|Year : 2011 | Volume
| Issue : 2 | Page : 167-173
Coumarin protects 7,12-dimethylbenz(a)anthracene-induced genotoxicity in the bone marrow cells of golden Syrian hamsters
Nagarethinam Baskaran, Duraisamy Rajasekaran, Shanmugam Manoharan
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar, Tamil Nadu, India
|Date of Submission||02-Apr-2011|
|Date of Acceptance||12-May-2011|
|Date of Web Publication||23-Aug-2011|
Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar - 608 002, Tamil Nadu
Source of Support: University Grants Commission (UGC), New Delhi, Conflict of Interest: None
| Abstract|| |
Aim : Aim of the present study was to evaluate the antigenotoxic effect of coumarin by measuring the frequencies of micronuclei and the degree of DNA damage in the bone marrow cells of hamster treated with 7,12-dimethylbenz(a)anthracene (DMBA). Materials and Methods: Genotoxicity was induced in experimental hamsters by single intraperitoneal injection of DMBA (30 mg/kg b.w.). The frequency of micronucleated polychromatic erythrocytes (MnPCEs) and DNA damage were assessed in the bone marrow cells of experimental hamsters. The status of lipid peroxidation, antioxidants and phase I and II detoxification agents were utilized as biochemical end points to assess the dose-dependent antigenotoxic potential of coumarin in DMBA-induced genotoxicity. Results: Increase in micronuclei frequency was accompanied by increase in tail length, percent of tail DNA, tail movement and olive tail movement in the bone marrow cells of hamsters treated with DMBA alone. A significant increase in the levels of thiobarbituric acid reactive substances, antioxidants and phase I and II detoxification agents were also noticed in hamsters treated with DMBA alone. Oral pretreatment of coumarin at a dose of 100 mg/kg b.w to hamsters treated with DMBA significantly decreased the frequency of MnPCEs and DNA damage in the bone marrow cells. Also, coumarin restored the status of biochemical variables in the plasma and liver of hamsters treated with DMBA. Conclusions: Present study demonstrated the antigenotoxic effect of coumarin in DMBA-induced genotoxicity. The antigenotoxic potential of coumarin is probably due to its antioxidant potential and modulating effect on detoxification cascade during DMBA-induced genotoxicity.
Keywords: Antioxidants, coumarin, DNA damage, genotoxicity, micronucleus
|How to cite this article:|
Baskaran N, Rajasekaran D, Manoharan S. Coumarin protects 7,12-dimethylbenz(a)anthracene-induced genotoxicity in the bone marrow cells of golden Syrian hamsters. Int J Nutr Pharmacol Neurol Dis 2011;1:167-73
|How to cite this URL:|
Baskaran N, Rajasekaran D, Manoharan S. Coumarin protects 7,12-dimethylbenz(a)anthracene-induced genotoxicity in the bone marrow cells of golden Syrian hamsters. Int J Nutr Pharmacol Neurol Dis [serial online] 2011 [cited 2019 Jun 16];1:167-73. Available from: http://www.ijnpnd.com/text.asp?2011/1/2/167/84209
| Introduction|| |
Humans and animals are constantly exposed to several toxic substances in day to day life. Genotoxicity studies in rodents could help to evaluate the genotoxic potential of the test agents to humans.  Measurement of micronuclei frequency could help to assess the cancer risk since altered micronuclei frequency indicates genomic instability in the target tissues. The rodent erythrocyte micronucleus (MN) assay in peripheral blood or bone marrow is routinely used to test the genotoxic potential of the test compound. Moreover, it is a more sensitive technique than any other cytogenetic assays due to increased statistical power brought by increase in the number of cells analysed and scored.  Assessment of micronuclei frequency surrogates the measures of the structural and numerical chromosomal aberrations. 
Comet assay is commonly employed to biomonitor DNA damage in various mammalian cells.  Comet assay reveal single cell DNA migration pattern, appeared in agarose gel electrophoresis under alkaline conditions. Comet assay, a rapid, repetitive, simple, versatile, visual, cost-effective and sensitive assay, is used to detect DNA lesions such as strand breaks, alkali labile sites, cross-links and incomplete excision repair sites. 
Exposure to environmental carcinogenic chemicals or their metabolites generates excessive reactive oxygen species, which interact with many cellular constituents, including lipids, protein and DNA. Experimental studies have reported 7,12-dimethylbenz(a)anthracene (DMBA)-induced gene mutations, chromosomal aberrations and other types of genotoxicity.  DMBA-induced genotoxicity is therefore commonly employed to assess the antigenotoxic potential of natural products and synthetic entities.
