Nomega-nitro-L-arginine methylester ameliorates myocardial toxicity induced by doxorubicin.

Abstract

The effects of Nomega-nitro-L-arginine methylester (L-NAME) and L-arginine 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.

Full text

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Journal of Biochemistry and Molecular Biology, Vol. 36, No. 6, November 2003, pp. 593-596
© KSBMB & Springer-Verlag 2003
Nω ω-Nitro-L-Arginine Methylester Ameliorates Myocardial Toxicity
Induced by Doxorubicin
Mahmoud Ahmed Mansour*, Ayman Gamal El-Din, Mahmoud N. Nagi,
Othman A. Al-Shabanah and Abdullah M. Al-Bekairi
Department of Pharmacology, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
Received 15 April 2003, Accepted 10 June 2003
The effects of Nω ω-nitro-L-arginine methylester (L-NAME)
and L-arginine on cardiotoxicity that is induced by
doxorubicin (Dox) were investigated. A single dose of Dox
15 mg/kg i.p. induced
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.
cardiotoxicity, manifested
Keywords: Doxorubicin, L-NAME, L-arginine, Oxidative
stress, CPK, rat
Introduction
Doxorubicin (Dox), an anthracyclin antibiotic, is primarily
used in the treatment of a variety of human tumors, including
breast cancer, the small cell carcinoma of the lung, and acute
leukemia (Blum and Carter, 1974). However, the clinical use
of Dox has been seriously restricted because of the cardiotoxic
side effects (Singal et al., 1987). Consequently, there is great
interest in expanding the clinical usefulness of Dox by
developing new agents in order to reduce its cardiotoxicity
(Al-Shabanah et al., 1998a). Therefore, the administration of
various agents with Dox has been reported. N-acetylcysteine
(Doroshow et al., 1981), iron chelator of desferrioxamine (Al-
Harbi et al., 1992), probucol (Singal et al., 1995), captopril
(Al-Shabanah et al., 1998a), and thymoquinone (Al-Shabanah
et al., 1998b; Nagi and Mansour, 2000) have all been shown
to reduce cardiotoxicity that is induced by Dox in animals.
The cardiotoxic effect that is induced by Dox has been
attributed to various mechanisms. These include inhibition of
protein synthesis (Buja et al., 1973), changes in adrenergic
functions (Tong et al., 1991), alteration in sarcolemmal
calcium transport (Singal and Pierce, 1986), and lipid
peroxidation (Myers et al., 1977). However, the current
suggestion is that cellular damage that is induced by Dox is
mediated via the formation of free radicals (Siveski-Iliskovic
et al., 1994; Morishima et al., 1998; Xu et al., 2001). Tissues
with less developed antioxidant defense mechanisms, such as
the heart, are therefore highly susceptible to injury that is
induced by free radical generation (Doroshow, 1983).
Nitric oxide (NO) is one of the smallest biologically active
molecules that are produced from L-arginine by nitric oxide
synthase (NOS) (de-Belder and Radomiski, 1994). There are
three isoforms of the nitric oxide synthase: the endothelial
type (eNOS), the neuronal type (nNOS), and the isoform that
is expressed de novo by the exposure to proinflammatory
cytokines, the inducible type (iNOS). Under pathological
conditions, iNOS catalyzes an inadequate quantity of
inducible nitric oxide (iNO). The overproduction of iNO has
*Present address
Department of Biochemistry, Faculty of Pharmacy, Al-Azhar Univer-
sity, Nasr City, Cairo-Egypt
*To whom correspondence should be addressed.
Tel: 202-508-42-37; Fax: 202-26-33-996
E-mail: mansour1960us@yahoo.com

