Pharmacology & Pharmacy, 2013, 4, 590-598
Published Online November 2013 (http://www.scirp.org/journal/pp)
http://dx.doi.org/10.4236/pp.2013.48085
Open Access PP
Comparative Assessment of Melatonin-Afforded
Protection in Liver, Kidney and Heart of Male Mice
against Doxorubicin Induced Toxicity
Abdullah A. Alghasham
Department of Pharmacology and Therapeutics, College of Medicine, Qassim University, Qassim, KSA.
Received September 10th, 2013; revised October 15th, 2013; accepted October 28th, 2013
Copyright © 2013 Abdullah A. Alghasham. This is an open access article distributed under the Creative Commons Attribution Li-
cense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Melatonin (MEL) was investigated for protection against the anthracycline antibiotic doxorubicin (Dox) that is well
known for its oxidative damage to various body organs. It was aimed to have a comparison of this protection to heart,
liver and kidney in the treated subjects. In this study, groups of mice were treated with Dox and melatonin and their in-
dividual or combined effects were evaluated by assessing lipidperoxidation, non-protein sulfhydryls (NP-SH) and ni-
trate/nitrite (NO) contents in these tissues. Plasma aminotransferases, LDH and CK-MB enzyme activities were meas-
ured. Moreover, these tissues were subject to histopathological assessment. MEL co-treatment significantly prevented
any rise in lipidperoxides more significantly in heart and liver as compared to kidney. In tandem, MEL prevented a de-
cline in GSH that was observed by Dox alone in liver and kidney. Dox significantly increased total NO levels in all the
tissues. Melatonin at both dose levels could not afford protection against nitrosative stress. MEL in combination treat-
ment provided significant (P < 0.01) decline in CK-MB at both the doses and only 5 mg/kg dose significantly prevented
a rise in LDH activity and prevented any histopathological change. Melatonin, probably by behaving as an antioxidant
prevented Dox-induced lipidperoxidation in heart, liver and kidney tissues and a decline in NP-SH. However, admini-
stration of MEL is able to decrease parameters of oxidative, and nitrosative stress in heart and liver more effectively
than kidney.
Keywords: Melatonin; Doxorubicin; Heart; Liver; Kidney; Lipidperoxidation; Non-Protein Sulfhydryls; Nitric Oxide;
Plasma Enzymes; Mice
1. Introduction
Doxorubicin (Dox), an anthracycline antibiotic, is pri-
marily used in the treatment of a variety of pathological
states including breast cancer, small cell carcinoma of
lung, and acute leukemia [1]. The clinical usage of this
agent is restricted due to its toxic manifestations to heart
[2], liver and kidney [3,4] which are attributed to its re-
dox activation to a semiquinone intermediate resulting in
generation of superoxide radicals [5,6]. Consequently,
there is immense interest in escalating the clinical use-
fulness of Dox by developing new adjuncts that could
result in diminution of its toxicity. Hence, the admini-
stration of a variety of antioxidants with Dox has been
reported. N-acetylcysteine [7], desferrioxamine [8], pro-
bucol [9], captopril [10,11], and thymoquinone [12,13]
have all been shown to reduce Dox induced cardiotoxic-
ity in experimental models. The reported mechanisms by
which these cardiotoxic effects are manifested include
alteration in sarcolemmal calcium transport [14], and
lipid peroxidation [15] mediated via the formation of free
radicals [16-18]. Tissues with less developed antioxidant
defense mechanisms, especially the heart, are therefore
highly susceptible to injury that is induced by free radical
generation [19]. An increasing evidence indicates that
NO mediates a plethora of actions through cGMC-inde-
pendent mechanisms [20-24]. Nitric oxide (NO•), which
is synthesized by a family of nitric oxide synthase (NOS)
and all of them have been localized in the kidney [25]
may lead to production of reactive nitrogen species
(RNS), such as peroxynitrite (ONOO−), peroxynitrous
acid, nitryl chloride (NO2Cl) and nitrogen dioxide radical
() [26] resulting in nitrosative stress. A target for
•
2
NO

Comparative Assessment of Melatonin-Afforded Protection in Liver, Kidney and Heart of Male Mice
against Doxorubicin Induced Toxicity
591
NO is sulfhydryl groups on proteins, to form S-nitro-
sothiol (SNOs) compounds. Reasonable levels of sulfhy-
dryls are thought to be more relevant in offering protec-
tion against free radical production during treatment with
Dox.
