSH in kidney tissue from l-NAME
treated rats. Treatment of rats with TQ decreased the elevated
creatinine and increased GSH to normal levels. TQ inhibited the in vitro
production of superoxide radical in enzymatic and non-enzymatic
systems. In conclusion, TQ is effective in protecting rats against lNAME-induced hypertension and renal damage possibly via antioxidant
activity. Copyright 2007 John Wiley & Sons, Ltd.
PMID: 17236176 [PubMed - indexed for MEDLINE]
3: Bioorg Med Chem. 2006 Dec 15;14(24):8608-21. Epub 2006
Sep 12.

Synthesis, dihydrofolate reductase inhibition, antitumor
testing, and molecular modeling study of some new 4(3H)quinazolinone analogs.
Al-Rashood ST, Aboldahab IA, Nagi MN, Abouzeid LA, AbdelAziz AA, Abdel-Hamide SG, Youssef KM, Al-Obaid AM, ElSubbagh HI.
Department of Pharmaceutical Chemistry, College of Pharmacy, King
Saud University, PO Box 2457, Riyadh 11451, Saudi Arabia.
In order to produce potent new leads for anticancer drugs, a new
series of quinazoline analogs was designed to resemble methotrexate
(MTX, 1) structure features and fitted with functional groups believed
to enhance inhibition of mammalian DHFR activity. Molecular modeling
studies were used to assess the fit of these compounds within the
active site of human DHFR. The synthesized compounds were
evaluated for their ability to inhibit mammalian DHFR in vitro and for
their antitumor activity in a standard in vitro tissue culture assay
panel. Compounds 28, 30, and 31 were the most active DHFR
inhibitors with IC(50) values of 0.5, 0.4, and 0.4microM, respectively.
The most active antitumor agents in this study were compounds 19,
31, 41, and 47 with median growth inhibitory concentrations (GI(50))
of 20.1, 23.5, 26.7, and 9.1microM, respectively. Of this series of
compounds, only compound 31 combined antitumor potency with
potent DHFR inhibition; the other active antitumor compounds (19, 41,
and 47) all had DHFR IC(50) values above 15microM, suggesting that
they might exert their antitumor potency through some other mode of
action. Alternatively, the compounds could differ significantly in uptake
or concentration within mammalian cells.
PMID: 16971132 [PubMed - indexed for MEDLINE]
4: Eur J Pharmacol. 2006 Aug 14;543(1-3):40-7. Epub 2006 Jun
3.

Neuroprotective effects of thymoquinone against transient
forebrain ischemia in the rat hippocampus.
Al-Majed AA, Al-Omar FA, Nagi MN.
Department of Pharmacology, College of Pharmacy, King Saud
University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
[email protected]
Increasing evidence demonstrates that oxidative stress plays an
important role in brain injury in experimental models of brain
ischemia. Thymoquinone, the main constituents of the volatile oil from
Negella sativa seeds, is reported to possess strong antioxidant
properties. Hence, the present study was undertaken to evaluate the
neuroprotective effect of thymoquinone against transient forebrain
ischemia-induced neuronal damage in the rat hippocampus. Rats were
divided randomly into five groups: control, sham, ischemia,
thymoquinone and ischemia+thymoquinone. Transient forebrain
ischemia was induced with bilateral occlusion of both common carotid
arteries for 10 min followed by 7 days of reperfusion. Thymoquinone
was administered (5 mg/kg/day p.o.) 5 days before ischemia and
continued during the reperfusion time. Animals were sacrificed, and
brain tissues were isolated for histopathological examination.
Hippocampal tissues were also used for determination of
malondialdehyde levels, an end product of lipid peroxidation;
glutathione (GSH) levels, a key antioxidant and the activities of the
antioxidant enzymes catalase and superoxide dismutase (SOD).
Thymoquinone and its metabolite thymohydroquinone were tested as
inhibitors of the in vitro non-enzymatic lipid peroxidation induced by
iron-ascorbate in the hippocampal homogenate. Forebrain ischemiareperfusion neural injury in rats was demonstrated by
histopathological observation, which revealed significant neural cell
death in the hippocampus CA1 area 7 days post-ischemia (77% cell
loss). Additionally, forebrain ischemia-reperfusion oxidative injury in
rats was demonstrated by a significant increase in malondialdehyde
and a significant decrease in GSH contents, catalase and SOD activities
in the hippocampal tissue compared to the control or sham-operated
groups. Pretreatment of thymoquinone attenuated forebrain ischemiainduced neuronal damage manifested by significantly decreasing the
number of dead hippocampal neuronal cells (24% in thymoquinonetreated versus 77% for ischemia, P<0.001), which confirm the
protective role of thymoquinone in ischemia-reperfusion injury. Also,
pretreatment of ischemic rats with thymoquinone decreased the
elevated levels of malondialdehyde and increased GSH contents,
catalase and SOD activities to normal levels. Thymoquinone and
thymohydroquinone inhibited the in vitro non-enzymatic lipid
peroxidation in hippocampal homogenate induced by iron-ascorbate.
The IC50 for thymoquinone and thymohydroquinone were found to be
12 and 3 microM respectively. This suggests that the protection of
thymoquinone and its metabolite involve increased resistance to
oxidative stress. In conclusion, thymoquinone is effective in protecting
rats against transient forebrain ischemia-induced damage in the rat
hippocampus. This spectacular protection makes thymoquinone a
promising agent in pathologies implicating neurodegenaration such as
cerebral ischemia.
PMID: 16828080 [PubMed - indexed for MEDLINE]
5: Neurochem Res. 2006 May;31(5):611-8. Epub 2006 May 23.

