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Journal of Applied Sciences Research, 6(3): 245-253, 2010
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Studies on the Chemopreventive Potential of Melatonin on 7,12dimethylbenz(a)anthracene Induced Mammary Carcinogenesis in Rats
Sankaran Mirunalini, Kandhan Karthishwaran, Ganesan Dhamodharan and Mohan Shalini
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Tamil
Nadu, India.
Abstract: Chemoprevention using drugs is a promising approach to control the mortality and occurrence
of cancer that accounts for millions of death worldwide. Melatonin (N-acetyl-5-methoxytryptamine), a
hormone of the pineal gland produced from the amino acid tryptophan in minute quantities has been
known to be a chemoprventive, anticancer agent in vitro and in vivo studies. DMBA (7, 12dimethylbenz(a)anthracene) has been shown to act via generation of reactive oxygen species (ROS) as
tumor promoter and also involved in the generation of free radicals. Many natural and synthetic
compounds exhibit potent cancer chemopreventive property as observed in the last few decades. Most of
them are known to exert their effects by quenching reactive oxygen, inhibiting lipid peroxidation. DMBA
is a potent carcinogenic agent that induces mammary cancer. Here we evaluate the chemopreventive
potential of melatonin on experimental animal models, female albino W istar rats were randomized into
four groups. Group I animals served as control without any treatment. Group II animals received a dose
of 5mg/kg body weight of DMBA orally at weekly intervals for one month. Group III animals received
a daily intraperitoneal administration of melatonin 5mg/ml per animals for 15 days prior to the first oral
administration of DMBA and continued for a month and Group IV animals received a daily intraperitoneal
administration of melatonin 5mg/ml beginning the next day after first DMBA administration for one
month. At the end of the experiment the rats were killed, blood, liver, kidney and mammary tissues were
taken for biochemical and histological studies. As a marker for liver function, the activity of gamma
glutamyl transpeptidase (GGT) was measured in the serum. To assess the lipid peroxidation and the
antioxidant status in tissues, the levels of thiobarbituric acid reactive substances (TBARS), superoxide
dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), ascorbic acid
(vitamin C) and á–tocopherol (vitamin E) were measured. DMBA administration inhibited body weight
and enhanced macroscopically detectable tumors with a increase in serum GGT and TBARS levels.
DMBA treatment decreased both enzymic and non-enzymic antioxidant status. Melatonin administration
significantly curtailed tumor development and counteracted all the biochemical effects. This indicates that
melatonin should be considered as an adjuvant drug in the treatment of neoplastic disease
Key words: melatonin, mammary carcinogenesis, gamma glutamyl transpeptidase, antioxidants, lipid
peroxidation
INTRODUCTION
7, 12-dimethylbenz(a)anthracene (DMBA) is a
potent carcinogenic polycyclic aromatic hydrocarbon
(PAH) used to induce mammary cancers in animal
models. DMBA consistently produces carcinomas in
animal models. Sources of PAHs are widely distributed
in our environment and are implicated in various types
of cancer. Enzymatic activation of PAHs leads to the
generation of active oxygen species such as peroxides
and superoxide anion radicals, which induce oxidative
stress in the form of lipid peroxidation [1 ,2 ]. The
consequences of the damage initiated by these
metabolic by products affect a large range of biological
reactions, like increases in mutation rate, alteration of
cellular membrane composition, structural proteins,
metabolic, detoxifying enzymes and cellular signaling
proteins[3 ] . DNA damage induced by oxidative stress
and/or deficient DNA repair may have etiological or
prognostic role in cancer. Free radicals play an
important role in tumor promotion by direct chemical
reaction or alteration of cellular metabolic process and
their scavengers represent inhibitors at different stages
of carcinogenesis.
Corresponding Author: Dr.S.Mirunalini, Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai
University, Annamalainagar, Tamil Nadu, India.608002
Mobile: 0091 9442424438, Off: 04144-238343 ext-221.
E-mail: [email protected]
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J. Appl. Sci. Res., 6(3): 245-253, 2010
D espite abundant info rmatio n
ab out
its
etiopathogenesis and early detection, effective
therapeutic modalities for patients with advanced stages
of the disease are still needed. Accumulating evidence
derived from laboratory studies and study cohorts
drawn from the general population have led to the
search for “chemoprotective” agents to attenuate the
risk of breast cancer based on the observation that
most human cancer are associated with a long period
of latency. Chemoprevention using drugs is a promising
approach to control the mortality and occurrence of
cancer that accounts millions of death W orld W ide [4 ].