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594 Mahmoud Ahmed Mansour et al.
been implicated in the pathogenesis of a variety of
inflammatory and immnunologically-mediated diseases
(Misko et al., 1993).
Reportedly, NO can rapidly combine with superoxide to
form peroxynitrite. Peroxynitrite is potent and versatile; it can
attack a wide range of biological targets (Pryor and Squadrito,
1995). A previous report showed that aminoguanidine, an
inducible nitric oxide synthase inhibitor, protects against
cardiotoxicity that is induced by Dox (Mostafa et al., 1999).
Therefore, the goal of the present study was to evaluate the
role of NO in the cardiotoxicity that is induced by Dox. This
aim will be achieved by the study of the effect of L-arginine as
a substrate for the NO formation, and L-NAME as the non-
specific nitric oxide synthase inhibitor on the cariotoxicity that
is induced by Dox.
Materials and Methods
Materials
(St. Louis, USA), while doxorubicin (Dox) was obtained from
Farmitalia (Milan, Italy). Thiobarbituric acid (TBA) was a product
of Fluka (Buchs, Switzerland). All of the remaining chemicals were
of the highest grade commercially available.
L-arginine and L-NAME were purchased from Sigma
Animals
all of the experiments. They were obtained from the Experimental
Animal Care Center of King Saud University, Riyadh, KSA. The
animals were maintained under standard conditions of a temperature
of 24± 1oC and 55± 5% relative humidity with a regular 12h light/
12h dark cycle. They were allowed free access to standard laboratory
food (Purina Chow) and water.
Male swiss albino rats, weighing 200-250g, were used in
Experimental protocol
six groups of 10 rats each. The first group (control) received
vehicles that were used for Dox (physiological saline solution, i.p.).
Throughout the duration of the experiments, the second and third
groups received L-arginine (70 mg/kg/d p.o.) and L-NAME in their
drinking water (4 mg/kg/d p.o.), respectively. The calculated doses
of L-arginine and L-NAME were based on the average daily intake
of water. The fourth group was injected with Dox (15 mg/kg i.p).
The fifth group received L-arginine (70 mg/kg/d p.o.) in their
drinking water for 5 consecutive days before and 3 days after the
Dox injection (15 mg/kg i.p.). The last group received L-NAME (4
mg/kg/d p.o.) in their drinking water for 5 consecutive days before
and 3 days after the Dox injection. Based on the preliminary data
from our laboratory, the selected concentrations of L-arginine and
L-NAME and the schedule of doses were chosen.
On the third day after the beginning of the Dox injection, blood
samples were drawn from the orbital plexus under light ether
anesthesia into non-heparinized tubes. Serum was separated by
centrifugation for 5 min at 4,000 rpm and stored at −20oC until
analysis.
The animals were divided at random into
Isolation and preparation of heart homogenates
collection of blood samples, the rats were sacrificed by cervical
dislocation. The upper abdomen was opened and the heart was
Following the
quickly isolated, washed with saline, blotted dry on filter paper, and
weighed. Thereafter, 10% (w/v) homogenate of the heart was made
in ice-cold saline using a Branson sonifier (250, VWR Scientific,
Danbury, USA).
Measurement of serum biochemical parameters
creatine phosphokinase (E.C. 2.7.3.2) was determined according to
the method of Gruber, 1978.
Serum
Determination of lipid peroxides and catalase activity in heart
homogenates
Tissue lipid peroxides (malondialdehyde (MDA)
production) in the heart tissue homogenates were determined as a
thiobarbituric acid-reactive substance (Ohkawa et al., 1979). The
absorbance was measured spectro-photometrically at 532nm and the
concentration is expressed as nmol MDA/g tissues. Catalase activity
was measured in the heart homogenate according to Higgins et al.,
1978. The tissue levels of the acid soluble thiols, mainly reduced
glutathione (GSH), was determined colorimetrically at 412nm
(Ellman, 1959).
Statistical analysis
statistical comparison between the different groups was performed
using a one-way analysis of the variance (ANOVA) followed by a
Tukey-Kramer multiple comparison test to judge the difference
between the various groups.
Data are expressed as (means ± SEM). A
Results
Effect of L-NAME and L-arginine on the elevated serum
creatine phosphokinase activity induced by Dox
demonstrates the effect of L-arginine (70 mg/kg/d p.o.) and L-
NAME (4 mg/kg/d p.o.) on the elevated CPK level that is
induced by a single injection of Dox (15 mg/ kg i.p.).
The intraperitoneal administration of Dox caused cardiac
toxicity in all of the rats. Serum CPK was significantly
increased, to 6-fold of the control value. Pretreatment of the
animals with L-NAME 5 days before and 3 days after a single
injection of Dox significantly reduced the elevated activity of
CPK by 78%. However, the oral administration of L-arginine
(70 mg/kg/d p.o.) did not reduce the cardiac toxicity that was
induced by the Dox administration.
Table 1
Effect of L-NAME and L-arginine on Dox-induced
changes in lipid peroxides in heart homogenates
shows the effect of the oral supplementation of L-arginine (70
mg/kg/d p.o.) and L-NAME (4 mg/kg/d p.o.) on the elevated
lipid peroxides that are induced by a single injection of Dox
(15 mg/kg i.p.).
A single injection of Dox induced a significant increase in
lipid peroxides, measured as malondialdhyde (MDA).
However, there was no difference in the reduced glutathione
or catalase activity. Pretreatment with L-NMAE (4 mg/kg/d
p.o.) prevented a rise in lipid peroxides. However, the
administration of L-arginine (70 mg/kg/d p.o.) produced no
significant decrease in the elevated level of MDA.
Table 1

