|Year : 2014 | Volume
| Issue : 4 | Page : 1040-1044
Effect of melatonin on antioxidant status and circadian activity rhythm during hepatocarcinogenesis in mice
Devi Verma1, Onn Haji Hashim2, Jaime Jacqueline Jayapalan2, Perumal Subramanian1
1 Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar, Tamil Nadu, India
2 University of Malaya Centre for Proteomics Research; Department of Molecular Medicine, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia
|Date of Web Publication||9-Jan-2015|
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar 608 002, Tamil Nadu, India
Source of Support: None, Conflict of Interest: None
Aim: Alteration of circadian systems can cause cancer and affects its development and response to therapeutics. The present study investigates whether cancer can disrupt circadian locomotor rhythms and evaluated the influence of melatonin (MLT) and oxaliplatin on the levels of antioxidants and circadian locomotor activity rhythms in N-nitrosodiethylamine (NDEA)-induced liver tumor in Indian field mouse (Mus booduga).
Materials and Methods: Effects of NDEA, NDEA, and MLT, as well as NDEA and oxaliplatin, on levels of mice liver marker enzymes and antioxidants and their circadian locomotor activity rhythm were assessed.
Results: Treatment of mice with NDEA caused significant alteration of their liver marker enzymes [aspartate transaminase and alanine transaminase; P< 0.05 Duncan's multiple range test (DMRT) test] antioxidant levels (superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase; P< 0.05, DMRT test] and circadian locomotor activity rhythm, which were abrogated when the animals were also given MLT or the anticancer drug, oxaliplatin.
Conclusion: Our study demonstrated that the circadian clock was disturbed by hepatocarcinogenesis and the effects could be reversed by the chronobiotic, MLT.
Keywords: Antioxidant enzymes, circadian rhythm, hepatocellular carcinoma, melatonin, MLT, NDEAN, nitrosodiethylamine
|How to cite this article:|
Verma D, Hashim OH, Jayapalan JJ, Subramanian P. Effect of melatonin on antioxidant status and circadian activity rhythm during hepatocarcinogenesis in mice. J Can Res Ther 2014;10:1040-4
|How to cite this URL:|
Verma D, Hashim OH, Jayapalan JJ, Subramanian P. Effect of melatonin on antioxidant status and circadian activity rhythm during hepatocarcinogenesis in mice. J Can Res Ther [serial online] 2014 [cited 2019 Nov 14];10:1040-4. Available from: http://www.cancerjournal.net/text.asp?2014/10/4/1040/138227
| > Introduction|| |
Reactive oxygen species (ROS) have been implicated in the development of cancer.  In hepatocellular carcinoma, which has been classified as the fourth most common cause of cancer mortality in the world, ROS-mediated damage occurs in the liver and is associated with the impairment of protein functions located in cell membrane. The involvement of free radicals in the pathogenesis of liver injury has been investigated for many years in a few well-defined experimental systems.  ROS could directly affect deoxyribonucleic acid (DNA) and cause mutations randomly and hence cell cycle genes when affected leading to cancer. Furthermore, ROS could affect proteins and cell membranes and thus the cells are more prone to the development of cancer. 
Oxidative stress, due to elevated ROS production, could cause oxidative damage to nucleic acids, proteins, and lipids. Among these biomolecules, lipids are more susceptible to oxidative damage or peroxidation. Superoxide dismutase (SOD) is an intracellular enzyme which acts as a catalyst in the process of dismutation of superoxide into oxygen and hydrogen peroxidase. Catalase (CAT) in a homotetrameric enzyme ferriheme-containing enzyme, converting H 2 O 2 into O 2 and H 2 O. Glutathione peroxidase (GPx) is an extracellular enzyme that catalyses the conversion of organic hydroperoxides, lipid hydroperoxides, and H 2 O 2 into harmless molecules. Glutathione-S-transferase (GST) possesses peroxidase activity and participates in the reduction of fatty acid hydroperoxides and participates in the reduction of fatty acid hydroperoxides to nontoxic alcohols. ,,
Melatonin (MLT), a naturally occurring hormone, is known to scavenge the highly toxic hydroxyl radical, the peroxynitrite anion, superoxide, and singlet oxygen.  MLT has a potent direct chain-breaking antioxidant activity and has protective properties against oxidative stress. , It is produced by the pineal gland in mammals and humans and is responsible for maintaining sleep. MLT transduces environmental lighting information into signals and also participates in several other important physiological functions, such as the control of seasonal reproduction in animals. 
Circadian rhythms are endogenously generated and occur with a periodicity of approximately 24 h. The biological clock regulates daily rhythms of physiology and behavior, enabling organisms to anticipate periodic changes in the environment, and to develop important adaptive mechanisms. Circadian dysfunction contributes to many clinical conditions, including sleep disorders, gastrointestinal diseases, metabolic syndrome, inflammation, and cancer.  Mice deficient in the circadian clock gene, period 2, are known to be cancer prone.  Numerous studies, since 1990s, have shown that circadian system alterations are not only risk factors for tumor incidence but are also related to progression of tumors.  Circadian clock is also known to modify the extent of toxicity of 40 anticancer drugs, including cytostatics, cytokines, and targeted biological agents, in mice, rats, and humans. 