Melatonin (N-acetyl-5-methoxytryptamine) a hormone
of the pineal gland produced from the amino acid
tryptophan in minute quantities has been known to be
a chemopreventive agent in
in vivo studies and
experimental animal models. It is soluble in lipids and
in aqueous environments and is also reported as a
highly efficient free radical scavenger and act as a
strong general antioxidant[5] . Moreover M elatonin acts
on the target cells either directly on G-protein
receptors, such as MT1R, MT2R and MT3R that
modulate several intracellular messengers such as
cAMP, cGMP and ca 2 +[6 ] . In addition to its potential on
anti tumor activity melatonin has proved to modulate
the effects of cancer chemotherapy, by enhancing its
therapeutic efficacy and reducing its toxicity. The
increase chemotherapeutic efficacy by melatonin may
depend on two main mechanisms, namely prevention of
chem o therapy-induced lymphocyte dam age and
antioxidant effect, which has been proved to amplify
cytotoxic actions of the chemotherapeutic agents
against cancer cells[7 ] . However, the clinical results
available at present, with melatonin and chemotherapy
in the treatment of human neoplasm are generally
limited. Considering the potential antitumor/antioxidant
activity of melatonin, the present investigation was
carried out to study on the chemopreventive potential
of melatonin against 7,12-dimethylbenz(a)anthracene
induced mammary carcinogenesis in female albino
W istar rats.
accordance with the guidelines of National Institute of
Nutrition, ICMR, Hyderabad, India and by the Animal
Ethical Committee, Annamalai University (Reg. No.
486/160/1999 CPCSEA).
Chemicals: 7, 12-dimethylbenz(a)anthracene was
purchased from Sigma Chemical Company, St.Louis,
MO,USA. M elatonin was obtained from Sisco Research
Laboratories, Mumbai, India. All other chemicals
including solvents used in the study were of high
purity and of analytical grade.
Experimental Design: Group I animals served as
control without any treatment. To induce mammary
carcinogenesis, rats received 5mg/kg body weight of
DMBA in 1 mL corn oil orally once in a week with a
total dose of 20 mg/kg body weight for 4 weeks. Corn
oil is used as ‘promoter of carcinogenesis in this
animal model[8] . Melatonin was dissolved in ethanol
which had previously been diluted 20 times with
Phosphate buffered saline (PBS) (Group II). Group III
animals were received a daily intraperitoneal
administration of melatonin 5mg/mL per animal per
day for 15 days before the first oral administration of
DMBA and continued for a month. Group IV rats
received melatonin (5mg/mL) beginning the next day
after first DMBA administration for one month [9 ].
Animals were monitored everyday. After the
termination of the experiment, all animals were
anesthetized with 24 mg/kg ketamine chloride and
sacrificed by cervical dislocation after an overnight
fast. The liver, kidney and mammary tissues were
dissected out, weighed, cleared off blood and
immediately transferred to beakers containing ice-cold
solution. A 10% tissue homogenate were prepared
using appropriate buffer. Another aliquot of tissues
were stored in 10% formalin for histopathological
studies. The tissues were sectioned using microtome,
dehydrated in alcohol, embedded in paraffin, sectioned
and stained with hematoxylin and eosin. Serum was
collected from the blood after centrifugation at 1000g
for 15 minutes for various biochemical estimation.
M ATERIALS AND M ETHODS
Biochemical Assays: As marker enzyme for liver
function in serum, the activity of gamma glutamyl
transferase (GGT) (expressed as IU/L plasma), was
assessed[1 0 ] . To asses lipid peroxidation and antioxidant
status in liver, kidney and mammary tissues, the levels
of thiobarbituric acid reactive substances (TBARS)
(expressed as nmol/100g tissue [1 1 ] and GSH levels
(expressed as mg/100mg tissue [1 2] were measured. In
addition, the activity of superoxide dismutase (SOD) [1 3 ],
catalase (CAT)[14 ] glutathione peroxidase (GPx) [1 5 ] ,
ascorbic acid (vitamin C)[1 6 ] and á–tocopherol (vitamin
E)[1 7 ] were also estimated. Enzyme activity was
Animals: Female rats of the W istar albino strain (120
days old) weighing 100g, were procured from
Tamilnadu veterinary science institute, Chennai, India,
were used. The animals (six per group) were fed with
standard pellet diet (Hindustan Lever Limited, Mumbai,
India) and water ad libitum throughout the experimental
period and replenished daily. The animals were housed
in well ventilated large polypropylene cages under
controlled conditions of light (12 hr light/12 hr dark),
humidity (50%) and ambient temperature (30+/-2 0 C).