Immediate and delayed treatments with curcumin prevents
forebrain ischemia-induced neuronal damage and oxidative
insult in the rat hippocampus.
Al-Omar FA, Nagi MN, Abdulgadir MM, Al Joni KS, Al-Majed AA.
Department of Pharmacology, College of Pharmacy, King Saud
University, P.O. Box 2457, 11451 Riyadh, Kingdom of Saudi Arabia.
Oxidative stress is believed to contribute to neurodegeneration
following ischemic injury. The present study was undertaken to
evaluate the possible antioxidant neuroprotective effect of curcumin
(Cur) on neuronal death of hippocampal CA1 neurons following
transient forebrain ischemia in rat. Treatment of Cur (200 mg/kg/day,
i.p.) at three different times (immediately, 3 h and 24 h after
ischemia) significantly (P<0.01) reduced neuronal damage 7 days
after ischemia. Also, treatment of ischemic rats with Cur decreased the
elevated levels of MDA and increased GSH contents, catalase and SOD
activities to normal levels. In the in vitro, Cur was as potent as
antioxidant (IC(50) = 1 microM) as butylated hydroxytoluene. The
present study demonstrates that curcumin treatment attenuates
forebrain ischemia-induced neuronal injury and oxidative stress in
hippocampal tissue. Thus treatment with curcumin immediately or
even delayed until 24 h may have the potential to be used as a
protective agent in forebrain ischemic insult in human.
PMID: 16770732 [PubMed - indexed for MEDLINE]
6: J Biochem Mol Biol. 2003 Nov 30;36(6):593-6.
Nomega-nitro-L-arginine methylester ameliorates myocardial
toxicity induced by doxorubicin.
Mansour MA, El-Din AG, Nagi MN, Al-Shabanah OA, Al-Bekairi
AM.
Department of Pharmacology, College of Pharmacy, King Saud
University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
[email protected]
The effects of Nomega-nitro-L-arginine methylester (L-NAME) and Larginine on cardiotoxicity that is induced by doxorubicin (Dox) were
investigated. A single dose of Dox 15 mg/kg i.p. induced
cardiotoxicity, manifested biochemically by a significant elevation of
serum creatine phosphokinase (CPK) activity [EC 2.7.3.2]. Moreover,
cardiotoxicity was further confirmed by a significant increase in lipid
peroxides, measured as malon-di-aldehyde (MDA) in cardiac tissue
homogenates. The administration of L-NAME 4 mg/kg/d p.o. in
drinking water 5 days before and 3 days after the Dox injection
significantly ameliorated the cardiotoxic effects of Dox, judged by the
improvement in both serum CPK activity and lipid peroxides in the
cardiac tissue homogenates. On the other hand, the administration of
L-arginine 70 mg/kg/d p.o. did not protect the cardiac tissues against
the toxicity that was induced by the Dox treatment. The findings of
this study suggest that L-NAME can attenuate the cardiac dysfunction
that is produced by the Dox treatment via the mechanism(s), which
may involve the inhibition of the nitric oxide (NO) formation. L-NAME
may, therefore, be a beneficial remedy for cardiotoxicity that is
induced by Dox and can then be used to improve the therapeutic index
of Dox.
PMID: 14659079 [PubMed - indexed for MEDLINE]
7: Pharmacol Res. 2003 Dec;48(6):631-5.
Protective effect of arabic gum against acetaminophen-induced
hepatotoxicity in mice.
Gamal el-din AM, Mostafa AM, Al-Shabanah OA, Al-Bekairi AM,
Nagi MN.
Department of Pharmacology, College of Pharmacy, King Saud
University, PO Box 2457, Riyadh 11451, Saudi Arabia.
Overdose of acetaminophen, a widely used analgesic drug, can result
in severe hepatotoxicity and is often fatal. This study was undertaken
to examine the effects of arabic gum (AG), which is commonly used in
processed foods, on acetaminophen-induced hepatotoxicity in mice.
Mice were given arabic gum orally (100 g l(-1)) 5 days before a
hepatotoxic dose of acetaminophen (500 mg kg(-1)) intraperitoneally.
Arabic gum administration dramatically reduced acetaminopheninduced hepatotoxicity as evidenced by reduced serum alanine (ALT)
and aspartate aminotransferase (AST) activities. Acetaminopheninduced hepatic lipid peroxidation was reduced significantly by arabic
gum pretreatment. The protection offered by arabic gum does not
appear to be caused by a decrease in the formation of toxic
acetaminophen metabolites, which consumes glutathione, because
arabic gum did not alter acetaminophen-induced hepatic glutathione
depletion. Acetaminophen increased nitric oxide synthesis as measured
by serum nitrate plus nitrite at 4 and 6 h after administration and
arabic gum pretreatment significantly reduced their formation. In
conclusion, arabic gum is effective in protecting mice against
acetaminophen-induced hepatotoxicity. This protection may involve
the reduction of oxidative stress.
PMID: 14527829 [PubMed - indexed for MEDLINE]
8: J Biochem Mol Toxicol. 2002;16(6):273-8.
Melatonin inhibits the contractile effect of vanadate in the
isolated pulmonary arterial rings of rats: possible role of
hydrogen peroxide.
Nagi MN, Mansour MA, Al-Shabanah OA, El-Kashef HA.
Department of Pharmacology, College of Pharmacy, King Saud
University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia.
[email protected]
The effect and possible mechanism of action of vanadate on the
isolated pulmonary arterial rings of normal rats were studied.
Pulmonary arterial rings contracted in response to vanadate (0.1-1
mM) in a concentration-dependent manner. Preincubation of the
pulmonary arterial rings with 1 mM melatonin significantly reduced the
contractile effect of vanadate by more than 60%. Furthermore,
addition of hydrogen peroxide (50 microM) or enzymatic generation of
hydrogen peroxide by the addition of glucose oxidase (10 U/mL) to the
medium containing glucose produced remarkable increases in the
pulmonary arterial tension, 46.2 +/- 7.3 and 78.7 +/- 9.7 g tension/g
tissue, respectively. Similarly, incubation of the pulmonary arterial
rings with 1 mM melatonin significantly reduced the contractile
responses of the arterial rings to hydrogen peroxide and
glucose/glucose oxidase to 25.7 +/- 2.9 and 24.7 +/- 4.4 g tension/g
tissue, respectively. Vanadate, in vitro, significantly stimulated the
oxidation of NADH by xanthine oxidase, and the rate of oxidation was
increased by increasing either time or vanadate concentration.
Similarly, addition of melatonin to a reaction mixture containing
xanthine oxidase and vanadate significantly inhibited the rate of NADH
oxidation in a concentration-dependent fashion. The results of the
present study indicated that vanadate induced contraction in the
isolated pulmonary arterial rings, which was significantly reduced by
melatonin. Furthermore, the contractile effect of vanadate on the
pulmonary arterial rings may be attributed to the intracellular
generation of hydrogen peroxide. Copyright 2002 Wiley Periodicals,
Inc. J Biochem Mol Toxicol 16:273-278, 2002; Published online in
Wiley InterScience (www.interscience.wiley.com). DOI
10.1002/jbt.10049
PMID: 12481302 [PubMed - indexed for MEDLINE]
9: J Biochem Mol Toxicol. 2002;16(5):254-9.
Protective effect of arabic gum against cardiotoxicity induced
by doxorubicin in mice: a possible mechanism of protection.
Abd-Allah AR, Al-Majed AA, Mostafa AM, Al-Shabanah OA, Din
AG, Nagi MN.
Department of Pharmacology, College of Pharmacy, King Saud
University, P O Box 2457, Riyadh 11451, Saudi Arabia.
Arabic gum (AG) is a naturally occurring compound that has been
proposed to possess potent antioxidant activity. In this study, the
possible effects whereby AG could protect against cardiotoxicity
induced by doxorubicin (DOX) in mice were carried out. Administration
of single dose of DOX (15 mg/kg, i.p.) induced cardiotoxicity 72 h,
manifested biochemically by a significant elevation of serum creatine
kinase (CK) (EC 2.7.3.2). In addition, cardiotoxicity was further
confirmed by the significant increase in lipid peroxides measured as
malondialdehyde (MDA). Administration of AG (25 g/kg) orally for 5
days before and 72 h after DOX injection produced a significant
protection against cardiotoxicity induced by DOX. This was evidenced
by significant reductions in serum CK and cardiac lipid peroxides. The
effect of AG was examined on the superoxide anion radical generated
by enzymatic and nonenzymatic methods. The results indicate that AG
is a potent superoxide scavenger. The superoxide scavenging effect of
AG may explain, at least in part, the protective effect of AG against
cardiotoxicity induced by DOX. Copyright 2002 Wiley Periodicals, Inc.
PMID: 12439867 [PubMed - indexed for MEDLINE]
10: Comp Biochem Physiol C Toxicol Pharmacol. 2002
Jun;132(2):123-8.
Protective effect of aminoguanidine against nephrotoxicity
induced by cisplatin in normal rats.
Mansour MA, Mostafa AM, Nagi MN, Khattab MM, Al-Shabanah
OA.
Department of Pharmacology, College of Pharmacy, King Saud
University, P.O. Box 2457, Riyadh 11451, Saudi Arabia.
[email protected]
The effect of aminoguanidine (AG) on nephrotoxicity induced by
cisplatin (CDDP) was investigated. A single dose of CDDP (7.5 mg/kg
i.p.) induced nephrotoxicity, manifested biochemically by a significant
elevation in serum urea, creatinine and a severe decrease in serum
albumin. Moreover, marked increases in kidney weight, urine volume
and urinary excretion of albumin were observed. Nephrotoxicity was
further confirmed by a significant decrease in glutathione-Stransferase (GST, E.C. 2.5.1.18), glutathione peroxidase (GSH-Px,
E.C. 1.11.1.9) and catalase (E.C. 1.11.1.6) and a significant increase
in lipid peroxides measured as malondialdhyde (MDA) in kidney
homogenates. Administration of AG (100 mg/kg per day p.o.) in
drinking water 5 days before and 5 days after CDDP injection produced
a significant protection against nephrotoxicity induced by CDDP. The
amelioration of nephrotoxicity was evidenced by significant reductions
in serum urea and creatinine concentrations. In addition, AG tended to
normalize decreased levels of serum albumin. Urine volume, urinary
excretions of albumin and GST and kidney weight were significantly
decreased. Moreover, AG prevented the rise of MDA and the reduction
of GST and GSH-Px activities in the kidney. These results suggest that
AG has a protective effect on nephrotoxicity induced by CDDP and it
may therefore improve the therapeutic index of CDDP.
PMID: 12106889 [PubMed - indexed for MEDLINE]
11: Cell Biochem Funct. 2002 Jun;20(2):143-51.
Effects of thymoquinone on antioxidant enzyme activities, lipid
peroxidation and DT-diaphorase in different tissues of mice: a
possible mechanism of action.
Mansour MA, Nagi MN, El-Khatib AS, Al-Bekairi AM.
Department of Pharmacology, Faculty of Pharmacy, King Saud
University, Riyadh, Kingdom of Saudi Arabia. [email protected]
The present investigation focused, firstly, on the effects of oral
administration of thymoquinone (TQ) on antioxidant enzyme activities,
lipid peroxidation and DT-diaphorase activity in hepatic, cardiac and
kidney tissues of normal mice. Superoxide dismutase (SOD;
E.C:1.15.1.1), catalase (CAT; E.C:1.11.1.6), glutathione peroxidase
(GSH-Px; E.C:1.11.1.9), glutathione-S-transferase (GST;
E.C:2.5.1.18), and DT-diaphorase (E.C:1.6.99.2) enzyme activities in
each tissue type were determined. Treatment of mice with the
different doses of TQ (25, 50 and 100 mg kg(-1) day(-1) orally) for 5
successive days, produced significant reductions in hepatic SOD, CAT
and GSH-Px activities. In addition cardiac SOD activity was markedly
inhibited with the higher doses of TQ, (namely 50 and 100 mg kg(-1)).
Moreover, TQ (100 mg kg(-1)) significantly reduced hepatic and
cardiac lipid peroxidation as compared with the respective control
group. Conversely, TQ (50,100 mg kg(-1)) and TQ (100 mg kg(-1))
enhanced cardiac and renal DT-diaphorase activity respectively.
However, the selected doses of TQ neither produced any change in
GST activity nor influenced reduced glutathione content in all tissues
studied. TQ was tested, secondly, as a substrate for hepatic, cardiac
and renal DT-diaphorase of normal mice in the presence of NADPH.
Kinetic parameters for the reduction of TQ to dihydrothymoquinone
(DHTQ) indicated that DT-diaphorase of different tissues can efficiently
reduce TQ to DHTQ. K(m) and V(max) values revealed that hepatic
DT-diaphorase exhibited the higher values, while the lower values
were associated with renal DT-diaphorase. TQ and DHTQ were tested,
thirdly, as specific scavengers for superoxide anion (generated
biochemically) or as general scavengers for free radicals (generated
photochemically). The results revealed that TQ and DHTQ acted not
only as superoxide anion scavengers but also as general free radical
scavengers. The IC(50) for TQ and DHTQ in biochemical and
photochemical assays were in the nanomolar and micromolar range
respectively. Our data may explain at least partly the reported
beneficial in vivo protective effects of TQ through the combined
antioxidant properties of TQ and its metabolite DHTQ. Copyright 2002
John Wiley & Sons, Ltd.
PMID: 11979510 [PubMed - indexed for MEDLINE]
12: J Biochem Mol Toxicol. 2001;15(6):317-21.