The present study investigates whether circadian locomotor rhythms are chronically disrupted during hepatocarcinogenesis in a well-defined chronobiological model system of the Indian field mouse, Mus booduga. In the study, N-nitrosodiethylamine (NDEA), was used as a carcinogen to induce the liver cancer in the experimental mice, while oxaliplatin, an anticancer drug, was used to compare the effects of MLT.
| > Materials and Methods|| |
NDEA, MLT, and oxaliplatin were purchased from Sigma chemical company, St. Louis, MO, USA. All other chemicals and solvents used in the study were of analytical grade and were obtained from Sigma Chemical Company or Hi Media Laboratories, Mumbai, India.
Adult male mice (M. booduga) were captured in the paddy fields near XXX campus. Animals were kept in a temperature-controlled experimental cubicle maintained at 25 ± 2C. Food comprised of millets, maize, grains, and water available ad libitum. The animals (six per group) were housed in plastic cages under controlled conditions of light (12 h light/12 h dark). The study protocols were approved by the Animal Ethical Committee, XXX (Reg. No. 160/1999/CPCSEA). The animals were divided into four groups; group I (control), group II (NDEA treated, 20 mg/kg b.w., i.p. for 10 days), group III (NDEA + MLT dissolved in 0.1 mL of 10% ethanol, 0.5 mg/kg b.w., i.p.) and group IV (NDEA + oxaliplatin dissolved in 0.5 mL water 2 mg/kg b.w., i.p.). MLT and oxaliplatin were administered for 10 weeks (thrice a week).
After the experimental period (10 weeks) animals were sacrificed by cervical dislocation after an overnight fast. The liver was dissected out, weighed, cleared of blood, and immediately transferred to beakers containing ice-cold saline solution. Morphological changes, development of tumor in NDEA treated animals were noted. A 10% tissue homogenate was prepared using phosphate buffered saline buffer. Blood was collected (by sinoocular puncture) in heparinized tubes and plasma was separated by centrifugation at 1000 rpm for 15 min for further biochemical analysis. The packed cells were washed three times with physiological saline; 0.5 mL of the erythrocyte fraction was lyzed with 2.5 mL phosphate buffer (pH 7.4). The hemolysate was separated by centrifugation at 2500 rpm for 15 min at 10C. The activity of liver function in plasma, the activity of aspartate transaminase (AST) and alanine transaminase (ALT) (expressed as IU/L plasma), was assessed.  In addition, the activities of SOD,  CAT,  glutathione peroxidase (GPx),  and GST  were assessed in erythrocyte fraction.
Statistical analysis was performed by one-way analysis of variance followed by Duncan's multiple range test (DMRT) using SPSS software package 9.05. Results were expressed as mean ± standard deviation from six rats in each group and P < 0.05 were expressed as significant.
After the experimental period (10 weeks) circadian locomotor activity rhythm studies were performed in Department of Animal Behavior and Physiology, Madurai Kamaraj University. Two sets of four groups (n = 6 in each group) of mice were subjected to (i) light:darkness 12:12 (L:D), with a programmable timer) and continuous darkness (DD) in light-tight and temperature controlled experimental cubicles. Dim red light of 610-700 nm was used, while feeding the animals under DD and the hours of routine care were varied. A magnet-activated switch was used to record running-wheel revolutions. The wheel revolutions were picked up by a computerized chronobiology recording system (software version 6.3, Aldys Technologies Pvt. Ltd. Pune). Actograms were constructed and double plotted in a manner that is now routine in chronobiological research. 
| > Results|| |
The biochemical variables associated with oxidative stress in different groups of mice were studied and the effect of MLT on liver marker enzymes and several indicators of liver and blood antioxidant status were assessed. The activities of antioxidants (SOD, CAT, GPx, and GST) were markedly decreased in NDEA treated group than all other groups [Figure 1]a-d. Administration of MLT or oxaliplatin to animals (P < 0.05) significantly increased the antioxidant levels compared to NDEA-induced group. The antioxidant levels were decreased significantly (P < 0.05) in MLT-treated compared with oxaliplatin-treated mice. In NDEA-treated animals, AST and ALT activities were found to be significantly increased compared with controls. In MLT-treated mice significantly decreased of AST and ALT activities were seen. No significant difference was observed between MLT- and oxaliplatin-treated groups [Figure 1]e.