The animals used in the present study were kept in
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expressed as units as follows: SOD (enzyme required
for 50% inhibition of nitroblue tetrazolium reduction
mm/mg protein), CAT (µmol of hydrogenperoxide
utilized/min/mg protein), GPx activity (µmol of GSH
utilized/min/mg protein), vitamin C (mmol/gm wet
tissue) and vitamin E (mmol/mg protein).
cyclic aromatic hydrocarbon (PAH) is widely present
in our environment and are implicated in various types
of cancer. Sources of PAHs include industrial and
domestic oil furnaces, gasoline and diesel engines [1 ].
In the present study, DMBA-treated rats exhibited
a significant inhibition of body growth, macroscopically
detectable tumors, with increased levels of serum GGT.
DMBA treatment increased TBARS levels in liver,
kidney and mammary tissues, while the activities of
SOD, CAT, GPx, vitamin C, vitamin E were decreased.
The generation of reactive oxygen species (ROS) and
the peroxidation of membrane lipids are well associated
with the initiation of carcinogenesis affecting the
normal biochemical process, which further leads to the
reduction of body weight[2 0 ,2 1 ] . These results agree with
the hypothesis that the administration of DMBA to rats
brought changes in body weight and enzyme activities
which may serve as markers to evaluate the
chemopreventive role of compounds of a potential
clinical use.
Melatonin is a highly conserved naturally occurring
molecules that is present virtually in all organism
tested from bacteria to mammals. However, it is a
nutrient and has been identified in bananas, tomatoes,
cucumbers and beetroots [2 2 ] . The present result indicates
that its administration significantly curtailed mammary
tumor developm ent and counteracted all the
biochemical effects of DMBA. Several studies reported
in the literature support the efficacy of melatonin to
prevent the development of mammary cancer. For
example, melatonin, in vivo, prevents the promotion
and growth of spontaneous or chemically induced
mammary tumors in rodents, whereas in vitro
melatonin inhibits breast cancer cell proliferation and
invasiveness. Moreover, melatonin hypothesis suggests
that decreased melatonin production may increase
breast adenocarcinomas had lower nocturnal plasma
levels of melatonin than healthy women.
There are several mechanisms by which melatonin
can exert its oncostatic actions in rat mammary gland:
(a) by its direct action on the mammary tissues and on
the activation of estrogen receptor for DNA binding [23] ;
(b) by enhancing its free radical scavenging
/antioxidant activity[24 ] ; (c) by enhancing immune
mechanisms in body[2 5 ] ; (d) by reducing the uptake of
key factors for tumor growth and tumor growth
signaling molecules (e.g. linoleic acid) [2 6 ] ; Additionally,
however melatonin kills neoplastic cells in vivo via the
induction of apoptotic process preventing apoptosis in
normal cells [2 7 ] .
Gamma glutamyl transferase activities in serum are
indexes of liver damage. Elevated activities of GGT
observed in the DMBA-treated group are indicative of
DMBA- induced hepatic damage and subsequent
leakage of these enzymes into circulation [2 8] . GGT act
Statistical Analysis: Statistical analysis of results was
performed and data were presented as mean ± S.D.
Differences between groups were assessed by analysis
of variance (ANOVA) using SPSS software package
followed by DMRT. A value of P<0.05 was considered
to indicate a significant differences between groups.
Results: Figure 1 presents the effect of melatonin on
carcinogen-induced changes in the body weight of
control and experimental animals. DMBA brought
about a significant impairment in body growth, which
was partially counteracted by the administration of
melatonin.
Macroscopically detectable tumors developed in the
DM BA-treated rats, an effect prevented by melatonin
administration. Histopathological examination of the
liver in group II shows mononuclear inflammatory
infiltrate around portal triad, kidney shows glomeruli
with cellular proliferation and tubular epithelial damage
and mammary tissues shows highly invasive tumor
cells surrounding the residual and expanding into
adjacent stroma were seen. These histopathological
features were greatly reduced in rats treated with
DMBA plus melatonin. The tissues of melatonin
pretreated or control rats did not differ in
morphological features (Fig.2 (i), (ii), (iii).