Beneficial effect of nitric oxide synthase inhibitor on
hepatotoxicity induced by allyl alcohol.
Alam K, Nagi MN, Al-Shabanah OA, Al-Bekairi AM.
Department of Biochemistry, Faculty of Medicine, Aligarh Muslim
University, Aligarh, India. [email protected]
The effect of aminoguanidine (a selective inhibitor of inducible nitric
oxide synthase) on allyl alcohol-induced liver injury was assessed by
the measurement of serum ALT and AST activities and
histopathological examination. When aminoguanidine (50-300 mg/kg,
i.p.) was administered to mice 30 min before a toxic dose of allyl
alcohol (75 microL/kg, i.p.), significant changes related to liver injury
were observed. In the presence of aminoguanidine the level of ALT
and AST enzymes were significantly decreased. All symptoms of liver
necrosis produced by allyl alcohol toxicity almost completely
disappeared when animals were pretreated with aminoguanidine at
300 mg/kg. Depletion of hepatic glutathione as a consequence of allyl
alcohol metabolism was minimal in mice pretreated with
aminoguanidine at 300 mg/kg. It was found that the inhibition of
toxicity was not due to alteration in allyl alcohol metabolism since
aminoguanidine did not effect alcohol dehydrogenase activity both in
vivo and in vitro. Copyright 2001 John Wiley & Sons, Inc.
PMID: 11835631 [PubMed - indexed for MEDLINE]
13: Res Commun Mol Pathol Pharmacol. 1999;106(3):193-202.
Protective effect of aminoguanidine against cardiovascular
toxicity of chronic doxorubicin treatment in rats.
Mostafa AM, Nagi MN, Al Rikabi AC, Al-Shabanah OA, El-Kashef
HA.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, Kingdom of Saudi Arabia.
The cardiotoxicity-induced by chronic treatment of doxorubicin have
recently be attributed to free radical formation and/or release of nitric
oxide. In the present study, an already established rat model of
doxorubicin -induced cardiotoxicity was used. Doxorubicin in a total
cumulative dose of 15 mgkg(-1) I.P. given in six equal injections over
two week period was administered. After three weeks of doxorubicin
administration, the blood pressure, serum lactate dehydrogenase, lipid
peroxides, asites fluid and mortality rate were significantly increased.
Doxorubicin-induced cardiotoxicity was further confirmed by examining
the histopathology of heart sections. Myocardial fibres necrosis with
prominent acute inflammatory cells were observed in rats hearts
treated with doxorubicin. Aminoguanidine, an inhibitor of nitric oxide
synthase, 100 mgkg(-1) injected every other day for two week was
given concurrently with doxorubicin. Aminoguanidine given
concurrently with doxorubicin return blood pressure, lactate
dehydrogenase and lipid peroxides to normal control values.
Furthermore, aminoguanidine reduces the mortality rate, ascites fluid
formation- induced by doxorubicin and improved the histopathology of
rats hearts treated with doxorubicin. In conclusion, inhibition of nitric
oxide formation may be beneficial in protecting rat hearts against
doxorubicin- induced cardiotoxicity.
PMID: 11485049 [PubMed - indexed for MEDLINE]
14: Pharmacol Res. 2000 Mar;41(3):283-9.
Protective effect of thymoquinone against doxorubicin-induced
cardiotoxicity in rats: a possible mechanism of protection.
Nagi MN, Mansour MA.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, 11451, Saudi Arabia.
Administration of thymoquinone (10 mg kg(-1)day(-1), p.o.) with
drinking water starting 5 days before a single injection of doxorubicin
(15 mg kg(-1)i.p.) and continuing during the experimental period
ameliorated the doxorubicin-induced cardiotoxicity in rats. This
protection was evidenced from the significant reduction in serum
enzymes: lactate dehydrogenase elevated level, 24 h and creatine
phosphokinase elevated levels, 24 h and 48 h after doxorubicin
administration. The cardiotoxicity of doxorubicin has been suggested
to result from the generation of superoxide free-radical. The protective
action of thymoquinone was examined against superoxide anion
radical either generated photochemically, biochemically or derived
from calcium ionophore (A23187) stimulated polymorphonuclear
leukocytes. The results indicate that thymoquinone is a potent
superoxide radical scavenger, scavenging power being as effective as
superoxide dismutase against superoxide. In addition thymoquinone
has an inhibitory effect on lipid peroxidation induced by
Fe(3+)/ascorbate using rat heart homogenate. The superoxide
scavenging and anti-lipid peroxidation may explain, in part, the
protective effect of thymoquinone against doxorubicin-induced
cardiotoxicity. 2000 Academic Press@p$hr Copyright 2000 Academic
Press.
PMID: 10675279 [PubMed - indexed for MEDLINE]
15: Life Sci. 2000;66(3):265-70.
Protective effect of aminoguanidine, a nitric oxide synthase
inhibitor, against carbon tetrachloride induced hepatotoxicity
in mice.
Al-Shabanah OA, Alam K, Nagi MN, Al-Rikabi AC, Al-Bekairi AM.
Department of Pharmacology, College of Pharmacy, King Saud
University, Saudi Arabia. [email protected]
The present study was undertaken to evaluate the effect of
aminoguanidine (AG) on carbon tetrachloride (CCl4)-induced
hepatotoxicity. Treatment of mice with CCl4 (20 microl/kg, i.p.)
resulted in damage to centrilobular regions of the liver, increase in
serum aminotransferase and rise in lipid peroxides level 24 hours after
CCl4 administration. Pretreatment of mice with AG (50 mg/kg, i.p.) 30
minutes before CCl4 was found to protect mice from the CCl4-induced
hepatic toxicity. This protection was evident from the significant
reduction in serum aminotransferase, inhibition of lipid peroxidation
and prevention of CCl4-induced hepatic necrosis revealed by
histopathology. Aminoguanidine, a relatively specific inhibitor of
inducible nitric oxide synthase, did not inhibit the in vitro lipid
peroxidation. Taken together, these data suggest a potential role of
nitric oxide as an important mediator of CCl4-induced hepatotoxicity.
PMID: 10666002 [PubMed - indexed for MEDLINE]
16: Eur J Cancer Prev. 1999 Oct;8(5):435-40.
Inhibition of benzo(a)pyrene-induced forestomach
carcinogenesis in mice by thymoquinone.
Badary OA, Al-Shabanah OA, Nagi MN, Al-Rikabi AC, Elmazar
MM.
Department of Pharmacology and Toxicology, College of Pharmacy, AlAzhar University, Nasr City, Cairo, Egypt.
The modulating effect of thymoquinone (TQ) on benzo(a)pyrene (BP)induced forestomach tumours was investigated in female Swiss albino
mice, receiving oral administration of BP at a dose of 1 mg twice
weekly for 4 weeks. Administration of 0.01% of TQ in drinking water 1
week before, during and after BP treatment until the end of the
experiment resulted in significant suppression of BP-induced
tumourigenesis when compared with the group receiving BP alone. TQ
inhibited both BP-induced forestomach tumour incidence and
multiplicity by 70% and 67%, respectively. Lipid peroxide
accumulation and decreased glutathione (GSH) content and
glutathione-S-transferase (GST) and DT diaphorase activities were
observed in the liver of BP-treated tumour-bearing mice. TQ alone
showed a significant induction in the enzyme activities of hepatic GST
and DT diaphorase. Mice treated with TQ along with BP showed almost
normal hepatic lipid peroxides and GSH levels, and normal enzyme
activities compared to the control group. The present data may
indicate the potential of TQ, the main constituent of the volatile oil of
Nigella sativa seed, as a powerful chemopreventive agent against BPinduced forestomach tumours in mice. The possible modes of action of
TQ may be through its antioxidant and anti-inflammatory activities,
coupled with enhancement of detoxification processes.
PMID: 10548399 [PubMed - indexed for MEDLINE]
17: Pharmacol Res. 1999 Aug;40(2):159-63.
The protective action of thymol against carbon tetrachloride
hepatotoxicity in mice.
Alam K, Nagi MN, Badary OA, Al-Shabanah OA, Al-Rikabi AC, AlBekairi AM.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, 11451, Saudi Arabia.
The protective action of thymol (paramethyl-isopropyl-phenol) was
investigated against carbon tetrachloride (CCl(4))-induced
hepatotoxicity in male Swiss albino mice. The CCl(4)at a dose of 20
microl kg(-1)produced damage to liver cells and was followed by the
significant increase (P<0.001) in serum alanine aminotransferase
(ALT) activity and hepatic lipid peroxidation after 24 h. The
hepatocellular necrosis was further confirmed by histopathological
examination of liver section. Oral administration of thymol in a single
dose (300 mg kg(-1)) resulted in significant (P<0.05) amelioration of
CCl(4)-induced hepatotoxicity. Thymol also inhibited lipid peroxidation
induced by CCl(4)in vivo. The protection offered by thymol was also
evident from histopathology photomicrograph. In a separate in vitro
assay, thymol inhibited the non-enzymatic lipid peroxidation of normal
mice liver homogenate induced by Fe(3+)-ascorbate. The present
study suggests that thymol protects the liver against CCl(4)-induced
toxicity and the protection may be mediated through its ability to
inhibit lipid peroxidation. However, other interactions between thymol
and CCl(4)remains to be elucidated. 1999 Academic Press. Copyright
1999 Academic Press.
PMID: 10433875 [PubMed - indexed for MEDLINE]
18: Biochem Mol Biol Int. 1999 Jan;47(1):153-9.
Thymoquinone protects against carbon tetrachloride
hepatotoxicity in mice via an antioxidant mechanism.
Nagi MN, Alam K, Badary OA, al-Shabanah OA, al-Sawaf HA, alBekairi AM.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, Saudi Arabia.
Thymoquinone (TQ) is the major active component of the volatile oil of
Nigella sativa seeds. The effects of TQ on carbon tetrachloride (CCl4)induced hepatotoxicity was investigated in male Swiss albino mice.
Carbon tetrachloride (20 microliters/Kg, i.p.) injected into mice,
induced damage to liver cells and was followed by the increase in
serum alanine aminotransferase (ALT) activity after 24 h. Oral
administration of TQ in a single dose (100 mg/Kg) resulted in
significant (p < 0.001) protection against the hepatotoxic effects of
CCl4. TQ was tested as a substrate for mice hepatic DT-diaphorase in
the presence of NADH. TQ appears to undergo reduction to
dihydrothymoquinone (DHTQ). Reduction rates as a function of protein
(liver homogenate) and substrate (TQ) concentrations are reported. An
apparent K(m) of 0.1 mM and an apparent Vmax of 74 mumol/min/g
liver were measured. TQ and DHTQ inhibited the in vitro nonenzymatic lipid peroxidation in liver homogenate (induced by Fe(3+)ascorbate) in a dose dependent manner. In this in vitro model DHTQ
was more potent in comparison with TQ and butylated hydroxytoluene
(BHT). The IC50 for DHTQ, TQ and BHT were found to be 0.34, 0.87
and 0.58 microM respectively. The data suggest that the in vivo
protective action of TQ against CCl4-induced hepatotoxicity may be
mediated through the combined antioxidant properties of TQ and its
metabolite DHTQ.
PMID: 10092955 [PubMed - indexed for MEDLINE]
19: J Exp Clin Cancer Res. 1998 Jun;17(2):193-8.
Thymoquinone protects against doxorubicin-induced
cardiotoxicity without compromising its antitumor activity.
al-Shabanah OA, Badary OA, Nagi MN, al-Gharably NM, alRikabi AC, al-Bekairi AM.
Dept. of Pharmacology, College of Pharmacy, King Saud University,
Riyadh, Saudi Arabia.
Doxorubicin (DOX) has a wide spectrum of antitumor activity with
dose-related cardiotoxicity as a major side effect. This cardiotoxicity
has been suggested to result from the generation of oxygen-free
radicals. The objective of the present study was to investigate the
influence of the antioxidant, thymoquinone (TQ) on cardiotoxicity and
antitumor activity of DOX in mice. TQ (8 mg/kg/day, p.o.)
administered with drinking water starting 5 days before a single i.p.
injection of DOX (20 mg/kg) and continuing during the experimental
period ameliorated the DOX-induced cardiotoxicity in mice. This finding
was evidenced by significant reductions in serum lactate
dehydrogenase and creatine kinase elevated levels and further
supplemented by histopathological examination of cardiac tissue. TQ
did not alter the plasma and heart DOX levels as monitored by
fluorometric analysis. In in vivo study on mouse Ehrlich ascites
carcinoma tumor, it could then be shown that TQ does not interfere
with the antitumor activity of DOX. The current data support TQ as a
potentially selective cytoprotective agent, which may ameliorate
cardiotoxicity without decreasing DOX antitumor activity.
PMID: 9700580 [PubMed - indexed for MEDLINE]
20: Can J Physiol Pharmacol. 1997 Dec;75(12):1356-61.
Thymoquinone ameliorates the nephrotoxicity induced by
cisplatin in rodents and potentiates its antitumor activity.
Badary OA, Nagi MN, al-Shabanah OA, al-Sawaf HA, alSohaibani MO, al-Bekairi AM.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, Saudi-Arabia.
The effects of thymoquinone (TQ) on cisplatin-induced nephrotoxicity
in mice and rats were studied. Oral administration of TQ (50 mg/L in
drinking water) for 5 days before and 5 days after single injections of
cisplatin (5 mg/kg, i.v., in rats and 7 or 14 mg/kg, i.p., in mice)
greatly ameliorated cisplatin-induced nephrotoxicity in both species. In
rats, i.v. cisplatin caused 4- and 5-fold elevations in serum urea and
creatinine, a 235% increase in urine volume, a 41% increase in kidney
weight, 8.5-fold decrease in creatinine clearance, and extensive
histological damage 5 days after treatment. In mice, similar alterations
in kidney function were observed. TQ-induced amelioration of cisplatin
nephrotoxicity was evident by significant reductions in serum urea and
creatinine and significant improvement in polyuria, kidney weight, and
creatinine clearance. The protective effects of TQ against cisplatininduced nephrotoxicity in the rat were further confirmed by
histopathological examination. To evaluate the possible modification of
the antitumor activity of cisplatin by TQ, we studied their interaction in
Ehrlich ascites carcinoma (EAC) bearing mice. The results revealed
that TQ potentiated the antitumor activity of cisplatin. The current
study suggests that TQ may improve the therapeutic index of cisplatin.
PMID: 9534946 [PubMed - indexed for MEDLINE]
21: Nephron. 1997;77(4):435-9.
Effect of L-histidinol on cisplatin nephrotoxicity in the rat.
Badary OA, Nagi MN, Al-Sawaf HA, Al-Harbi M, Al-Bekairi AM.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, Saudi Arabia.
The effect of L-histidinol (LHL) on the acute nephrotoxicity produced
by cisplatin (CDDP; 6 mg/kg, i.v.) was investigated in the rat.
Intraperitoneal administration of LHL (100 mg/kg x 5 doses, 2 h apart)
starting 2 h prior to CDDP single injection produced significant
protection of renal function. The attenuation of nephrotoxicity was
evidenced by significant reductions in serum urea and creatinine
concentrations, decreased polyuria, reduction in body weight loss,
marked reduction in urinary fractional sodium excretion and
glutathione-S-transferase (GST) activity, and increased urine/serum
creatinine ratio as well as increased creatinine clearance. LHL
significantly ameliorated the toxic renal biochemical changes induced
by CDDP. Renal lipid peroxides, glutathione levels and GST activity
showed a marked tendency towards the normal values. Accumulation
of platinum in renal tissues was significantly decreased in the presence
of LHL. It is concluded that LHL can act as a nephroprotectant, and it
is suggested that it would have beneficial effects on the kidney in
clinical settings.
PMID: 9434066 [PubMed - indexed for MEDLINE]
22: Biochem Mol Biol Int. 1995 Jul;36(3):633-8.
Spectrophotometric assay for superoxide dismutase based on
the nitroblue tetrazolium reduction by glucose-glucose oxidase.
Nagi MN, al-Bekairi AM, al-Sawaf HA.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, Saudi Arabia.
A new spectrophotometric assay of superoxide dismutase (SOD) is
described. The assay is based on the SOD-mediated inhibition in the
rate of nitroblue tetrazolium reduction to the blue formazan at alkaline
pH. The optimized assay of SOD is performed in 50 mM glycine-NaOH
buffer, pH 9.5, at 25 degrees C. The SOD concentration is determined
from the V/v ratio of rates measured in the absence (V) or the
presence (v) of SOD. One unit of SOD has been defined as the
concentration that decrease the rate to 50% (V/v = 2). The assay is
simple, sensitive, uses commercially available reagents, rapid and
easy to perform and could be used routinely for monitoring superoxide
dismutase levels in purified protein fractions.
PMID: 7549963 [PubMed - indexed for MEDLINE]
23: Biochem Mol Biol Int. 1994 Sep;34(2):233-8.