Biomarkers, which assist in diagnosing the diseases, are measured and evaluated as an indicator of normal physiologic processes, pathologic processes, or pharmacologic responses to a therapeutic intervention.  Oxidative stress plays an important role in the pathogenesis of many diseases including cancer and can cause oxidative damage to the structure and functions of DNA, protein and lipids. Overproduction of reactive oxygen species (ROSs) can induce abnormalities in the chromosome structure and functions.  Mammalian cells are however endowed with sophisticated enzymatic and non-enzymatic antioxidant defense mechanism to prevent oxidative DNA damage. An imbalance in oxidant and antioxidant status has been reported in DMBA-induced genotoxicity. 
Liver plays a vital role in the detoxification of xenobiotics and carcinogens and thus measurement of phase I and II detoxification agents could help to assess the antigenotoxic potential of the test compounds.  Phase I and II detoxification enzymes play crucial role in the metabolic activation and detoxification of carcinogenic metabolites, respectively, and thereby reducing DNA damage and mutation. Several studies documented the status of liver detoxification agents in DMBA-induced genotoxicity. 
Coumarin, a member of the benzopyrone family of compounds, are found throughout the plant kingdom and at high levels in some essential oils, particularly cinnamon bark oil, cassia leaf oil and lavender oil. Coumarin is also found in many plants and natural food products, such as citrus fruits, tomatoes, vegetables and green tea. Coumarin has long been used in hand soaps, detergents, face creams, toothpastes, shampoos, body lotions and perfumes at concentrations usually extending from 0.01 to 0.8%.  Coumarin has diverse pharmacological properties including antithrombotic, anti-inflammatory, antiviral, antioxidant, immunomodulatory and anti-tumor properties.  It has been shown that oral administration of 65 and 130 mg/kg/day coumarin by gavage for 7 days did not induce micronuclei in the bone marrow cells of male and female ICR mice.  To the best of our knowledge, there were no experimental studies on the antigenotoxic effect of coumarin in DMBA-induced genotoxicity. The present study was therefore undertaken to investigate the antigenotoxic effect of coumarin in DMBA-induced genotoxicity in the bone marrow cells of golden Syrian hamsters.
| Materials and Methods|| |
Twenty-four male golden Syrian hamsters, 8 weeks old, weighing 80-120 g, were obtained from National Institute of Nutrition, Hyderabad, India, and maintained in Central Animal House, Rajah Muthiah Medical College and Hospital, Annamalai University. The animals were housed in polypropylene cages and provided standard pellet diet and water ad libitum. The animals were maintained under controlled conditions of temperature and humidity with a12 h light-dark cycle.
DMBA, coumarin, colchicine, Giemsa and May-Grόnwald's stains were purchased from Sigma Aldrich Chemical Pvt. Ltd., Bangalore, India. All other chemicals used were of analytical grade.
The Institutional Animal Ethics Committee (Reg. No.: 160/1999/CPCSEA), Annamalai University, Annamalai Nagar, approved the experimental design. A total number of 24 animals were divided into four groups and each group contained six animals. Group 1 animals were served as control. Group 3 animals were pretreated with coumarin (100 mg/kg b.w. p.o.) for 5 days and were intraperitoneally injected with DMBA (30 mg/kg b.w.) on fifth day after 2 h of administration of coumarin. Group 2 animals were given intraperitoneal injection of DMBA (30 mg/kg b.w.) on fifth day. Group 4 animals were pretreated with coumarin (100 mg/kg b.w. p.o.) alone for 5 days and did not receive DMBA. ,, All the animals were sacrificed after 24 h of DMBA injection by cervical dislocation for the assessment of micronucleus frequency and DNA damage.
Bone marrow micronucleus test
Bone marrow micronucleus test was carried out according to the method of Schmid.  The femur bones removed from the golden Syrian hamsters were cleaned and the content was flushed into the tube containing 1 ml of calf serum and was centrifuged at 500 Χ g for 10 min. The obtained pellet was suspended with few drops of fresh serum and slides were prepared and air dried for 18 h. After drying, the slides were stained with May-Grόnwald stain followed by Giemsa stain. The frequency of micronucleated polychromatic erythrocytes (MnPCEs) in each group was calculated by scoring 2500 polychromatic erythrocytes (PCEs) per animal. 
Single cell gel electrophoresis assay/Comet assay
The single cell gel electrophoresis (comet) assay, a rapid, simple and reliable technique, was used to assess the DNA damage in the bone marrow cells.  The femur bone marrow cells were flushed into Hank's balanced salt solution (HBSS) and then filtered through a 50 μm nylon filter. The cells were counted and diluted to arrive a final suspension of 50,000-100,000 cells/ml. The mixture of 10 μl bone marrow cells and 200 μl of 0.5% low melting point agarose was layered onto pre-coated slides, which contain 1% normal melting point agarose and then covered with a cover slip. The slides were placed in the chilled lysing solution containing 2.5 M NaCl, 100 mM Na 2+ EDTA, 100 mM Tris-HCL, pH 10 and 1% DMSO, 1% Triton X 100 and 1% sodium sarcosinate, for 1 h at 4°C and followed by alkaline buffer (pH > 13) for 20 min. The electrophoresis was carried out for 20 min, at 25 V and 300 mA. The slides were stained with 50 μl of ethidium bromide (20 μg/ml) and analysed under fluorescence microscope. The images (25 cells/slide) were viewed under high performance Nikon camera.