Figures and tables summarize the effect of
melatonin on carcinogen-induced changes on liverrelated enzyme, lipid peroxidation and antioxidant
status. As shown in Fig. 3, the parameter of liver
damage, i.e., serum GGT activity, exhibited comparable
changes after experimental manipulation. DMBA
treatment augmented them, while melatonin partially
counteracted the effect of DMBA. In all the three
tissues liver, kidney and mammary tissues, DMBA
brought about a significant increase of TBARS levels
(P<0.05) when compared to control animals. The effect
was almost fully and partially counteracted by
melatonin in groups III and IV (fig. 4). Activities of
enzymic and non-enzymic antioxidants in liver, kidney
and mammary tissues were augmented during
carcinogenesis, an effect fully reversed by melatonin in
group III and partially in group IV (tables 1, 2 and 3).
Discussion: Chemoprevention involving the use of
synthetic or natural products to inhibit or reverse the
carcinogenic process is an effect approach to control
cancer (18, 19) 7,12-dimethylbenz(a)anthracene, a poly
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Table 1: Effect of m elatonin on enzym ic and non-enzym ic antioxidants in the liver of control and experim ental anim als
Param eters
Group I Control
Group II D M BA
Group III M el + D M BA
Group IV D M BA +M el
SO D
6.37 ± 0.43a
3.80 ± 0.32 b
5.99 ± 0.47 a
4.92 ± 0.42 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CAT
48.94 ± 4.73 a
24.17 ± 2.12 b
46.15 ± 2.02 a
32.45 ± 2.18 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Gpx
5.90 ± 0.29a
3.10 ± 0.21 b
5.67 ± 0.37 a
4.43 ± 0.28 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GSH
6.98 ± 0.42a
4.15 ± 0.21 b
6.15 ± 0.29 a
4.76 ± 0.23 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Vitam in C
3.50 ± 0.32a
1.08 ± 0.09 b
3.05 ± 0.26 a
2.57 ± 0.19 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Vitam in E
4.79 ± 0.43a
1.53 ± 0.15 b
4.52 ± 0.31 a
3.23 ± 0.25 c
Table 2: Effect of m elatonin on enzym ic and non-enzym ic antioxidants in the kidney of control and experim ental anim als
Param eters
Group I Control
Group II D M BA
Group III M el + D M BA
Group IV D M BA +M el
SO D
5.63 ± 0.39a
2.12 ± 0.19 b
5.29 ± 0.32 a
3.75 ± 0.24 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CAT
40.72 ± 3.86 a
17.25 ± 1.14 b
35.80 ± 2.81 a
23.42 ± 1.82 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Gpx
3.64 ± 0.27a
0.75 ± 0.06 b
3.43 ± 0.24 a
1.82 ± 0.16 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GSH
4.65 ± 0.41a
1.69 ± 0.08 b
3.69 ± 0.31 a
2.18 ± 0.21 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Vitam in C
1.72 ± 0.11a
0.25 ± 0.01 b
1.65 ± 0.11 a
0.86 ± 0.05 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Vitam in E
2.17 ± 0.18a
0.56 ± 0.03 b
1.96 ± 0.15 a
0.98 ± 0.05 c
Table 3: Effect of m elatonin on enzym ic and non-enzym ic antioxidants in the m am m ary tissues of control and experim ental anim als
Param eters
Group I Control
Group II D M BA
Group III M el + D M BA
Group IV D M BA +M el
SO D
10.61 ± 0.95 a
6.25 ± 0.44 b
10.05 ± 0.76 a
8.00 ± 0.56 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------CAT
60.00 ± 3.74 a
30.00 ± 2.73 b
55.00 ± 4.73 a
46.33 ± 3.47 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Gpx
8.50 ± 0.68 a
5.80 ± 0.32 b
8.01 ± 0.63 a
6.92 ± 0.46 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GSH
8.48 ± 0.75 a
5.90 ± 0.49 b
8.10 ± 0.62 a
7.00 ± 0.47 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Vitam in C
3.24 ± 0.31a
1.42 ± 0.09 b
3.20 ± 0.29 a
2.50 ± 0.23 c
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Vitam in E
5.25 ± 0.46a
3.10 ± 0.23 b
5.10 ± 0.36 a
4.10 ± 0.29 c
Fig. 1: Effect of melatonin on body weight of control and experimental animals
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Fig. 2(i): Histopathological examination of liver tissues of control and experimental animals
Fig. 2(ii): Histopathological examination of kidney tissues of control and experimental animals
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Fig. 2(iii): Histopathological examination of mammary tissues of control and experimental animals
Fig. 3: Activity of melatonin on GGT in serum of control and experimental animals
as useful markers for hepatic metastasis from breast
and colon primaries. Melatonin administration to
DMBA-treated animals significantly decreased GGT
activities indicating a significant cytoprotective effect
on the hepatocytes [2 9 ].
