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Study on the antiradiation role of melatonin: an investigation on induced oxidative stress in mice by radiomimetic drug cyclop

Study on the Antiradiation Role of Melatonin: An Investigation on Induced Oxidative
Stress in Mice by Radiomimetic Drug Cyclophosphamide
K. Manda and A.L. Bhatia*
Laboratory of Neurobiology and Aging,
Department of Zoology,
University of Rajasthan,
Jaipur-302 004, India

Abstract:
Clinical studies have demonstrated an altered pineal function in cancer patients. Owing to the
documented antineoplastic activity of the pineal gland, these anomalies could have a prognostic significance.
This study was carried out to monitor the effect of higher blood levels of melatonin, the most important pineal
hormone, which could be applied in relation to the response to chemotherapy in human neoplasms.
Cyclophosphamide is a commonly used chemotherapeutic drug and well-known mutagen and clastogen. It is an
alkylating agent, producing highly active carbonium ion, which reacts with the extremely electron-rich area of
the nucleic acids and proteins. The present study aimed to investigate the protective effect of melatonin against
cyclophosphamide induced oxidative stress in mice tissues. Lipid peroxidation, Reduced glutathione (GSH),
Glutathione disulphide (GSSG), Glutathione peroxidase (GSH-Px) and serum phosphatase level have taken as
endpoints. Twenty days oral administration with melatonin (0.25 mg/Kg body weight) followed by an acute
treatment with cyclophosphamide (75mg/kg b.w.) inhibited the radiomimetic drug-induced augmented level of
lipid peroxidation, Blood GSSG and acid phosphatase. Cyclophosphamide induced depletion in the level of
GSH, GSH-Px and alkaline phosphatase is ameliorated significantly by melatonin administration. The findings
support the results showing melatonin as a free radical scavenger, and singlet oxygen quencher. Results clearly
indicate the antioxidative properties of melatonin against the radiomimetic drug which could be effectively used
selectively for the protection of normal tissue during chemotherapy.
KEY WORDS: Melatonin, cyclophosphamide, lipid peroxidation, glutathione, antioxidants.
* Corresponding author: Tel. +91-141-2711304; Fax: +91-141-2701137.
E-ma (A.L. Bhatia)

1. Introduction:

Lipid peroxidation is an important outcome of the oxidative stress. Lipid peroxidation in vivo is a fundamentally deteriorative reaction that is involved in aging processes, atherosclerosis and cancer [1-3]. Lipid peroxidation brings about several changes in the biological membrane [4]. It is a highly destructive process to cellular organelles as well as to the organism as a whole. The loss of biochemical function and/or structural architecture leads to damage or death of cells [5]. The glutathione antioxidant system plays a fundamental role in cellular defense against reactive free radicals and other oxidant species [6] (Meister and Andersen, 1983). Depletion of GSH results in enhanced lipid peroxidation [7]. Excessive lipid peroxidation can cause increased GSH consumption [8]. Melatonin, the major secretory product of the pineal gland has been shown to participate in a number of physiological processes such as regulation of reproduction, sleep, mood and behavior, circadian rhythms [9-12]. Melatonin is also now known to be an antioxidant. It detoxifies a variety of free radicals and reactive oxygen intermediates including the hydroxyl radical, peroxynitrite anion, singlet oxygen and nitric oxide [13]. Many studies show that melatonin inhibits tumor growth both in vivo and in vitro [14, 15]. The interest in melatonin was significantly increased after the discovery of the antioxidant potential of the molecule, both in vitro and in vivo [16, 17]. To produce an antioxidative effect, melatonin is absorbed by the body and becomes available in the tissues which are exposed to oxidative stress because melatonin crosses all morphophysiological barriers, e.g., the blood-brain barrier, placenta, and distributes throughout the cell. This increases the
efficacy of melatonin as antioxidant increases [18]. Melatonin is evidently effective against age-
induced changes in the level of MDA, GSH, GSSG, GSH-Px and phosphatase activity and it was
reported that melatonin afford significant protection against age-induced oxidative stress in mice
which was measured in the terms of lipid peroxidation and glutathione [19,20]. As age advances, the
nocturnal production of melatonin decreases in animals of various species, including humans [10, 16].
Cyclophosphamide is a commonly used chemotherapeutic drug and well-known mutagen and
clastogen [21]. It is an alkylating agent, producing the highly active carbonium ion, which reacts with
the extremely electron-rich area of nucleic acids and proteins [22]. Since melatonin has demonstrated
excellent antioxidative and anti-aging properties, there is a likely possibility that melatonin would
protect against the toxicity of cyclophosphamide i.e. an elevated level of melatonin in body may prove
prophylactic against damage. If it so, it would further validate hitherto debated role of melatonin
against several drug induced oxidative stress.
2. Materials and Methods
2.1 Animals