Evidence for superoxide radical production by a simple
flavoprotein: glucose oxidase.
al-Bekairi AM, Nagi MN, Shoeb HA, al-Sawaf HA.
Department of Pharmacology, College of Pharmacy, King Saud
University, Riyadh, Kingdom of Saudi Arabia.
Nitroblue tetrazolium was reduced to blue formazan during the
oxidation of glucose by glucose oxidase. The rate of blue color
formation was dependent on the concentrations of glucose, nitroblue
tetrazolium and glucose oxidase. The rate of the reaction was
negligible below pH 8.4, but sharply increased with increasing pH. The
reduction of nitroblue tetrazolium was inhibited by superoxide
dismutase, consistent with the participation of superoxide anion radical
in the reaction.
PMID: 7849633 [PubMed - indexed for MEDLINE]
24: Biochem J. 1992 Oct 1;287 ( Pt 1):91-100.

Evidence that beta-hydroxyacyl-CoA dehydrase purified from
rat liver microsomes is of peroxisomal origin.
Cook L, Nagi MN, Suneja SK, Hand AR, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06030.
The present study provides strong evidence that the previously
isolated hepatic microsomal beta-hydroxyacyl-CoA dehydrase (EC
4.2.1.17), believed to be a component of the fatty acid chainelongation system, is derived, not from the endoplasmic reticulum, but
rather from the peroxisomes. The isolated dehydrase was purified over
3000-fold and showed optimal enzymic activity toward betahydroxyacyl-CoAs or trans-2-enoyl-CoAs with carbon chain lengths of
8-10. The purified preparation (VDH) displayed a pH optimum at 7.5
with beta-hydroxydecanoyl-CoA, and at 6.0 with betahydroxystearoyl-CoA. Competitive-inhibition studies suggested that
VDH contained dehydrase isoforms, and SDS/PAGE showed three
major bands at 47, 71 and 78 kDa, all of which reacted to antibody
raised to the purified preparation. Immunocytochemical studies with
anti-rabbit IgG to VDH unequivocally demonstrated gold particles
randomly distributed throughout the peroxisomal matrix of liver
sections from both untreated and di-(2-ethylhexyl) phthalate-treated
rats. No labelling was associated with endoplasmic reticulum or with
the microsomal fraction. Substrate-specificity studies and the use of
antibodies to VDH and to the peroxisomal trifunctional protein
indicated that VDH and the latter are separate enzymes. On the other
hand, the VDH possesses biochemical characteristics similar to those
of the D-beta-hydroxyacyl-CoA dehydrase recently isolated from rat
liver peroxisomes [Li, Smeland & Schulz (1990) J. Biol. Chem. 265,
13629-13634; Hiltunen, Palosaari & Kunau (1989) J. Biol. Chem. 264,
13536-13540]. Neither enzyme utilizes crotonoyl-CoA or cis-2-enoylCoA as substrates, but both enzymes convert trans-2-enoyl substrates
into the D-isomer only. In addition, the VDH also contained betaoxoacyl-CoA reductase (beta-hydroxyacyl-CoA dehydrogenase)
activity, which co-purified with the dehydrase.
PMID: 1417796 [PubMed - indexed for MEDLINE]
25: Arch Biochem Biophys. 1992 Feb 14;293(1):71-8.