Melatonin a main hormone of pineal gland is impli-
cated in a variety of physiological processes. Regulation
of endocrine rhythms [27], antigonadotropic effects [28],
neuroprotective effects, and stimulation of the immune
function [29] have been reported to be affected by mela-
tonin. The reports on its in vitro effects have shown that
melatonin functions as an antioxidant, i.e. a scavenger of
the hydroxy radical and peroxy radical [30,31]. It has
also been shown that melatonin affords substantial pro-
tection against the oxidative destruction of lipids. An-
other study [32] has demonstrated that melatonin dose
dependently attenuated the increase in both ROS and
lipid peroxidation by acetaminophen.
Based on the rationale that melatonin functions as an
antioxidant and scavenge hydroxy and peroxy radicals
[30,31] the experiments were designed to explore mela-
tonin in vivo using biochemical parameters like lipidper-
oxides, glutathione, and nitric oxide levels as indicators
of free radical status in the heart, liver and kidney tissues
of mice treated with doxorubicin (adriamycin) known to
be a free radical producing drug. Similarly, plasma ami-
notransferases as appraise of hepatic damage, LDH as a
measure of kidney function and CK-MB, cardiac marker,
enzyme activities were measured. Furthermore, histopa-
thological assessment of these tissues was performed to
find possible evidence. Relative protection offered by
melatonin to these organs was also assessed.
2. Material and Methods
2.1. Animals
Experimental design and use of animals were approved
by a local ethical committee (Experimental animal han-
dling, care and use committee of College of Medicine,
Qassim University, Qassim, Saudi Arabia). Male Swiss
albino (SWR) mice, weighing 25 - 30 g were used. The
animals were housed in groups and were kept at con-
trolled temperature (22˚C ± 1˚C), relative humidity (50%)
and light cycle (7:0 am - 7:0 pm). Food and water were
made freely accessible.
2.2. Drugs and Chemicals
Doxorubicin (Dox; adriamycin) was obtained from Far-
mitalia (Milan, Italy). Thiobarbituric acid (TBA) was a
product of Fluka (Buchs, Switzerland). For determination
of nitrates and nitrites, N-(1-Naphthyl)-ethylenediamine
dihydrochloride (NEDD), sulfanilamide (SULF), vana-
dium(III) chloride (VCl3) were all purchased from
Sigma-Aldrich. Solid VCl3 was stored in the dark under
vacuum. All other chemicals and reagents used in this
study were of analytical reagent grade procured from
commercial sources.
In all experiments Dox was injected intraperitoneally
(i.p.) 30 min after melatonin. Two dose levels for mela-
tonin were tested in vivo. The sacrifice timing of the
treatment groups was kept at 6 hours after the last dosage.
The reason for selecting this time point was our prelimi-
nary work where maximal response of melatonin was
found to be between 5 - 6 hours of the treatment with the
same dose regimen by i.p. route. A total of 72 mice were
randomly assigned to 2 sets. One set of six experimental
groups of mice was treated as follows: 1) vehicle control
(Saline); 2) doxorubicin 2.5 mg/kg/day, for consecutive
five days [33]; 3) melatonin 1 mg/kg, for consecutive
five days; 4) melatonin 1 mg/kg, for consecutive five
days followed by doxorubicin 2.5 mg/kg/day; 5) mela-
tonin 5 mg/kg, for consecutive five days; 6) melatonin 5
mg/kg, for consecutive five days followed by doxorubi-
cin 2.5 mg/kg/day.
Six hours after the last dosage, animals from each
group were killed by cervical dislocation. In the first set,
abdomen was incised and the liver, kidney and heart tis-
sues from each group were excised of the body. A part of
freshly isolated tissue was cut and used for the analysis
of glutathione contents. The other part of these tissues
were immediately frozen by using HistoFreeze™ (Fisher
Scientific Company, Pittsburgh, PA, USA) and kept at
−40˚C until used for the determination of lipid peroxides
or NO as a measure for the level of free radicals. From
the second set of treatment groups, blood was collected
under light ether anesthesia and plasma was isolated to
use in assessment of enzyme activities. These animals
were then killed by cervical dislocation and their liver,
kidney and heart tissues were preserved in buffered for-
malin and used in histological studies after processing.