Male Swiss albino mice were selected from an inbred colony and maintained on standard mice feed
(Hindustan Lever Ltd., New Delhi) and water ad labitum. Mice were maintained at constant
temperature (22±1 ºC) and light (12L: 12D).

2.2 Chemicals

Glutathione and thiobarbituric acid were purchased from Sigma, USA, melatonin from Aristo
Pharmaceuticals Ltd, India and cyclophosphamide from Asta Medica AG, Frankfurt, Germany. All
other chemicals used in the study have been of analytical grade.

2.3 Experimental Protocol

Swiss albino mice of 6-8 week age group were divided into three groups (10 animals in each group):
The first group served as normal (did not receive any treatment). Second group (Experimental) was
administered the melatonin (0.1 mg/Kg body weight/day) orally for 15 days (every day at 10 AM) and
then administered an acute intra-peritoneal dose of cyclophosphamide (75 mg/kg body weight) on 15th
day (two hour after the last dose of melatonin). The third group (control) was sham treated with
melatonin and then administered an acute dose of cyclophosphamide on 15th day.
Mice were killed humanly by cervical dislocation after 24 hrs of treatment or cyclophosphamide
administration. Various organs i.e., brain, spleen liver, lungs, kidney and testes were removed for
biochemical estimation of lipid peroxidation and GSH. Blood was collected by cardiac puncture for
the estimation of serum phosphatase, GSH, GSSG and GSH-Px.

2.4 Biochemical assay

Lipid peroxidation was assessed biochemically by determining the level of malondialdehyde (MDA),
hydroperoxide and conjugated dienes [23]. GSH was measured as described by Ellman and Archs [24]
using 5, 5-dithiobis- (2-nitrobenzoic acid) (DTNB) reagent. GSSG was measured by masking GSH
with 2-vinylpyridine by the same procedure of GSH estimation. GSH-Px estimation was carried out by
the method of Hochstein and Utley [25]. Serum phosphatases were estimated by commercially
accessible kits (Span Diagnostics Ltd. India).
The values are expressed as mean± S.E.M of 10 animals. The difference between various groups has
been analyzed by Student’s t-test.
3. Results:
It is evident from tables that, cyclophosphamide treated mice (control) have a significantly (P<0.001)
higher value of MDA content (Table-1), hydroperoxide (Table-2) and conjugated diene (Table-3), and
lower value of GSH (Table-4), in relation to normal mice. Such cyclophosphamide-induced changes in
lipid peroxidation and GSH level have been checked by the administration of melatonin, as it is
evident by the values of MDA content, hydroperoxide and conjugated diene in melatonin treated group
which have been closer to the normal values. The cyclophosphamide-induced depleted level of GSH
was found significantly compensated in melatonin treated group.

Table 5 shows the cyclophosphamide-induced changes in the level of blood GSH, GSSG, GSH-Px and
phosphatase activity. The activities of antioxidant enzymes, GSH-Px and alkaline phosphatase were
significantly inhibited in blood following cyclophosphamide exposure. However, the levels of GSSG
and acid phosphatase had risen after drug administration. Melatonin treatment lowered significantly
(p<0.001) blood GSH levels as well as GSH-Px and alkaline phosphatase activities in comparison to
those treated only with cyclophosphamide (control). The cyclophosphamide-induced increase in the
acid phosphatase activity and GSSG level were inhibited significantly (p<0.001) in melatonin treated
group. A profound decline in GSH/GSSG ratio was reported in cyclophosphamide-treated mice
whereas melatonin treated group showed almost near the normal level.