Depletion of rat hepatic glutathione and inhibition of
microsomal trans-2-enoyl-CoA reductase activity following
administration of a dec-2-ynol and dec-2-ynoic acid.
Nagi MN, Suneja SK, Cook L, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06030.
The effects of administration of dec-2-ynol and dec-2-ynoic acid on the
hepatic glutathione (GSH) content and hepatic microsomal trans-2enoyl-CoA reductase activity were examined in rat. Both compounds,
when administered ip, caused a marked depletion of GSH levels and a
corresponding inactivation of trans-2-enoyl-CoA reductase activity in
both a time- and dose-dependent manner. The dec-2-ynoic acid
caused greater hepatotoxicity than dec-2-ynol based on serum alanine
transaminase activity. Based on the observations that (a) the alcohol
did not interact with GSH in the presence or absence of cytosol, (b)
the spectral manifestation of the interaction between GSH and the
alcohol occurred only when NAD+ was added to the reaction mixture
containing the cytosol and reactants, and (c) a similar absorbance
spectrum was obtained following the interaction between aldehyde and
GSH, it was concluded that dec-2-ynol is converted to an electrophile,
dec-2-ynal, which causes depletion of GSH. The decrease in GSH
content following administration of the acid appears to be due to
activation of the acid to the electrophile, dec-2-ynoyl CoA, which then
interacts with GSH, resulting in its depletion, based on the in vitro
observations that (a) the acid did not interact with GSH in the
presence or absence of cytosol, and (b) the spectral manifestation of
interaction between GSH and dec-2-ynoyl CoA occurred both
nonenzymatically and enzymatically in the presence of rat liver
glutathione S-transferase (Sigma). Bovine serum albumin stimulated
the enzymatic reaction. Comparable to the effects on GSH were the
effects of dec-2-ynol, dec-2-ynal, dec-2-ynoic acid, and dec-2-ynoyl
CoA on the microsomal trans-2-enoyl-CoA reductase activity in vitro.
While the alcohol had no effect on the enzyme activity, its electrophilic
product, the aldehyde, was a potent inhibitor. Similarly, the acid did
not inhibit the enzyme activity unless the acid was present at high
concentration; however, its electrophilic product, the CoA thioester,
was a very potent inhibitor at very low concentration.
PMID: 1731641 [PubMed - indexed for MEDLINE]
26: Prog Lipid Res. 1992;31(1):1-51.

The fatty acid chain elongation system of mammalian
endoplasmic reticulum.
Cinti DL, Cook L, Nagi MN, Suneja SK.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06030.
Much has been learned about FACES of the endoplasmic reticulum
since its discovery in the early 1960s. FACES consists of four
component reactions, requires the fatty acid to be activated in the
form of a CoA derivative, utilizes reducing equivalents in the form of
NADH or NADPH, is induced by a fat-free diet, resides on the
cytoplasmic surface of the endoplasmic reticulum, appears to function
in concert with the desaturase system and appears to exist in multiple
forms (either multiple condensing enzymes connected to a single
pathway or multiple pathways). FACES has been found in all tissues
investigated, namely, liver, brain, kidney, lung, adrenals, retina, testis,
small intestine, blood cells (lymphocytes and neutrophils) and
fibroblasts, with one exception--the heart has no measurable activity.
Yet, much more needs to be learned. The critical, inducible and ratelimiting condensing enzyme has resisted solubilization and purification;
the purification of the other components has met with limited success.
We know nothing about the site of synthesis of each component of
FACES. How is each component enzyme integrated into the
endoplasmic reticulum membrane? Is there a single mRNA directing
synthesis of all four components or are there four separate mRNAs?
How are elongation and desaturation coordinated? What is (are) the
physiological regulator(s) of FACES--ADP, AMP, IP3, G-proteins,
phosphorylation, CoA, Ca2+, cAMP, none of these? The molecular
biology of FACES is only in the fetal stage of development. We are only
scratching the surface--it is an undiscovered country.
PMID: 1641395 [PubMed - indexed for MEDLINE]
27: J Neurochem. 1991 Jul;57(1):140-6.

Decreased long-chain fatty acyl CoA elongation activity in
quaking and jimpy mouse brain: deficiency in one enzyme or
multiple enzyme activities?
Suneja SK, Nagi MN, Cook L, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06030.
Using long-chain fatty acyl CoAs (arachidoyl CoA and behenoyl CoA), a
decrease in overall fatty acid chain elongation activity was observed in
the quaking and jimpy mouse brain microsomes relative to controls.
Arachidoyl CoA (20:0) and behenoyl CoA (22:0) elongation activities
were depressed to about 50% and 80% of control values in quaking
and jimpy mice, respectively. Measurement of the individual enzymatic
activities of the elongation system revealed a single deficiency in
enzyme activity; only the condensation activity was reduced to the
same extent as total elongation in both quaking and jimpy mice. The
activities of the other three enzymes, beta-ketoacyl CoA reductase,
beta-hydroxyacyl CoA dehydrase, and trans-2-enoyl CoA reductase, in
both mutants were similar to the activities present in the control
mouse. In addition, the activities of these three enzymes were more
than two to three orders of magnitude greater than the condensing
enzyme activity in all three groups, establishing that the condensing
enzyme catalyzes the rate-limiting reaction step of total elongation.
When the elongation of palmitoyl CoA was measured, only a 25%
decrease in total elongation occurred in both mutants; a similar
percent decrease in the condensation of palmitoyl CoA also was
observed. The activities of the other three enzymes were unaffected.
These results support the concept of either multiple elongation
pathways or multiple condensing enzymes.
PMID: 2051161 [PubMed - indexed for MEDLINE]
28: Lipids. 1991 May;26(5):359-63.

Do rat kidney cortex microsomes possess the enzymatic
machinery to desaturate and chain elongate fatty acyl-CoA
derivatives?
Suneja SK, Nagi MN, Cook L, Osei P, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06030.
Rat kidney cortex microsomal preparations were unable to catalyze
delta 9, delta 6 and delta 5 desaturation of stearoyl-coenzyme A
(CoA), linoleoyl-CoA and dihomo-gamma-linolenoyl-CoA, respectively.
The kidney cortex microsomal fraction, however, did catalyze the
malonyl-CoA dependent fatty acyl-CoA elongation. The biochemical
properties of palmitoyl-CoA elongation were studied as a function of
protein concentration, time, reduced nicotinamide adenine dinucleotide
phosphate (NADPH), malonyl-CoA and substrate concentrations; of the
substrates investigated, delta 6,9,12-18:3 was the most active. Unlike
what was observed in the hepatic system, a high-carbohydrate, fatfree diet did not induce kidney fatty acid chain elongation. All
intermediate kidney cortex microsomal reactions, i.e., beta-ketoacylCoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoylCoA reductase activities, were significantly higher (greater than one
order of magnitude) than the condensing enzyme activity, suggesting
that the rate-limiting step in total elongation is the initial condensation
reaction. Contrary to other reports, the results suggest that the kidney
cannot synthesize arachidonic acid needed for eicosanoid production.
PMID: 1895882 [PubMed - indexed for MEDLINE]
29: Biochim Biophys Acta. 1990 Jan 16;1042(1):81-5.

Enzyme site-specific changes in hepatic microsomal fatty acid
chain elongation in streptozotocin-induced diabetic rats.
Suneja SK, Osei P, Cook L, Nagi MN, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06032.
The hepatic microsomal fatty acid chain elongation of palmitoyl-CoA
and gamma-linolenoyl-CoA was diminished by 40-50% in male
Sprague-Dawley rats made diabetic for 2 and 4 weeks following the
intravenous administration of a single dose (65 mg/kg) of
streptozotocin. Analysis of the activities of the four enzymatic
components showed that only one enzyme, the condensing enzyme,
which catalyzes the initial and rate-limiting step in chain elongation,
was altered by the diabetic state. Both chain elongation and
condensation activities were depressed to the same extent, whereas
beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and
trans-2-enoyl-CoA reductase activities were the same as the values
obtained with non-diabetic controls. 2 week administration of 10 units
of insulin per day to rats which were diabetic for a 2-week period
resulted in the reversal of the reduced palmitoyl-CoA elongation and
condensation activities to control values. However, neither the
condensation nor the elongation of gamma-linolenoyl was reversed by
the insulin treatment. These results support the notion of multiple
condensing enzymes or chain elongation systems.
PMID: 2297524 [PubMed - indexed for MEDLINE]
30: Biochem Biophys Res Commun. 1989 Dec 29;165(3):1428-34.

Evidence for two separate beta-ketoacyl CoA reductase
components of the hepatic microsomal fatty acid chain
elongation system in the rat.
Nagi MN, Cook L, Suneja SK, Peluso PS, Laguna JC, Osei P, Cinti
DL.
Department of Pharacology, University of Connecticut Health Center,
Farmington 06032.
The hepatic microsomal fatty acid chain elongation system can utilize
either NADPH or NADH. Elongation activity, measured as the rate of
malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fatfree diet and by bovine serum albumin (BSA) when either cofactor was
employed. When the intermediate products were determined, it was
observed that in the presence of BSA and NADPH, the predominant
product was the saturated elongated fatty acid, whereas in the
presence of BSA and NADH, the major intermediate was the betaketoacyl derivative. Employing beta-ketostearoyl CoA as substrate,
BSA markedly inhibited NADH-supported beta-ketoacyl CoA reductase
activity and stimulated NADPH-supported activity. Furthermore, the
sum of the NADH-dependent and NADPH-dependent betaketoreductase activities approximated the activity obtained when both
cofactors were present in the incubation medium, suggesting the
existence of two beta-ketoacyl CoA reductases, one using NADH and
the other NADPH.
PMID: 2692567 [PubMed - indexed for MEDLINE]
31: Biochem Biophys Res Commun. 1989 Oct 31;164(2):927-33.