2.3. Estimation of Lipid Peroxides
The method described by Ohkawa et al., [34] was used
with some modifications. Malondialdehyde (MDA) was
measured as an indicator of lipidperoxidation. Liver,
heart or kidney tissues were thawed and homogenized in
aqueous KCl solution, supplemented with 0.5% butylated
hydroxytoluene, by using Ultra-Turrax® (Janke and Kun-
kel GmbH & Co. KG IKA-WERK Staufen) homoge-
nizer at 20 × 1000 rpm for few seconds. The aliquots of
tissue homogenate were immediately treated with 20%
acetic acid (pH 3.5) and sodium dodecyl sulphate was
added. The whole mixture was incubated with thiobarbi-
turic acid (0.8% aqueous) for one hour at 95˚C using ball
condensers and cooled to room temperature. After cen-
trifugation the pink clear layer was extracted and read on
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Comparative Assessment of Melatonin-Afforded Protection in Liver, Kidney and Heart of Male Mice
against Doxorubicin Induced Toxicity
592
a spectrophotometer against reagent blank. Malondial-
dehyde bis (dimethyl acetal) tetra ammonium salt was
used as an external standard.
2.4. Estimation of Non-Protein Sulfhydryl
Groups (NP-SH)
The tissue levels of the acid soluble thiols, mainly re-
duced glutathione (GSH) in liver, heart or kidney tissue
were measured as described by Sedlak and Lindsay [35].
The fresh tissues were immediately homogenized in ice
cold 0.02 M ethylenediaminetetraacetic acid disodium.
Aliquots of tissue homogenate were treated with 50%
w/v trichloroacetic acid while shaking, kept for 15 min
and centrifuged. Supernatant fractions were mixed with
Tris buffer pH 8.9; 5-5’-dithiobis-(2-nitrobenzoic acid)
(DTNB) was added. After mixing the contents, samples
were colorimetrically read at 412 nm within 5 min of the
addition of DTNB against reagent blank with no ho-
mogenate. Reduced glutathione was used as an external
standard.
2.5. Determination of Total Nitrate and Nitrite
(NO)
The level of NO in tissues was determined in the form of
total nitrate and nitrite the stable main metabolite of NO.
A colorimetric method adopted from Miranda et al. [36]
was used. The procedure started with deproteinization of
300 μl of tissue homogenate by the addition of an equal
amount of methanol. The mixture was shaken and set
aside in refrigerator till protein sedimentation. Then the
tubes were centrifuged at 3000 rpm for 10 - 15 min. An
aliquot of 300 μl of the clear supernatant was then aspi-
rated and mixed with an equal volume of VCl3 followed
by another 300 μl of a mixture containing equal amounts
of sulfanilic acid (SULF) and N-(1-Naphthyl)-ethyl-
enediamine (NEDD) premixed just prior to the assay.
Reagent blank was the same but using 300 μl of distilled
water in place of sample. Absorbance was measured at
540 nm using a spectrophotometer (UV1201; Schimadzu
Corporation, Japan) after 30 - 45 min incubation at 37˚C.
Kits for enzymes activities of glutamic-oxaloacetic
transaminase (AST, EC 2.6.1.1), glutamic-pyruvic trans-
aminase (ALT, EC 2.6.1.2), lactate dehydrogenase (LDH,
EC 1.1.1.27) from United Diagnostic Industry, Dammam
Saudi Arabia were used. Kits for the determination of
CK-MB were procured from Boehringer Mannheim,
GmbH, Mannheim, Germany.
2.6. Histopathological Procedure
Tissue from the liver, heart and kidney was excised from
each group and fixed in neutral buffered formalin solu-
tion for 24 h. After fixation, each sample was dehydrated,
cleared and embedded in paraffin wax. Sections of about
5 µm thickness were cut with an American optical rotary
microtome. These sections were stained with Harris’
hematoxylin and counter stained with eosin, using the
routine procedure [37]. The slides were examined under
the microscope for pathomorphological changes such as
necrosis, degeneration, vacuolization, inflammation, pa-
renchymal cell death, distortion, collagen formation, al-
teration in cell size, presence of nuclei, presence of Kupf-
fer cells, regeneration and severity of histopathological
state.