4. Discussion

Results obtained from this study indicate that the melatonin acts as prophylactic agent and renders
protection against cyclophosphamide-induced oxidative stress. Oxidative stress refers to the cytotoxic
consequence of reactive oxygen byproducts: superoxide anions and hydroxyl radicals which are
generated as metabolites of normal and aberrant metabolic processes that utilize molecular oxygen
[26]. Oxidative stress leads to lipid peroxidation, protein and carbohydrate oxidation and metabolic
disorders [27-29]. The products of lipid peroxidation such as MDA and 4- hydroxynonenal are toxic to
cells [30]. Lipid peroxidation within the membrane has a devastating effect on the functional state of
the membrane because it alters membrane fluidity, typically decreasing it and thereby allowing ions
such as Ca++ to leak into the cell. The peroxyl radical formed through lipid peroxidation attacks
membrane protein and enzymes and reinitiates lipid peroxidation.
The preservation of cellular membrane integrity depends on protection or repair mechanism capable of
neutralizing oxidative reactions. In present study the reduction in MDA equivalents and elevation in
GSH level in the melatonin-treated animals suggests that melatonin may scavenge the free radicals
generated during oxidative stress. GSH with its sulfhydryl group functions in the maintenance of
sulfhydryl groups of other molecules (especially proteins), as a catalyst for disulfide exchange
reactions, and in the detoxification of foreign compounds, hydrogen peroxide and free radicals. When
GSH acts as a reducing agent, its SH becomes oxidized and forms a disulfide link with other
molecules of GSH. GSSG, in turn, can be reduced to GSH by the action of GSSG reductase, in a
reaction using NADPH. NADPH is recycled by glucose-6 phosphate dehydrogenase via the pentose
phosphate pathway, which is particularly important in red blood cells [31].
The GSSG/GSH ratio may be a sensitive indicator of oxidative stress. GSH-Px is also the major
antioxidative enzyme, which decomposes H2O2 to H2O molecules. By doing so, it reduces the
formation of hydroxyl radicals [31]. The cyclophosphamide induced the inhibition of GSH-Px and
decreased GSH/GSSG ratio in mice blood. Melatonin treatment before cyclophosphamide treatment
could maintain their level to near the normal values.
An increase in serum acid phosphatase activity has been noticed after cyclophosphamide
administration. Acid phosphatase is localized in cellular lysosomes. An enhanced Golgi activity and
peroxidation of lysosomal membranes due to cyclophosphamide possibly resulted in the efflux of the
enzymes and hence caused an increase in acid phosphatase levels. But melatonin treatment could
check in the levels of both, the lipid peroxidation and acid phosphatase. In addition, alkaline
phosphates activity decreased in cyclophosphamide-treated mice. Alkaline phosphatase plays an
important role in maintenance of cellular permeability and acts on monophosphoesters. Damage to cell
membrane caused by cyclophosphamide may be the reason for declined activity of alkaline
phosphatase. The sustained melatonin level in the blood has evidently checked the decline through its
lipid peroxidation preventive action on membrane. Since, the antioxidative mechanisms of melatonin
include singlet oxygen quenching, free radical scavenging and chain breaking during lipid
peroxidation [17].
The results of present study may also be corroborated by the findings of Kaya et al (1999) who
demonstrated that melatonin inhibit the cyclophosphamide-induced augmentation in the level of lipid
peroxidation in Wistar rat [32]. They have also reported that cyclophosphamide induced inhibition of
GSH-Px activity is ameliorated by melatonin. While evaluating the anti-aging role, melatonin was
found to lower GSSG levels and maintain glutathione in its reduced state [19]. The protection by
melatonin against oxidative stress induced by several antitumoral drugs and antibiotics has also been
reported [33, 34]
The present finding that melatonin ameliorates the depletion of GSH, GSH-Px and alkaline
phosphatase activities and reduces the level of GSSG, lipid peroxidation and acid phosphatase in mice
make it a potential prophylactic and preventive agent against oxidative stress.