Evidence for two separate beta-ketoacyl CoA reductase
components of the hepatic microsomal fatty acid chain
elongation system in the rat.
Nagi MN, Cook L, Suneja SK, Peluso PS, Laguna JC, Osei P, Cinti
DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06032.
The hepatic microsomal fatty acid chain elongation system can utilize
either NADPH or NADH. Elongation activity, measured as the rate of
malonyl CoA incorporation into palmitoyl CoA, was enhanced by a fatfree diet and by bovine serum albumin (BSA) when either cofactor was
employed. When the intermediate products were determined, it was
observed that in the presence of BSA and NADPH, the predominant
product was the saturated elongated fatty acid, whereas in the
presence of BSA and NADH, the major intermediate was the betaketoacyl derivative. Employing beta-ketostearoyl CoA as substrate,
BSA markedly inhibited NADH-supported beta-ketoacyl CoA reductase
activity and stimulated NADPH-supported activity. Furthermore, the
sum of the NADH-dependent and NADPH-dependent betaketoreductase activities approximated the activity obtained when both
cofactors were present in the incubation medium, suggesting the
existence of two beta-ketoacyl CoA reductases, one using NADH and
the other, NADPH.
PMID: 2573353 [PubMed - indexed for MEDLINE]
32: Anal Biochem. 1989 Jun;179(2):251-61.

Spectrophotometric assay for the condensing enzyme activity
of the microsomal fatty acid chain elongation system.
Nagi MN, Cook L, Suneja SK, Osei P, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06032.
A rapid and simple spectrophotometric method was developed to
measure the activity of the condensing enzyme component of the
microsomal fatty acid chain elongation system. The intermediate
product of the condensation reaction is the beta-ketoacyl CoA which
exists in two tautomeric forms, i.e., keto and enol. The addition of
bovine serum albumin (BSA) to a cuvette cell containing a betaketoacyl CoA derivative resulted in the formation of a 303-nm
absorbance peak, characteristic of enolate formation. The betaketoacyl CoAs with carbon chain length of 6 to 18 interacted with BSA
to produce the 303-nm peak; acetoacetyl CoA was the only beta-keto
compound tested which did not interact with BSA to produce the peak.
Other compounds which were unaffected by BSA included CoA, free
beta-keto acid, beta-hydroxyacyl CoA, acyl CoA, trans-2-enoyl CoA,
and malonyl CoA. BSA could not be replaced by ovalbumin;
furthermore, denatured (boiling) BSA could not induce the 303-nm
peak. The specific activity of the condensing enzyme measured by the
spectrophotometric method compares favorably with the activity
obtained by the radioactive method. The apparent extinction
coefficient (epsilon) for the absorbance peak generated by the betaketo thioester varied from 5 to 30 mM-1 cm-1 depending on the betaketo derivative. The spectrophotometric procedure can be used in the
determination of the condensing enzyme activity in not only hepatic
microsomes but also in kidney and brain microsomes both of which
have significantly lower activity. The advantages of the novel method
over the radioactive method are that (i) it does not involve the use of
radioactive compounds, (ii) it is much less cumbersome and
significantly less costly, and (iii) it is rapid and easy to perform.
PMID: 2774174 [PubMed - indexed for MEDLINE]
33: J Biol Chem. 1989 Apr 25;264(12):6844-9.

Topography of rat hepatic microsomal enzymatic components
of the fatty acid chain elongation system.
Osei P, Suneja SK, Laguna JC, Nagi MN, Cook L, Prasad MR,
Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06032.
The orientation of the condensing enzyme, the beta-hydroxyacyl-CoA
dehydrase, and the trans-2-enoyl CoA reductase within the rat liver
microsomal membrane was investigated by the use of impermeant
inhibitors of enzyme activity: trypsin, chymotrypsin, subtilisin,
mercury-dextran, and anti-beta-hydroxyacyl-CoA dehydrase IgG. The
activity of the condensing enzyme was inhibited more than 70% by
various proteases and was completely inhibited by 80 microM
mercury-dextran. Similar results were obtained for the trans-2-enoylCoA reductase activity. On the other hand, in the absence of
detergent, proteases inhibited beta-hydroxyacyl-CoA dehydrase
activity by 25-40%, while in the presence of detergent the inhibition
increased to 65-90%. Furthermore, anti-beta-hydroxyacyl-CoA
dehydrase IgG, which in the absence of detergent produced no
inhibition, in the presence of detergent inhibited beta-hydroxyacyl-CoA
dehydrase activity by more than 80%; under identical conditions,
preimmune IgG caused a 13% inhibition. Microsomes used throughout
this study displayed greater than 90% latency with respect to
mannose-6-phosphatase activity, indicating that the microsomes were
intact. Latency was not affected by the proteases, by mercury-dextran,
or by the presence of the enzyme assay components. These results
suggest that both the condensing enzyme and the reductase are
present on the cytoplasmic surface of the membrane, whereas the
beta-hydroxyacyl-CoA dehydrase is embedded in the microsomal
membrane.
PMID: 2540164 [PubMed - indexed for MEDLINE]
34: Arch Biochem Biophys. 1989 Feb 15;269(1):264-71.

Disruption of rat hepatic microsomal electron transport chains
by the selenium-containing anti-inflammatory agent Ebselen.
Nagi MN, Laguna JC, Cook L, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06032.
The influence of Ebselen, an organoselenium anti-inflammatory agent,
on the two electron transport chains present in rat liver microsomes
has been studied. At low micromolar concentrations, Ebselen markedly
inhibited the flow of reducing equivalents from NADPH-cytochrome
P450 reductase to both its natural electron acceptor, cytochrome
P450, and its artificial electron acceptor, cytochrome c. Similarly, the
microsomal NADH-cytochrome c reductase system consisting of
cytochrome b5 and its flavoprotein, NADH-cytochrome b5 reductase,
was also significantly inhibited by Ebselen. The inhibition appears to be
due to the inability of the reduced pyridine nucleotide to transfer
electrons to the flavin (FAD and/or FMN) in the flavoprotein reductase.
This was shown with the purified NADPH-cytochrome P450 reductase,
which in the presence of Ebselen was not converted to the
semiquinone form following the addition of NADPH. The addition of
Ebselen to a suspension of hepatic microsomes from either untreated
or phenobarbital-treated rats did not result in any spectral change
characteristic of type I, type II, or reverse type I.
PMID: 2916842 [PubMed - indexed for MEDLINE]
35: Arch Biochem Biophys. 1989 Feb 15;269(1):272-83.