3. Statistical Analysis
GraphPad InStat® 3.10, version 1.3.2 was employed for
statistical inference. Data are expressed as mean ± SEM.
The one-way analysis of variance (ANOVA) followed by
Tukey-Kramer multiple comparisons is used to analyze
the data. P < 0.05 is considered to be statistically signifi-
cant.
4. Results
4.1. Effect of Melatonin on Tissue
Lipidperoxides
Dox increased lipidperoxide contents significantly (P <
0.001) in heart, liver and kidney tissues when used alone
in mice for consecutive five days. Treatment with mela-
tonin was not different from control group. Co-treatment
with melatonin in combination groups prevented rise in
levels of lipid peroxides significantly at 5 mg/kg dose.
This protection was more pronounced in heart and liver
tissue being significant at P < 0.001. Whereas, protection
afforded by melatonin was significant at P < 0.05 in the
kidney tissue (Table 1).
4.2. Effect of Melatonin on Tissue Non-Protein
Sulfhydryl Contents
Dox treatment for five days declined NP-SH contents
significantly (P < 0.001) in all the three tissues analyzed.
On the other hand GSH levels remained essentially at
base levels close to the control group in heart, liver and
kidney tissues after treatment with melatonin. Similarly,
melatonin at 1 mg/kg dose failed to prevent any change
in GSH contents that were significantly reduced by Dox.
Melatonin co-treatment at 5 mg/kg dose level protected
heart and liver tissue significantly (P < 0.001). The pre-
vention in GSH decline was not significant in kidney
tissue at both of the doses of melatonin (Table 2).
4.3. Effect of Melatonin on Serum Nitrite and
Nitrates (NO)
Dox treatment for consecutve five days increased NO i
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Comparative Assessment of Melatonin-Afforded Protection in Liver, Kidney and Heart of Male Mice
against Doxorubicin Induced Toxicity
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593
Table 1. Effect of melatonin on lipid peroxidation (nmol/g wet tissue) induced by doxorubicin in liver, kidney and heart tissue
of mice.
Group Treatment Heart Kidney Liver
1 Vehicle control 267.8 ± 6.25 212.55 ± 5.84 268.45 ± 7.51
2 Doxorubicin 403.42 ± 7.21*** 326.05 ± 8.68*** 392.18 ± 8.06***
3 Melatonin 1 mg/kg 296.18 ± 8.25# 210.75 ± 9.12# 272.16 ± 6.99#
4 Melatonin 1 mg/kg, +doxorubicin 400.10 ± 6.46***@ 320.11 ± 8.70***@ 378.90 ± 7.12***
5 Melatonin 5 mg/kg 301.30 ± 7.75*# 231.21 ± 11.45# 288.60 ± 8.18$
6 Melatonin 5 mg/kg + doxorubicin 321.18 ± 8.12***# 255.64 ± 7.72*# 348.45 ± 7.95***#
Six animals were used in each treatment group. Values are expressed as Mean ± SEM. Treatments were given daily for consecutive five days. Statistical
analysis was done by One Way ANOVA followed by Tukey-Kramer multiple comparisons test in each column independently. All the groups were compared to
control. *P < 0.05; **P < 0.01 and ***P < 0.001. #P < 0.001 and $P < 0.01 compared to doxorubicin alone; @P < 0.001 Group 6 compared to Group 4.
Table 2. Effect of melatonin and doxorubicin on non-protein sulfhydryl contents (nmol/100mg wet tissue) in liver, kidney and
heart tissue in mice.
Group Treatment Heart Kidney Liver
1 Vehicle control 56.45 ± 3.11 42.45 ± 2.49 66.32 ± 3.16
2 Doxorubicin 28.42 ± 2.57*** 31.40 ± 2.69 43.18 ± 2.91***
3 Melatonin 1 mg/kg 53.10 ± 2.61***# 44.10 ± 3.15∆ 64.26 ± 2.90#
4 Melatonin 1 mg/kg, +doxorubicin 30.45 ± 2.78 32.70 ± 2.91 55.92 ± 2.78∆
5 Melatonin 5 mg/kg 52.15 ± 2.70# 40.01 ± 2.33 61.11 ± 3.12$
6 Melatonin 5 mg/kg + doxorubicin 41.49 ± 2.85**∆ 37.55 ± 2.15 48.44 ± 2.70**
Six animals were used in each treatment group. Values are expressed as Mean ± SEM. Treatments were given daily for consecutive five days. Statistical
analysis was done by One Way ANOVA followed by Tukey-Kramer multiple comparisons test in each column independently. All the groups were compared to
control. *P < 0.05; **P < 0.01 and ***P < 0.001. #P < 0.001, $P < 0.01 and ∆P < 0.05 compared to doxorubicin alone; @P < 0.001 Group 6 compared to Group 4.