DNA damage, as reflected by % DNA in tail (tail intensity), tail length, tail moment (product of tail DNA/total DNA by the center of gravity) and olive tail moment (the product of the distance between the barycenters of the head and tail and the proportion of DNA in the tail) of the stored images, was investigated from 25 cells per treatment using CASP software ( http://casp.sourceforge.net ).
Blood samples were collected from the jugular vein of experimental animals at 8.00 am in the research laboratory of Department of Biochemistry and Biotechnology, Annamalai University. Blood samples were collected into heparinized tubes. Plasma was separated by centrifugation at 1000 Χ g for 15 min. The buffy coat was removed and the packed cells were washed three times with physiological saline. Liver tissues from animals were washed with ice cold saline and homogenized using appropriate buffer in an all glass homogenizer with Teflon pestle and used for biochemical estimations.
Lipid peroxidation was estimated as evidenced by the formation of thiobarbituric acid reactive substances (TBARSs). TBARS in plasma and tissues were assayed by the method of Yagi  and Ohkawa et al0. respectively. The activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in plasma and liver was determined according to the methods of Kakkar et al., Rotruck et al. and Sinha,  respectively. Reduced glutathione (GSH) level in the liver was determined by the method of Beutler and Kelley.  The levels of cytochrome P450 and b5 in the liver were determined according to the method of Omura and Sato.  The activity of glutathione-s-transferase (GST) in liver was assayed by the method of Habig et al. Glutathione reductase (GR) activity in the liver was assayed by the method of Carlberg and Mannervik. 
The data are expressed as mean ± SD. Statistical comparisons were performed by one way analysis of variance (ANOVA), followed by Duncan's multiple range test (DMRT). The results were considered statistically significant if the p values were 0.05 or less.
| Results|| |
[Table 1] shows the frequency of MnPCEs in the bone marrow cells of control and experimental hamsters in each group. Hamsters treated with DMBA alone [Group 2] showed highest frequency of MnPCEs as compared to control hamsters [Group 1]. Oral pretreatment of coumarin to DMBA-treated hamsters [Group 3] significantly decreased the frequency of MnPCEs. Oral pretreatment of coumarin alone to hamsters [Group 4] displayed no significant difference in MnPCEs frequency as compared to control hamsters.
|Table 1: Frequency of MnPCEs in control and experimental animals in each group|
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[Table 2] shows the severity of DNA damage (% DNA in tail, tail length, tail moment, olive tail moment) in the bone marrow cells of control and experimental hamsters in each group. We observed an increase in DNA tail length, tail moment, % DNA in tail and olive tail moment in hamsters treated with DMBA alone [Group 2]. Oral pretreatment of coumarin significantly protected DNA damage in DMBA-treated hamsters [Group 3]. Hamsters treated with coumarin alone [Group 4] and control hamsters [Group 1] displayed no significant difference in % DNA in tail, tail length, tail moment and olive tail moment.
|Table 2: Changes in the levels of DNA damage (% DNA in tail, tail length, tail moment and olive tail moment) in the hamsters bone marrow cells|
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The appearance of comet during single cell gel electrophoresis in control and experimental hamsters is shown in [Figure 1]. We noticed a comet head with long tail in hamsters treated with DMBA alone [Group 2]. The length of tail was significantly smaller in DMBA + coumarin-treated hamsters [Group 3]. Hamsters treated with coumarin alone [Group 4] and control hamsters [Group 1] displayed similar pattern of comet appearance.
|Figure 1: Representative photographs depict the extent of DNA damage in control (a), DMBA-treated hamsters (b), DMBA + Coumarin treated hamsters (c) and coumarin alone treated hamsters (d) (× 40 magnification)|
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[Table 3] and [Table 4] show the status of TBARS and enzymatic antioxidants (SOD, CAT, GPx) in plasma and liver, respectively, of control and experimental hamsters in each group. TBARS and enzymatic antioxidants were increased in hamsters treated with DMBA alone [Group 2] as compared to control hamsters. Oral pretreatment of coumarin significantly decreased the levels of TBARS and enzymatic antioxidants activities in hamsters treated with DMBA [Group 3]. No significant difference was observed between control hamsters [Group 1] and coumarin alone treated hamsters [Group 4].