5. Acknowledgement

One of the authors [K. Manda] is thankful to Council of Scientific and Industrial Research, Human
Resources Development Group, Government of India for the fellowship.
6. References

1. Muscari M.R., Caldarera C.M. and Guarnieri C., Age-dependent production of mitochondrial hydrogen peroxide, lipid peroxides and fluorescent pigments in the rat heart. Basic Research in Cardiology; 85:172-178, (1990). Atheroma begins at birth. In: Kummerow FA eds. Metabolism of Lipids as Related to Atherosclerosis. Springfield, IL, USA, 18-25, (1965). P., Peroxidant states and tumor production. Science, 227: 337-381, (1985) 4. Leyko W, Bartosz G., Membranes effect of ionizing radiation and hyperthermia. Int J. Radiat 5. Kale R.K, Sitaswad S.L., Radiation induced lipid Peroxidation in liposomes. Radiat Phys 6. Meister A., Andersen M.E., Glutathione. Ann Rev Biochem, 52, 711-760, (1983). 7. Anderstam B., Vaca, C., Ringdahl M.H. Lipid peroxide level in a murine adenocarcinoma exposed to hyperthermia: The role of glutathione depletion. Radiat Res, 132, 296-300, (1992). Glutathione depleting agents and lipid peroxidation. Chem Phys Lipids; 45: Melatonin and human reproduction. Ann Med, 30, 103-108, (1998). 10. Waldhauzer F., Kovacs J. and Reiter E., Age-related changes in melatonin levels in humans and its potential consequences for sleep disorders, Exp Gerontol, 33, 759-772, (1998). 11. Zhdanova I.V., Cantor M.L., Leclair O.U., Behavioral effect of melatonin treatment in non- human primates. Sleep Res Online, 1, 114-118, (1998). 12. Yu H.S., Tsin A.T.C. and Reiter, R.J., Melatonin: history, biosynthesis and assay methodology. In: Yu HS and Reiter RJ eds, Melatonin, Biosynthesis, Physiological Effects, and Clinical Applications. CRC Press, Boca Raton, 1-16, (1993). 13. Tan, D.X., Manchester L.C., Reiter R.J., Qi, W.B., Karbownik, M. and Calvo, J.R., Significance of melatonin in antioxidative defense system: reactions and products. Biol Signals Recept., 9, 137-159, (2000). 14. Blask, D.E., Melatonin in oncology. In: Yu, HS and Reiter RJ eds. Melatonin, Biosynthesis, Physiological Effects, and Clinical Applications. CRC Press: Boca Raton, 447-475, (1993). 15. Anisimov, V.N., Popovich I.G., Zabezhinski M.A., Melatonin and colon carcinogenesis: I. Inhibitory effects of melatonin on development of intestinal tumors induced by 1, 2-dimethylhydrazine in rats. Carcinogenesis; 18, 1549-1553, (1997). 16. Reiter R.J., Tan D.X., Manchester L.C. and Qi W., Biochemical reactivity of melatonin with reactive oxygen and nitrogen species: a review of evidence. Cell Biochem Biophys, 34: 237-256(2001). 17. Allegra, M., Reiter R.J., Tan D.X., Gentile, G., Tesoriere, L. and Livrea M.A., The chemistry of melatonin’s interaction with reactive species. J Pineal Res 2003, 34, 1-10. 18. Reiter R.J., Functional pleiotropy of the neurohormone melatonin: antioxidant protection and neuroendocrine regulation. Front Neuroendocrinol; 16, 383-415, (1995).
19. Manda K. and Bhatia A.L., Melatonin-induced reduction in age-related accumulation of oxidative damage in mice. Biogerontology; 4(3),133-139, (2003 a).
20. Manda K. and Bhatia A.L., Melatonin’s anti-aging role: A study on LPO in mice tissues. Ind J Mohn G.R. and Ellenberger., Genetic effect of cyclophosphamide, ifosfamide and trofosfamide. Mutat Res, 32, 334-360, (1976). 22. Schneider E.L., Sternberg H. and Tice R.R., The analysis of cellular replication. Proc. Natl. Acad. Sci. USA., 74, 2041-2044 (1977). 23. Bueg J.A. and Aust S.D., Methods in Enzymology, Vol. 52, Academic Press, New York, 302- 24. Ellman G.L and Archs., Tissue sulfhydril groups. Biochem Biophys; 82, 70-77, (1959). 25. Hochstein P and Utley H., Hydrogen peroxide detoxification by glutathione peroxidase and catalase in rat liver homogenate. Mol Pharmacol; 4, 574-579, (1968). 26. Sies H and Stahl W., Vitamin E and C, β-carotene and other carotenoids as antioxidants. Am 27. Halliwell B., Free radicals, antioxidants and human disease: curiosity, cause or consequence? Lancet; 344, 721–724, (1994). 28. Sies H., Oxidative stress. Academic Press, London;1-8, (1985). 29. Pryor W.A and Godber, S.S., Noninvasive measures of oxidative stress status in humans. Free Rad Biol Med; 10, 177-84, (1991). 30. Raleigh J.E., Radioprotection of membranes. Phamacol Ther, 39, 109-113, (1985). 31. Gul M., Kutay F.Z., Temocin S. and Hanninen, O., Cellular and clinical implication of glutathione. Ind J Exp Biol, 38, 625-634, (2000). 32. Kaya H, Oral B, Ozguner F, Tahan V, Babar Y, Delibas N and Zentralbl.????, The effect of melatonin application on lipid peroxidation during cyclophosphamide therapy in female rats. Zentralbl Gynakol, 121, 499-502, (1999). 33. Lopez-Gonzalez M.A., Guerrero J.M., Torronteras R, Osuna C, Delgado F., Ototoxicity caused by aminoglycosidase is ameliorated by melatonin without interfering with the antibiotic capacity of the drugs. J Pineal Res, 28, 26-33, (2000). 34. Lopez-Gonzalez M.A., Guerrero J.M., Torronteras R., Osuna C., Delgado F., Ototoxicity caused by cisplatine is ameliorated by melatonin and other antioxidants. J Pineal Res, 28, 73-80, (2000). Table-1: MDA content (n mol/g wet tissue) in mice tissues 24 hours post cyclophosphaphamide
(CP) treatment with and without melatonin. Values ± S.E.M. of 10 animals in each group.