levels significantly (P < 0.001 in heart and liver and P <
0.01 in kidney tissue). Melatonin at 1 mg/kg dose could
not afford protection against Dox in combination treat-
ment and NOlevels remained close to Dox alone. How-
ever, melatonin 5 mg/kg prevented rise in NO contents
but was not significantly low and did not reach to control
group in any of the tissues analyzed (Table 3).
4.4. Effect of Melatonin on Plasma Biochemical
Parameters
Melatonin alone did not have any significant effect on
plasma enzyme activities at both dose levels. Dox treat-
ment for consecutive five days significantly elevated
aminotransferases, LDH and CK-MB. In the combined
treatment groups, melatonin treatment significantly pre-
vented any rise in CK-MB activities at both the dose lev-
els. Melatonin at 5 mg/kg was found to decrease LDH
activities significantly (P < 0.05). However, both the
doses of melatonin failed to avert any rise in activities of
aminotransferases in plasma of Dox treated groups (Ta-
ble 4).
4.5. Effect of Melatonin on Doxorubicin Induced
Histological Changes
Low dose of melatonin in combination with Dox has
shown mild symptoms of fine granulated and eosinic
cytoplasm with leucocytic infiltration and hyperchroma-
cia in the cardiac tissue of mice. This shows a relative
failure of melatonin 1 mg/kg dose to prevent any change
in the heart tissue histological features (Figure 1). Simi-
larly the kidneys from the same treatment group showed
degenerative changes marked as cloudy swelling, tubular
cell necrosis and sloughing in the tubular lumens (Figure
2). Hepatocytic vacuolation with mononuclear inflam-
matory cell infiltration was noticed in this treatment
group (Figure 3). Co-treatment with melatonin 5 mg/kg
and Dox demonstrated significant protection to the car-
diac tissue that appeared almost normal in its architecture
with few myocardial cells showing hypertrophy. Slides
of kidney tissue from this group appeared normal in the
architecture except interstitial edema with mononuclear
cell infiltration. Liver tissue in this group appeared nor-
mal except few hepatocytes typify atrophied.
5. Discussion
A number of research reports indicate that melatonin
effectively attenuated Dox-induced cardiotoxicity [38],
nephrotoxicity [39] and hepatotoxicity [40] in experi-
mental models. It was suggested that melatonin has anti-
oxidant potential [41] and might play an important role in
the attenuation of free radical induced toxicity to heart,

Comparative Assessment of Melatonin-Afforded Protection in Liver, Kidney and Heart of Male Mice
against Doxorubicin Induced Toxicity
594
Table 3. Effect of Melatonin pretreatment on NO contents (μmol/g wet tissue) in heart, kidney and liver tissue of mice treated
with doxorubicin.
Group Treatment Heart Kidney Liver
1 Vehicle control 406 ± 41 724 ± 63 644 ± 41
2 Doxorubicin 705 ± 53*** 1044 ± 64** 898 ± 38***
3 Melatonin 1 mg/kg 422 ± 47$ 740 ± 55$ 656 ± 42$
4 Melatonin 1 mg/kg, +doxorubicin 742 ± 48*** 1048 ± 61** 848 ± 31***
5 Melatonin 5 mg/kg 428 ± 42$ 742 ± 49$ 658 ± 36$
6 Melatonin 5 mg/kg + doxorubicin 541 ± 41@ 868 ± 49 695 ± 39
Six animals were used in each treatment group. Values are expressed as Mean ± SEM. Treatments were given daily for consecutive five days. Statistical
analysis was done by One Way ANOVA followed by Tukey-Kramer multiple comparisons test in each column independently. All the groups were compared to
control. *P < 0.05; **P < 0.01 and ***P < 0.001. #P < 0.001, $P < 0.01 and ∆P < 0.05 compared to doxorubicin alone; @P < 0.05 Group 6 compared to Group 4.