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L-NAME and Cardiotoxicity Induced by Doxorubicin595
Discussion
In the present study, the cardiac damage that is induced by
Dox administration (15 mg/kg i.p.) was confirmed by a
significant increase in serum CPK. The cardiac toxicity was
also reflected in the heart tissue by the significant elevation of
lipid peroxides.
Pretreatment of the rats with L-NAME (4 mg/kg/d p.o.) or
L-arginine (70 mg/kg/d p.o.) for 10 days induced no changes
in the biochemical parameters that were measured. However,
providing L-NAME with the drinking water (5 consecutive d
before and then continuing for another 3 consecutive d) to the
Dox-treated animals produced an improvement in the cardiac
enzyme CPK when compared to the Dox-treated animals.
This improvement was evidenced in the serum since the
elevated level of the serum CPK activity was significantly
lowered. In addition, the attenuation of cardiac toxicity was
also observed in the heart tissue. L-NAME prevented a rise in
lipid peroxides, measured as MDA. However, pretreatment of
the rats with L-arginine (70 mg/kg/d p.o.) for 5 consecutive
days before and 3 days after the single dose of the Dox
injection did not protect the cardiac tissues against toxicity
that is induced by Dox.
The rationale for the L-NAME and L-arginine dose
schedule in this study was to maintain a study with sufficient
plasma concentration before, during, and after the critical
period of the Dox-induced cardiac toxicity. It is of critical
importance that the biochemical changes occur in the heart
within a few hours of the Dox administration. The time period
for our study was based on our own preliminary experiments
that showed the maximum cardiac injury that is induced by
Dox treatment.
NO was reported to be involved in the diverse physiological
and pathophysiological process, including the host immune
defense, vasoregulation, and pathogenesis of diabetes (Corbett
et al., 1992; Nathan, 1992; Choi et al., 2002). It was reported
that large amounts of NO and superoxide were produced in rat
hearts with experimental myocarditis (Ishiyama et al., 1997).
Nitric oxide reacts with superoxide and forms a peroxynitrite,
which is a powerful oxidant and causes tissue damage (Pryor
and Squadrito, 1995).
Many investigators reported that Dox may be capable of
generating the reactive oxygen species, thereby increasing the
lipid peroxidation that is initiated by the hydroxyl radicals that
are formed from the combination of superoxide and hydrogen
peroxide and free iron (Vivar et al., 1997; Myers, 1998). These
toxic free radicals cause myocardial damage (Doroshow, 1983;
Kaul et al., 1993). In the present study, the relation between
Dox treatment and cardiotoxicity (manifested by increasing
lipid peroxides) are positively correlated. However, it has been
demonstrated that acute and chronic Dox treatment, inhibiting
glutathione synthesis (Doroshow et al., 1981) and the severe
depletion of glutathione, is known to be associated with
increasing lipid peroxidation (Siveski-Iliskovic et al., 1994). In
the present study, no difference was discovered in the
endogenous glutathione level. This may be explained by the
difference in experimental design, since we measured
glutathione after 3 days of Dox treatment.
The results of the present study clearly demonstrate that L-
NAME provides protection against cardiotoxicity that is
induced by Dox treatment. Therefore, the amelioration of
cardiac toxicity, induced by Dox by the pretreatment with L-
NAME, may confirm the implication of NO in the cardiac
toxicity that is induced by Dox.
However, the reported antioxidant effects of L-NAME (Seif
El-Nasr and Fahim, 2001) may not be excluded. It is, therefore,
difficult to assess which of these properties of L-NAME is
responsible for this protective effect, since the mechanism of
cardiotoxicity that is induced by Dox is still uncertain.
Our results indicate that L-NAME is beneficial as a
protective agent against cardiotoxicity that is induced by Dox
in normal rats. Further studies are needed to elucidate the
mechanism(s) of protection and the effect of L-NAME on the
antitumor activity of Dox.
Table 1. Effect of L-NAME and L-arginine pretreatment on Dox induced changes on rat serum creatine phosphokinase activity and
lipid peroxides (MDA), glutathione content, and catalase activity in rat heart homogenates
ParametersControl L-NAME L-arginineDoxDox + L-NAME Dox + L-arginine
CPK (U/l) 156 ± 5192 ± 23144 ± 13 957 ± 264*** 211 ± 60##467 ± 82
MDA
(nmol/g tissue)
398 ± 44459 ± 58456 ± 48826 ± 101**538 ± 43#617 ± 63
GSH
(µmol/g tissue)
3.0 ± 0.1 2.5 ± 0.1 2.7 ± 0.22.7 ± 0.12.8 ± 0.23 ± 0.1
Catalase
(mmol/min/g)
5.8 ± 0.35 ± 0.45.2 ± 0.46.4 ± 0.4 5.5 ± 0.35.6 ± 0.2
All data represent mean values ± SEM.
L-arginine (70 mg/kg/d p.o.) and L-NAME (4 mg/kg/d p.o.) were given in drinking water for 5 consecutive days before and 3 days after
Dox administration. Blood samples were obtained 3 days after Dox (15 mg/kg i.p.).
Significant difference from control group. **P<0.01
Significant difference from Dox group. #P<0.05

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596 Mahmoud Ahmed Mansour et al.
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