Under LD cycle, the synchronization of locomotor rhythm to the LD cycle in NDEA-treated animals is affected and lesser amount of activity is seen than controls [[Figure 2]a and b, respectively]. MLT treatment appeared to significantly reverse the disturbed rhythms [Figure 2]c and this reversal is not predominantly seen in oxaliplatin-treated group [Figure 2]d. Under constant darkness, free running rhythm is noticed in controls [Figure 3]a and this free running rhythm is again noticeably disturbed during hepatocarcinogenesis due to treatment with NDEA [Figure 3]b. MLT-treated group showed robust rhythmicity [Figure 3]c and oxaliplatin-treated group improved the rhythmicity [Figure 3]d compared with NDEA-treated group [Figure 3]b.
|Figure 1: (a) Changes in the activity of superoxide dismutase in hemolysate. Values are given as mean ± standard deviation from six rats in each group. Values not sharing a common superscript letter differ significantly at P < 0.05 Duncan's multiple range test (DMRT); UA-one unit of activity was taken as the enzyme reaction, which gave 50% inhibition of Nitroblue tetrazolium (NBT) reduction in 1 min (b) Changes in the activity of catalase in hemolysate. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT); UB-μ mole of hydrogen peroxide consumed/minute (c) Changes in the activity of glutathione peroxidase in hemolysate. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT); Ub-μg of glutathione consumed/minute (d) Changes in the activity of glutathione-S- transferase in haemolysate. Values not sharing a common superscript letter differ significantly at P < 0.05 (DMRT); Uc-μ mole of Chlorodinitrobenz- reduced glutathione (CDNB-GSH) conjugated formed/min/mg Hb (e) Changes in the activities of aspartate transaminase and alanine transaminase; a and c indicate no significant difference between groups, whereas b indicates significant difference compared with other groups at P < 0.05 (DMRT)|
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|Figure 2: (a-d) Representative actograms demonstrate the wheel-running activity rhythms of Mus booduga maintained under light dark (L:D-12:12) cycles|
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|Figure 3: (a-d) Representative actograms demonstrate the wheel-running activity rhythms of M. booduga maintained under constant darkness (DD)|
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| > Discussion|| |
Oxidative stress is known to contribute to NDEA-induced tumorigenicity via mediating damages in DNA and cellular proteins.  Metabolic activation of NDEA could produce ROS which is capable of initiating damage in the cell.  The increased oxidative stress may alter the antioxidant system and, hence, could decrease SOD, CAT, GPx, and GST levels. The activities of GST and GPx in liver, decreased during carcinogenesis, and these effects are partially reversed by MLT as it could quench free radicals. , Furthermore, MLT could also participate in antioxidative defense by incapacitating toxic species , and by modulating the activities of enzymes that maintain the redox balance within the cells. , MLT could scavenge ROS, such as superoxide anion, hydroxyl radical, and peroxynitrite, could stimulate the expression of antioxidant enzymes (SOD, CAT, GPx, and GST), , and could also indirectly act as a stimulator of the important antioxidative enzyme SOD  and thus the elevation of antioxidant enzymes could be comparatively higher than oxaliplatin-treated group. In the present study, oxaliplatin was used to compare the effects of MLT. This anticancer drug is an alkylating agent that forms DNA adducts, which in turn are responsible for cell death.  It is rapidly and nonenzymatically biotransformed to other molecular species. 
MLT could cross all morphophysiological barriers, for example, the blood-brain barrier and placenta, and could be distributed throughout the cell. These features increase the efficacy of MLT as an antioxidant.  This chronobiotic (MLT) has been shown to prevent oxidative damage under a variety of experimental conditions. , Serum transaminases are sensitive indicators of hepatic injury. Several reports have shown an increase in the activities of AST and ALT during NDEA-induced hepatocarcinognesis. , NDEA-induced group showed elevated level of AST and ALT and administration of MLT restored the activities of these enzymes to near normal values, which may be attributed to the hepatoprotective role of MLT.
The circadian clock could play a fundamental role in liver physiology and chronodisruption is known to augment hepatocarcinogenesis.  Circadian rhythm alterations have been described in tumor tissue, tumor-bearing animals, and in cancer patients; the disruption of locomotor activity in NDEA-treated animals clearly indicates that the biological clock is affected and this could augment the development of liver tumor. MLT and oxaliplatin could reverse the disruption of locomotor rhythms and the presence of robust clock could aid in the prevention of tumorigenesis. As MLT is a chronobiotic (a compound capable on acting on the biological clock directly), it could normalize the disturbed locomotor rhythms more effectively than the anticancer drug, oxaliplatin. To conclude, the present data added evidences that rest-activity circadian cycle could be altered during carcinogenesis and the circadian pattern of rest-activity cycle could be used as a reference for the chronotherapy with anticancer drugs, at specific times in order to improve tolerability and efficacy with minimal side effects. Furthermore, the data generated in this nocturnal system (M. booduga) will form a basis to extrapolate the studies in a comprehensive manner, in other mammalian systems to tailor chronotherapeutic strategies for liver cancer.
| > Acknowledgments|| |
Financial assistance in the form of ICMR to PS (58/4/08-BMS dated 03.12.2010) is gratefully acknowledged. Visiting Professorship to PS at Department of Molecular Medicine, Faculty of Medicine, University of Malaya is gratefully acknowledged.
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[Figure 1], [Figure 2], [Figure 3]