Table 4. Effect of melatonin and doxorubicin on plasma biochemical parameters (U/L, Mean ± SEM) in mice.
Group Treatment ALT AST LDH CK-MB
1 Vehicle control 19.61 ± 0.98 14.54 ± 1.60 248.66 ± 18.23 149.65 ± 12.44
2 Doxorubicin 29.36 ± 1.95** 24.20 ± 1.21** 404.5 ± 12.45*** 325.35 ± 19.38***
3 Melatonin 1 mg/kg 21.33 ± 1.79∆ 16.58 ± 1.81∆ 261.35 ± 21.48# 168.90 ± 19.8#
4 Melatonin 1 mg/kg + doxorubicin 26.48 ± 1.98 23.32 ± 0.99** 361.32 ± 22.72** 238.05 ± 11.90**$
5 Melatonin 5 mg/kg 22.03 ± 1.69∆ 17.11 ± 1.51∆ 265.76 ± 21.70# 195.55 ± 10.69#
6 Melatonin 5 mg/kg + doxorubicin 23.36 ± 1.39 20.22 ± 2.05 308.95 ± 14.14∆ 239.80 ± 4.85**$
Six animals were used in each treatment group. Values are expressed as Mean ± SEM. Treatments were given daily for consecutive five days. Statistical
analysis was done by One Way ANOVA followed by Tukey-Kramer multiple comparisons test in each column independently. All the groups were compared to
control. *P < 0.05; **P < 0.01 and ***P < 0.001. #P < 0.001, $P < 0.01 and ∆P < 0.05 compared to doxorubicin alone; @P < 0.05 Group 6 compared to Group 4.
Figure 1. Micrograph showing granulated and eosinic cyto-
plasm, leucocytic infiltrate and hyperchromacia in cardiac
tissue in melatonin 1 mg/kg and Dox treated mice (H & E
×400).
liver and kidney that are produced by metabolic path-
ways of doxorubicin. In the present study, doxorubicin
treatment resulted in an increase of malondialdehyde
(MDA) in all the tissues analyzed. Heart tissue was most
susceptible to deleterious effects of Dox reflected by an
increase in levels of MDA, NO and elevated activity of
CK-MB with a concomitant decrease in the contents of
NP-SH. Furthermore, histological findings paralleled the
levels of MDA. Doroshow, [19] suggested that tissues
with less developed antioxidant defense mechanisms,
Figure 2. Micrograph showing degenerating cloudy swelling
and sloughing in tubular lumens in kidney tissue in mela-
tonin 1 mg/kg and Dox treated mice (H & E ×400).
especially the heart, are therefore highly susceptible to
free radical injury. In the present study melatonin effec-
tively prevented rise in the levels of these indicators of
oxidative stress in heart, liver and kidney tissues indi-
cated by a pronounced protection to the heart tissue. This
is in accordance with previous studies stating melatonin
as being very potent antioxidant having potential to scav-
enge hydroxyl radicals [41], peroxy radicals (ROO˚),
superoxide anions [41,42] and H2O2 [43]. Hardeland et
al., [41] has also shown melatonin to be more potent than
mannitol or glutathione in scavenging hydroxyl radical
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Comparative Assessment of Melatonin-Afforded Protection in Liver, Kidney and Heart of Male Mice
against Doxorubicin Induced Toxicity
595
Figure 3. Micrograph showing hepatic vacuolation with mo-
nonuclear inflammatory cell infiltrate in hepatic tissue in
melatonin 1 mg/kg and Dox treated mice (H & E ×400).
and also, more potent than vitamin E in sequestering
peroxyl radicals [44]. Another advantage associated with
melatonin is its distribution to sub-cellular compartments
because of its solubility in water as well as in lipids
making it readily available to afford protection [44]. Pre-
sent study strengthens the findings of Chen et al., [45]
who demonstrated free radical load being a cause of
myocardial infarction. In another study, Liu et al., [38]
has verified melatonin protecting against Dox induced
cardiotoxicity.