Tissues Normal
CP+Melatonin
a- Statistical difference with experimental (CP+Melatonin) ; b- Statistical difference
with normal. ∗- P<0.001; •- P< 0.01; #- insignificant.
Table-2: Hydroperoxide content (m mol/100g wet tissue) in mice tissues 24 hours post
cyclophosphamide (CP) treatment with and without melatonin. Values ± S.E.M. of 10 animals
in each group.

Tissues Normal
CP+Melatonin
a- Statistical difference with experimental (CP+Melatonin) ; b- Statistical difference
with normal. ∗- P<0.001; •P<-0.01; #- insignificant.
Table-3: Conjugated diene content (m mol/100g wet tissue) in mice tissues 24 hours post
cyclophosphamide (CP) treatment with and without melatonin. Values ± S.E.M. of 10 animals
in each group.

Tissues Normal
CP+Melatonin
a- Statistical difference with experimental (CP+Melatonin) ; b- Statistical difference
with normal. ∗- P<0.001; •-P<0.01; #- insignificant.
Table-4: GSH content (n mol/g) in mice tissues 24 hours post cyclophosphamide (CP) treatment
with and without melatonin. Values ± S.E.M. of 10 mice in each group.

Tissues Normal
CP+Melatonin
a- Statistical difference with experimental (CP+Melatonin) ; b- Statistical difference with
normal. ∗- P<0.001; •-P<0.01; #- insignificant.
Table 5: Variation in GSH, GSSG, GSH-Px and Phosphatase levels in murine blood 24 hours
post cyclophosphamide (CP) treatment with and without melatonin. Values ± S.E.M. of 10
animals in each group.

Parameters Normal
CP CP+Melatonin
148.504±2.32 a• 109.302±1.22 139.369±1.41 b∗ (µ mol/ml)
11.369±0.17 a∗ 50.573±0.61 20.504±∗0.38 b∗ (µ mol/ml)
GSH/GSSG ratio
13.062±1.35 a∗ 2.161±0.09 6.797±0.13 b∗ (U/gm Hb)
Acid phosphatase
2.462±0.03 a• 6.636±0.05 3.686±0.07 b∗ Alkaline
7.863±0.09 a• 3.601±0.05 6.347±0.12 b∗ phosphatase
a- Statistical difference with experimental (CP+Melatonin) ; b- Statistical difference with
normal. ∗- P<0.001; •-P<0.01; #- insignificant.

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