Reactive oxygen species generation is a major
factor involved in all steps of carcinogenesis, i.e.
initiation, promotion and progression [3 0 ] . Oxidant-
antioxidant balance impacts the rate of cell proliferation
and tumor cells generally display low levels of lipid
peroxidation which in turn can stimulate cell division
and promote tumor growth [31 ] . Oxidative stress induced
due to the generation of free radicals and/or decreased
antioxidant level in the target cell and tissues has been
sugge ste d to p la y a n im portant role in
carcinogenesis [3 2 ].
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Fig. 4: The level of TBARS in circulation and tissues of control and experimental animals
cancer initiation[3 9 ] . GSH serves as a marker for
evaluation of oxidative stress and it act as an
antioxidant at both intracellular and extra cellular
levels[4 0 ] . W e also observed decreased activities of GSH
in cancer-bearing animals. Melatonin increased the
GSH levels, which clearly suggest their antioxidant
property. Decreased levels of water-soluble antioxidant
in cancer-bearing animals may be due to the utilization
of antioxidants to scavenge free radicals. Ascorbic acid
is a good scavenger of free radicals and it protect
cellular membranes their by preventing degenerative
disease like cancer[4 1 ,4 2 ] . Decreased levels of water
soluble antioxidants in cancer bearing animals may be
due to the utilization antioxidants to scavenge free
radicals. Vitamin C undergoes a synergistic interaction
with tocopheroxyl radical in the regeneration of
á–tocopherol[4 3 ] . Vitamin E protects cell membranes
from oxidative damage initiated by carcinogens. The
free radical clearing capacity of vitamin E is due to the
localization of an unpaired electron on its conjugated
double bond.
Free radicals are involved both in the initiation as
well as promotion stage of tumorigenesis and their
biochemical reactions in each stage of the metabolic
process are associated with cancer development[3 3] . It is
evident from the results that increased level of LPO
was found in cancer bearing animals when compared
to control group. On the contrary, reduced level of
LPO was observed in melatonin treated animals
indicating that it is a potent free radical scavenger.
Both physiological and pharmacological concentration
of melatonin in vivo are and commonly effective in
reducing total oxidative burden with in organisms [3 4 ].
During the process of neutralizing toxic reactants,
melatonin protects proteins, lipids, mitochondrial DNA
and nuclear DNA from oxidative damage [3 5 ].
Antioxidants act as the primary line of defense
against ROS and suggest their usefulness in eliminating
the risk of oxidative damage induced during
carcinogenesis.
SOD and CAT acts as mutually
supportive antioxidative enzymes, which provide
protective defense against reactive oxygen species [3 6 ].
The present study reveals that the activity of SOD is
depleted in the cancer-bearing animals, which may be
due to altered antioxidant status caused by
carcinogenesis. A similar result was observed for CAT
in group II, which may be due to the utilization of
antioxidant enzymes in the removal of H 2 O 2 by
DMBA. GPx is an important defense enzyme which
catalyses the oxidation of GSH to GSSG at the
expense of H 2 O 2[3 7 ] . Decreased GPx activity was also
observed in cancerous conditions [3 8 ]. Our findings agree
well with this observation.
The non-enzymatic antioxidant systems are the
second line of defense against free radical damage.
GSH and GSH depended enzymes are involved in
scavenging the electrophilc moieties involve in the
Administration of melatonin significantly reversed
the alteration to near normal level in cancer-bearing
animals and substantially inhibited the breast tumor
incidence or decrease in initiation of tumorigenesis in
pretreated group. From the results it can be inferred
that melatonin positively modulated antioxidant activity
by quenching and detoxifying the free radicals induced
by DMBA. It is worth emphasizing the protective role
of melatonin against the side effects of chemo and/or
radiotherapy. Although majority of the studies involved
experimental animals, the results could suggest
applicability of melatonin in humans. Further
investigation on the anticancer mechanisms of
melatonin remains to be carried out in vitro studies.
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J. Appl. Sci. Res., 6(3): 245-253, 2010
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