|Table 3: Plasma TBARS and antioxidants in control and experimental animals in each group|
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|Table 4: Liver TBARS and antioxidants in control and experimental animals in each group|
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[Table 5] shows the activities of phase I [Cytochrome P450 and b5] and phase II [GST and GR and GSH] detoxification agents in the liver of control and experimental hamsters in each group. The activities of detoxification agents were significantly increased in hamsters treated with DMBA alone [Group 2] as compared to control hamsters. Oral pretreatment of coumarin to DMBA-treated hamsters [Group 3] brought back the status of detoxification agents to near normal range. No significant difference was observed between control hamsters [Group 1] and coumarin alone treated hamsters [Group 4].
|Table 5: Activities of phase I and phase II detoxifi cation enzymes and glutathione content in the liver of control and experimental animals in each group|
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| Discussion|| |
In vivo cytogenetic studies could help to screen and explore the genotoxicity of chemicals or drugs, which are harmful to humans. Previous studies from our laboratory demonstrated the antigenotoxic potential of natural products and their active constituents in DMBA-induced genotoxicity. ,, The present study investigated the antigenotoxic potential of coumarin in DMBA-induced genotoxicity. DMBA, a potent organ specific carcinogen, can induce several types of gene mutations. DMBA-induced H and N-ras mutations were demonstrated in experimental carcinogenesis. N-ras mutation has been shown to be the earliest event in DMBA-induced leukemogenesis.  In the present study, highest MnPCEs frequency was observed in the bone marrow cells of hamsters treated with DMBA alone. Profound studies demonstrated increased MnPCEs frequency in animals treated with DMBA. , Present results corroborate these findings. Oral pretreatment of coumarin at a dose of 100 mg/kg b.w. significantly reduced MnPCEs frequency in the bone marrow cells of DMBA-treated hamsters. Our results thus suggest that coumarin has potent anticlastogenic potential against DMBA-induced genotoxicity.
We measured the severity of DNA damage in the bone marrow cells of experimental hamsters in each group by correlating with the appearance of comet during single cell gel electrophoresis. Appearance of comet head with long tail indicates extensive DNA damage. In the present study, comet with head and long tail was noticed in hamsters treated with DMBA alone, which indicates severe DNA damage in the bone marrow cells. Oral pretreatment of coumarin at a dose of 100 mg/kg b.w. to DMBA-treated hamsters significantly reduced the formation of DNA tail. Our results suggest that coumarin significantly protected DNA damage (antigenotoxic potential) in the bone marrow cells of DMBA-treated hamsters during DMBA-induced genotoxicity.
Liver phase I and II detoxification agents play a crucial role in the metabolic activation and detoxification, respectively, of carcinogenic metabolites. Altered status of detoxification agents in liver thus indicates the insult by toxic foreign substances. Agents that modulate the activities of detoxification cascade to excrete the carcinogenic metabolites are thus considered to have potent antigenotoxic potential.  In the present study, the activities of phase I and II detoxification enzymes and GSH content were increased in hamsters treated with DMBA alone, which indicates that liver detoxification cascade was stimulated to detoxify the carcinogenic metabolite of DMBA. Oral pretreatment of coumarin restored the status of phase I and II detoxification agents in DMBA-treated hamsters, which suggest that coumarin might have inhibited the metabolic activation of DMBA and/or stimulated the excretion of ultimate carcinogenic metabolite of DMBA, dihydrodiol epoxide.
ROS has been implicated in the pathogenesis of several disorders, if they are excessively generated in the cells or tissues. Natural or synthetic substances, that act as inducer of detoxification cascade or antioxidant defense mechanism can reduce carcinogen-induced genotoxicity. , Mammalian cells with insufficient antioxidant potential are more prone to oxidative DNA damage.  Increased levels of lipid peroxidation by products (TBARS) accompanied by an increase in the activities of endogenous enzymatic antioxidants (SOD, CAT, GPx) confirm the oxidative stress in hamsters treated with DMBA alone. Oral pretreatment of coumarin brought back the status of plasma TBARS and antioxidant enzymes in DMBA-treated hamsters. Present results thus indicate the antioxidant potential of coumarin in DMBA-induced genotoxicity. The antioxidant potential relies on its coumarin nucleus, consists of an aromatic ring fused to a condensed lactone ring, which serve as nucleophiles to scavenge the reactive carcinogenic metabolites. 
| Conclusions|| |
Present study thus demonstrated the antigenotoxic potential of coumarin in DMBA-induced genotoxicity. The antigenotoxic potential of coumarin is probably due to its antioxidant potential and modulating effect on phase I and II detoxification cascade during DMBA-induced genotoxicity.
| Acknowledgement|| |
Financial support from University Grants Commission (UGC), New Delhi to Mr. N. Baskaran in the form of UGC-RGNF-SRF is gratefully acknowledged.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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