In the present study melatonin treatment prevented de-
cline in the contents of sulfhydryl contents in heart, liver
and kidney tissues. Hardeland et al., [41] has demon-
strated that melatonin is almost five times more potent in
scavenging hydroxyl radicals that might result in sparing
endogenous sulfhydryls and lead to restoration of their
level in all the tissues. Matsura et al., [46] has also dem-
onstrated that treatment with melatonin did not change
GSH levels in their study and as such our results are in
accordance with their results. In the present study, mela-
tonin alone did not change the sulfhydryl contents of the
tissue at both doses. It is suggested that since melatonin
is readily distributed in sub-cellular compartments mak-
ing it available being the first line of defense against free
radical load. In a recent study, Ozan et al., [39] have
demonstrated melatonin in protecting free radical in-
duced damage to kidney by cigarette smoke. They re-
ported melatonin to cause a decrease in malondialdehyde
(MDA) and GSH (glutathione) levels and avert structural
changes. The results of this study are consistent with the
findings of Oktem et al., [47] who demonstrated mela-
tonin to prevent an increase in MDA levels of renal tis-
sue and decrease in renal tissue SOD and GSH-Px activi-
ties and related it to its radical scavenging and anti-oxi-
dant properties.
There is sufficient evidence in the literature indicating
that melatonin protects liver against free radical load.
Matsura et al., [46] have shown that melatonin protected
against acetaminophen induced sever damage to mouse
liver and suggested that exogenously administered mela-
tonin exhibits a potent hepatoprotective effect against
acetaminophen induced hepatic damage via its antini-
trosative and anti-inflammatory activities in addition to
its antioxidant potential. Another study showed mela-
tonin effective in reducing MDA contents [48]. Sahna et
al., [48] demonstrated a parallel relation between MDA
and morphological changes in the heart tissue that was
prevented by pharmacological doses of melatonin. Oth-
man et al., [40] demonstrated that Dox induces lipidper-
oxidation in the liver and decreases GSH levels. They
attributed this effect to the oxidative stress that was pre-
vented by melatonin.
In the present study, Dox treatment induced the car-
diac, hepatic and renal damage as it was shown by
plasma biochemical markers that have indicated an in-
crease in their activities. Concomitant administration of
melatonin as an effective antioxidant demonstrated that
melatonin is a cytoprotective against Dox induced free
radical load. Cotreatment with melatonin replenished the
depleted GSH levels back to normal and furthermore,
decreased MDA to attain levels non-significantly differ-
ent from normal values in parallel to regression of he-
patic, cardiac and renal toxicity indicators.
Dox-induced cytoplasmic vacuolization and mito-
chondrial swelling was confirmed by Bellini and Solcia
[10]. It has been previously established that Dox induces
mitochondrial injury through generation of superoxide
anions. Results of the present study suggest that the pre-
vention of pathological changes by melatonin can be
attributed at least in part to the preservation of subcellu-
lar integrity of cardiac myocytes [49].
Liu et al., [38] also reported effects of melatonin on
Dox-induced cardiotoxicity and demonstrated it to be
protective. In the present study low dose of melatonin
failed to protect against Dox-induced toxicity. This could
be attributed to the low dose of melatonin used in this
study. In most of the studies referred here, bit high doses
are reported. However, plasma t½ of melatonin is short
(20 - 40 min), but tissue levels of melatonin remain high
enough than physiological levels even when plasma me-
latonin levels fall to the physiological levels [50].
In conclusion, melatonin, probably by behaving as an
antioxidant protected mice from Dox-induced lipidper-
oxidation and NO in heart, liver and kidney tissues and
prevented a decline in GSH which is a first barrier in
preventing oxidative and nitrosative damage. Results,
also, show that protection is more pronounced in heart
and liver as compared to kidneys. Moreover, results on
plasma aminotransferases, LDH and CK-MB indicated
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Comparative Assessment of Melatonin-Afforded Protection in Liver, Kidney and Heart of Male Mice
against Doxorubicin Induced Toxicity
596
that melatonin declined CK-MB more effectively pre-
venting any damage to heart. These findings support the
role of free radical induced toxicity by Dox. However,
administration of melatonin is able to decrease parame-
ters of oxidative, and nitrosative stress in heart and liver
more effectively than kidney.
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Abbreviations
MDA: Malondialdehyde;
NP-SH: non-protein sulfhydryls;
GSH: glutathione;
DTNB: 5-5’-dithiobis-(2 nitrobenzoid acid);
Dox: Doxorubicin;
NO: Nitric oxide.