Action of Ebselen on rat hepatic microsomal enzyme-catalyzed
fatty acid chain elongation, desaturation, and drug
biotransformation.
Laguna JC, Nagi MN, Cook L, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06032.
In the previous study, the organoselenium-containing antiinflammatory agent, Ebselen, was found to disrupt both hepatic
microsomal NADH- and NADPH-dependent electron transport chains.
In the current investigation, we focus on the action of Ebselen on three
separate metabolic reactions, namely, fatty acid chain elongation,
desaturation, and drug biotransformation, which utilize reducing
equivalents via these microsomal electron transport pathways. Both
NADH-dependent and NADPH-dependent chain elongation reactions
showed (i) that the condensation step was inhibited by Ebselen; all
three substrates, palmitoyl CoA (16:0), palmitoleoyl CoA (16:1), and
gamma-linolenyl CoA (18:3), were differentially affected by Ebselen;
for example, the apparent Ki's of Ebselen for the condensation of 16:0,
16:1, and 18:3 in the absence of bovine serum albumin (BSA)
preincubation were 7, 14, and 34 microM, and those in the presence of
BSA preincubation were 35, 62, and 150 microM, respectively,
supporting earlier data for multiple condensing enzymes; (ii) that the
beta-ketoacyl CoA reductase-catalyzed reaction step which appears to
receive electrons, at least in part, from the cytochrome b5 system,
was also markedly inhibited by varying Ebselen concentrations; and
(iii) that similar results were obtained with the dehydrase and the
enoyl CoA reductase. Hence, each of the four component steps was
significantly inhibited by Ebselen. Another important fatty acid
biotransformation reaction, delta 9 desaturation of stearoyl CoA to
oleoyl CoA, was significantly inhibited (90%) by 30 microM Ebselen.
This effect appeared to be directly related to the NADH-dependent
electron transport chain rather than to a direct action on the
desaturase enzyme. Last, Ebselen also inhibited both aminopyrine and
benzphetamine N-demethylations, two cytochrome P450-catalyzed
reactions, in untreated rats, in rats on a high carbohydrate diet, and in
phenobarbital-treated rats.
PMID: 2563645 [PubMed - indexed for MEDLINE]
36: Arch Biochem Biophys. 1988 Nov 15;267(1):1-12.
Dual action of 2-decynoyl coenzyme A: inhibitor of hepatic
mitochondrial trans-2-enoyl coenzyme A reductase and
peroxisomal bifunctional protein and substrate for the
mitochondrial beta-oxidation system.
Nagi MN, Cook L, Laguna JC, Cinti DL.
Department of Pharmacology, University of Connecticut Health Center,
Farmington 06032.
The present study was designed to determine the action of the 2acetylenic acid thioester on mitochondrial fatty acid chain elongation
and beta-oxidation. Addition of 2-decynoyl CoA to a rat liver
mitochondrial suspension resulted in a significant stimulation of the
rate of oxidation of NADPH and NADH. This enhanced oxidation rate
was not due to the mitochondrial trans-2-enoyl CoA reductasecatalyzed conversion of the 2-acetylenic acid thioester to the saturated
product, decanoate, as measured by gas-liquid chromatography. On
the contrary, the mitochondrial trans-2-enoyl CoA reductase activity
was markedly inhibited by the 2-acetylenic acid derivative, as
evidenced by the decrease in the reduction of trans-2-decenoyl CoA to
decanoic acid. Incubation of the mitochondrial fraction with either
NADPH or NADH and 2-decynol CoA resulted in the gas
chromatographic identification of three products: beta-ketodecanoate,
beta-hydroxydecanoate, and trans-2-decenoate. In the absence of
reduced pyridine nucleotide, a single product was formed and
identified as beta-ketodecanoate. Confirmation of the identity of this
product was obtained by the observation of the formation of the
Mg2+-enolate complex (303-nm absorbance peak). These results
suggest that, although the 2-decynoyl CoA is an inhibitor of
mitochondrial trans-2-enoyl CoA reductase activity, it is a substrate for
the mitochondrial trans-2-enoyl CoA hydratase (crotonase). This was
confirmed by incubation of 2-decynoyl CoA with commercially purified
liver mitochondrial crotonase. The beta-ketodecanoate is formed in a
two-step process: hydration of the 2-decynoyl CoA to an unstable enol
intermediate which undergoes rearrangement to the betaketodecanoyl CoA. Interestingly, although the mitochondrial crotonase
can utilize the 2-acetylenic acid thioesters, this was not the case for
the peroxisomal bifunctional hydratase which was markedly inhibited
by varying concentrations of 2-decynoyl CoA.
PMID: 3058034 [PubMed - indexed for MEDLINE]
37: Arch Biochem Biophys. 1987 Feb 1;252(2):369-81.
Source of the hepatic microsomal trans-2-enoyl CoA hydratase
bifunctional protein: endoplasmic reticulum or peroxisomes.
Ghesquier D, Cook L, Nagi MN, MacAlister TJ, Cinti DL.
The present study was designed to investigate the hepatic localization
of the microsomal bifunctional trans-2-enoyl CoA hydratase. Despite
the low activity (less than 10%) of peroxisomal marker enzymes in
isolated hepatic microsomes (acyl CoA oxidase (this study), catalase,
and urate oxidase (L. Cook, M. N. Nagi, J. Piscatelli, T. Joseph, M. R.
Prasad, D. Ghesquier, and D. L. Cinti, 1986, Arch. Biochem. Biophys.
245, 24-26), additional evidence in this study suggests that the
microsomal enzyme is derived from peroxisomes. For example, the
microsomal hydratase activity was associated with the ribosomal
fractions but not with the smooth endoplasmic reticulum. In addition,
when an extract of the peroxisomal enzyme was incubated with either
free ribosomes or membrane-bound ribosomes, marked binding was
observed with each of the fractions. Furthermore, the ease of release
of the bifunctional enzyme from both free ribosomes and membranebound ribosomes by only KCl suggests that the bound enzyme is not a
nascent protein. Labeling of liver tissue from DEHP-treated rats with
rabbit immune IgG made to the purified microsomal hydratase
followed by gold conjugated goat anti-rabbit IgG suggested a single
subcellular site for the bifunctional hydratase--the peroxisomal
organelle.
PMID: 3813543 [PubMed - indexed for MEDLINE]
38: Arch Biochem Biophys. 1987 Feb 1;252(2):357-68.
Biochemical and immunological identity of the hepatic
peroxisomal and microsomal trans-2-enoyl CoA hydratase
bifunctional protein.
Cook L, Ghesquier D, Nagi MN, Favreau LV, Cinti DL.
In the present study, the hepatic microsomal and peroxisomal
bifunctional trans-2-enoyl CoA hydratases were isolated and purified
from rats treated with 2% di-(2-ethylhexyl)phthalate for 8 days. These
two enzymes (microsomal and peroxisomal) were purified with the
identical purification procedures and had identical molecular masses of
76 kDa. A single band was observed on an electrophoretic gel of an
equimixture of the two proteins. Both preparations had identical pI's of
8.6 and pH optima of 6.0 for the dehydrogenase (reductase) and 7.5
for the hydratase activity. Two-dimensional gel analysis of an
equimixture of the two preparations showed only one band.
Ouchterlony double-diffusion analysis showed that an antibody raised
against the purified microsomal enzyme interacted at a point with the
peroxisomal enzyme, indicating immunologic identity. Western blot
analysis demonstrated that the antibody formed a single band with
total microsomal and peroxisomal fractions. The antibody inhibited the
enzymatic activities of both preparations in a similar manner.
Interestingly, the antibody had a markedly greater inhibitory effect on
the reductase activity of the two enzyme preparations, and a much
less inhibitory effect on the hydratase activity, suggesting that the
antigenic determinants reside at or near the catalytic site of the
reductase portion of the protein. These results suggest that the
microsomal and peroxisomal bifunctional proteins are identical.
PMID: 3545080 [PubMed - indexed for MEDLINE]
39: Biochem Biophys Res Commun. 1986 Oct 15;140(1):74-80.
Do rat hepatic microsomes contain multiple NADPH-supported
fatty acid chain elongation pathways or a single pathway?
Nagi MN, Cook L, Prasad MR, Cinti DL.
[2-14C]-trans-2-hexadecenoyl CoA (16:1) and [2-14C]-trans-2-cis8,11,14-eicosatetraenoyl CoA (20:4) were chemically synthesized and
employed as competitive substrates for the liver microsomal trans-2enoyl CoA reductase component of the fatty acid chain elongation
system. Both 7.5 microM and 15 microM 20:4 competitively inhibited
the reduction of 16:1 CoA to palmitoyl CoA. In addition, the reduction
of both substrates was identically inhibited to the same extent by the
acetylenic derivative, dec-2-ynoyl CoA. Furthermore, trypsin,
chymotrypsin and subtilisin inhibited trans-2-enoyl CoA reductase
activity when three different substrates were employed--16:1, 20:4
and trans-2-cis-11-octadecadienoyl CoA (18:2). These results are
consistent with the hypothesis of multiple condensing enzymes
connected to a single elongation pathway.
PMID: 3778460 [PubMed - indexed for MEDLINE]
40: J Biol Chem. 1986 Oct 15;261(29):13598-605.
Site of inhibition of rat liver microsomal fatty acid chain
elongation system by dec-2-ynoyl coenzyme A. Possible
mechanism of inhibition.
Nagi MN, Cook L, Ghesquier D, Cinti DL.
The present study examines the effect of the acetylenic thioester dec2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty
acid chain elongation pathway in rat liver. When the individual
reactions of the elongation system were measured in the presence of
delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity
was markedly inhibited (Ki = 2.5 microM), whereas the activities of the
condensing enzyme, the beta-ketoacyl-CoA reductase, and the betahydroxyacyl-CoA dehydrase were not affected. The absence of
inhibition of total microsomal fatty acid elongation was attributed to
the significant accumulation of the intermediates, beta-hydroxyacylCoA and trans-2-enoyl-CoA, without formation of the saturated
elongated product, indicating that the trans-2-enoyl-CoA reductasecatalyzed reaction was the only site affected by the inhibitor. The
nature of the inhibition was noncompetitive. In contrast to the delta 2
10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit
trans-2-enoyl-CoA reductase activity, suggesting that the mode of
inhibition was not via formation of the 2,3-allene derivative. Based on
the observation (a) that p-chloromercuribenzoate markedly inhibits
reductase activity, (b) that dithiothreitol protects the enzyme against
inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral
manifestation of the interaction between thiol reagents and delta 2 10
identical to 1-CoA depicting an absorbance peak similar to that of the
beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar
absorbance spectrum formed by the interaction between delta 2 10
identical to 1-CoA and liver microsomes, and (e) of the absence of
formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver
microsomes, we propose that delta 2 10 identical to 1-CoA inactivates
trans-2-enoyl-CoA reductase by covalently binding to a critical
sulfhydryl group at or in close proximity to the active site of the
enzyme.
PMID: 3759985 [PubMed - indexed for MEDLINE]
41: Arch Biochem Biophys. 1986 Jul;248(1):408-18.
Induction of rat liver mitochondrial fatty acid elongation by the
administration of peroxisome proliferator di-(2-
ethylhexyl)phthalate: absence of elongation activity in
peroxisomes.
Nagi MN, Cook L, Ghesquier D, Cinti DL.
The administration of di-(2-ethylhexyl)phthalate (DEHP)3 to male
Sprague-Dawley rats resulted in more than a threefold increase in
activity of acetyl CoA-dependent hepatic mitochondrial fatty acid
elongation. Peroxisomes obtained either from control or DEHP-treated
rats were not capable of elongating any of the fatty acyl CoAs tested.
Furthermore, the peroxisomes possessed no trans-2-enoyl CoA
reductase activity. Therefore, the elongation activity in the 7500g
fraction from both control and DEHP-fed animals can be attributed
totally to the mitochondria. Maximal incorporation of acetyl CoA
occurred in the presence of both NADH and NADPH, and octanoyl CoA
(8:0) and decanoyl CoA (10:0) were found to be optimal primers for
fatty acid elongation in both control and DEHP-treated animals. The
apparent Km for 8:0 CoA was 17 microM in both animal groups while
the Vmax was increased from 4.5 to 12.5 nmol/min/mg following
treatment. The apparent Km for 10:0 CoA was 10 microM in both
control and DEHP-treated groups while the apparent Vmax increased
from 2.5 to 10 nmol/min/mg; palmitoyl-CoA (16:0) was a very poor
primer for chain elongation. Although the acetyl CoA-dependent fatty
acid elongation was stimulated by DEHP treatment, the mitochondrial
trans-2-enoyl CoA reductase activity was unaffected. The
mitochondrial total elongation activity following DEHP-treatment using
8:0 CoA as primer was about two times higher than enoyl CoA
reductase activity using trans-2-decenoyl CoA (10:1). This was the
result of accumulation of intermediates, which were identified as trans2-10:1 (35%), beta-hydroxy 10:0 (25%), unidentified (15%), and
elongated saturated product 10:0 (24%). Elongation by one acetate
unit was found in both the control and DEHP-treated animals. The
results are discussed in terms of physiological significance.
PMID: 3729425 [PubMed - indexed for MEDLINE]
42: J Biol Chem. 1986 Jun 25;261(18):8213-7.
Evidence for multiple condensing enzymes in rat hepatic
microsomes catalyzing the condensation of saturated,
monounsaturated, and polyunsaturated acyl coenzyme A.
Prasad MR, Nagi MN, Ghesquier D, Cook L, Cinti DL.
The condensation of palmitoyl-CoA with malonyl-CoA by rat hepatic
microsomes was competitively inhibited by myristoyl-CoA, whereas it
was noncompetitively inhibited by palmitoleoyl and gamma-linolenoylCoA. Furthermore, the condensation of palmitoleoyl-CoA with malonylCoA was also noncompetitively inhibited by gamma-linolenoyl-CoA.
Replacement of normal diet by a fat-free high carbohydrate diet
resulted in 8-, 2.5-, and 2.3-fold increases in the condensation rates of
both palmitoyl- and myristoyl-CoA, palmitoleoyl-CoA, and gammalinolenoyl-CoA, respectively. On the other hand, administration of di(2-ethylhexyl)phthalate (DEHP) resulted in a 2-fold stimulation of the
condensation activities with myristoyl- and palmitoyl-CoA, while those
with palmitoleoyl- and gamma-linolenoyl-CoA decreased to about 83
and 63%, respectively. Similar results following dietary changes or
DEHP administration were obtained for total elongation activities.
Finally condensation activities of 16:0, 16:1, and gamma-18:3 CoA
were differently affected by the proteolytic enzyme, chymotrypsin. The
competitive substrate studies, those of dietary and DEHP
administration, and the differential action of chymotrypsin strongly
suggest the existence of at least three discrete condensing enzymes
catalyzing the condensation of saturated, monounsaturated, and
polyunsaturated acyl-CoAs. These studies also indicate that the
condensation reaction is the regulating and rate-limiting step of the
fatty acid chain elongation system.
PMID: 3722151 [PubMed - indexed for MEDLINE]
43: Arch Biochem Biophys. 1986 Feb 15;245(1):24-36.
Hepatic subcellular distribution of short-chain beta-ketoacyl
coenzyme A reductase and trans-2-enoyl coenzyme A
hydratase: 25- to 50-fold stimulation of microsomal activities
by the peroxisome proliferator, di-(2-ethylhexyl)phthalate.
Cook L, Nagi MN, Piscatelli J, Joseph T, Prasad MR, Ghesquier
D, Cinti DL.
The present study demonstrates unequivocally the existence of shortchain trans-2-enoyl coenzyme A (CoA) hydratase and beta-ketoacyl
CoA reductase activities in the endoplasmic reticulum of rat liver.
Subcellular fractionation indicated that all four fractions, namely,
mitochondrial, peroxisomal, microsomal, and cytosolic contained
significant hydratase activity when crotonyl CoA was employed as the
substrate. In the untreated rat, based on marker enzymes and heat
treatment, the hydratase activity, expressed as mumol/min/g liver,
wet weight, in each fraction was: mitochondria, 684; peroxisomes,
108; microsomes, 36; and cytosol, 60. Following di-(2ethylhexyl)phthalate (DEHP) treatment (2% (v/w) for 8 days), there
was only a 20% increase in mitochondrial activity; in contrast,
peroxisomal hydratase activity was stimulated 33-fold, while
microsomal and cytosolic activities were enhanced 58- and 14-fold
respectively. A portion of the cytosolic hydratase activity can be
attributed to the component of the fatty acid synthase complex.
Although more than 70% of the total hydratase activity was associated
with the mitochondrial fraction in the untreated rat, DEHP treatment
markedly altered this pattern; only 11% of the total hydratase activity
was present in the mitochondrial fraction, while 49 and 29% resided in
the peroxisomal and microsomal fractions, respectively. In addition, all
four subcellular fractions contained the short-chain NADH-specific
beta-ketoacyl CoA (acetoacetyl CoA) reductase activity. Again, in the
untreated animal, reductase activity was predominant in the
mitochondrial fraction; following DEHP treatment, there was marked
stimulation in the peroxisomal, microsomal, and cytosolic fractions,
while the activity in the mitochondrial fraction increased by only 39%.
Hence, it can be concluded that both reductase and hydratase
activities exist in the endoplasmic reticulum in addition to
mitochondria, peroxisomes, and soluble cytoplasm.
PMID: 3511853 [PubMed - indexed for MEDLINE]
44: Arch Biochem Biophys. 1983 Oct 1;226(1):50-64.
Biochemical properties of short- and long-chain rat liver
microsomal trans-2-enoyl coenzyme A reductase.
Nagi MN, Prasad MR, Cook L, Cinti DL.
This study describes the biochemical properties of the rat hepatic
microsomal NADPH-specific short-chain enoyl CoA reductase and
NAD(P)H-dependent long-chain enoyl CoA reductase. Of the substrates
tested, crotonyl CoA and trans-2-hexenoyl CoA are reduced by the
short-chain reductase only in the presence of NADPH. The trans-2octenoyl CoA and trans-2-decenoyl CoA appear to undergo reduction
to octanoate and decanoate, respectively, catalyzed by both enzymes;
64% conversion of the C8:1 is catalyzed by the short-chain reductase,
while 36% conversion is catalyzed by the long-chain enzyme. For the
C10:1 substrate, 45% is converted by the short-chain reductase, while
55% is reduced by the long-chain reductase. trans-2-Hexadecenoyl
CoA is a substrate for the long-chain enoyl CoA reductase only.
Reduction of C4 and C6 enoyl CoA's was unaffected by bovine serum
albumin (BSA), whereas BSA markedly stimulated the conversion of
C10 and C16 enoyl CoA's to their respective saturated product.
Reduction rates as a function of microsomal protein concentration,
incubation time, pH, and cofactors are reported including the apparent
Km and Vmax for substrates and cofactors. In general, the apparent
Km's for the substrates ranged from 19 to 125 microM. The apparent
Vmax for the short-chain enoyl CoA reductase was greatest with trans2-hexenoyl CoA, having a turnover of 65 nmol/min/mg microsomal
protein, while the apparent Vmax for the long-chain enzyme was
greatest with trans-2-hexadecenoyl CoA, having a turnover of 55
nmol/min/mg microsomal protein. With respect to electron input,
NADPH-cytochrome P-450 reductase, either alone, mixed with
phospholipid, or incorporated into phospholipid vesicles, possessed no
enoyl CoA reductase activity. Cytochrome c did not affect the NADPHdependent conversion of the trans-2-enoyl CoA. In addition, antiNADPH-cytochrome P-450 reductase IgG did not inhibit the reduction
of trans-2-hexadecenoyl CoA in hepatic microsomes. Finally, the
NADPH-specific short-chain and NAD(P)H-dependent long-chain enoyl
CoA reductases were solubilized and completely separated from
NADPH-cytochrome P-450 reductase by employing DE-52 column
chromatography. These studies demonstrate the noninvolvement of
NADPH-cytochrome P-450 reductase in either the short-chain (13) or
long-chain enoyl CoA reductase system. Thus, the role of NADPHcytochrome P-450 reductase in the microsomal elongation of fatty
acids appears to be at the level of the first reduction step.
PMID: 6416174 [PubMed - indexed for MEDLINE]
45: Biochem Biophys Res Commun. 1983 Jun 15;113(2):659-65.
Kinetic evidence for two separate trans-2-enoyl CoA reductases
in rat hepatic microsomes: NADPH-specific short chain- and
NAD(P)H-dependent long chain-reductase.
Prasad MR, Nagi MN, Cook L, Cinti DL.
The rat hepatic microsomal conversion of crotonyl- and hexenoyl CoA
to butyrate and hexanoate was supported only by NADPH, while both
NADH and NADPH were effective cofactors in the conversion of trans2-hexadecenoyl CoA to palmitate. Experiments using mixtures of longand short-chain enoyl-CoA substrates and competition experiments
support the conclusion that microsomes contain 2 distinct enoyl CoA
reductases, (1) a long chain enoyl CoA reductase capable of accepting
reducing equivalents from either NADH or NADPH, and (2) a NADPHspecific short chain enoyl CoA reductase.
PMID: 6870879 [PubMed - indexed for MEDLINE]
46: J Biol Chem. 1982 Dec 10;257(23):14333-40.
Evidence for a second microsomal trans-2-enoyl coenzyme A
reductase in rat liver. NADPH-specific short chain reductase.
Cinti DL, Nagi MN, Cook L, White RE.
Evidence for the existence of a previously unknown rat hepatic
microsomal reductase, short chain trans-2-enoyl-CoA reductase (SC
reductase) is presented. This reductase has a specific requirement for
NADPH, is unable to utilize NADH, and catalyzes the conversion of
crotonyl-CoA and trans-2-hexenoyl-CoA to butyric acid and hexenoic
acid at a rate of 5 and 65 nmol per min per mg of microsomal protein,
respectively. Highly purified NADPH cytochrome P-450 reductase
incorporated into liposomes prepared from dilauroyl
phosphatidylcholine in the presence or absence of cytochrome P-450
possesses no SC reductase activity. These liposomal preparations did,
however, catalyze mixed function oxidations of benzphetamine and
testosterone. Rabbit antibody to rat liver NADPH cytochrome P-450
reductase had little to no effect on the conversion of crotonyl-CoA and
trans-2-hexenoyl-CoA, suggesting that the SC reductase accepts
reducing equivalents directly from NADPH. When acetoacetyl-CoA was
incubated with hepatic microsomes and either NADH or NADPH, no
formation of butyrate was detected; however, when both cofactors
were present, a rate of formation of 3 nmol of butyrate was
determined per min per mg of microsomal protein. These results
suggest the presence of a previously unknown short chain betaketoreductase which catalyzes the reduction of short chain beta-keto
acids, only in the presence of NADH. Our results also indicate that the
electrons from NADH to the beta-ketoreductase bypass cytochrome
b5. The physiological significance is discussed in terms of lipogenesis
and ketone body utilization by the liver.
PMID: 6815194 [PubMed - indexed for MEDLINE]