Protective Effect of Jasminum grandiflorum Linn. On DMBA-induced Chromosomal...

[Full title: Protective Effect of Jasminum grandiflorum Linn. On DMBA-induced Chromosomal Aberrations in Bone Marrow of Wistar Rats]

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Life International Journal of Pharmacology ISSN 1811-7775 Life science alert ansinet Asian Network for Scientific Information

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International Journal of Pharmacology 2 (1): 106-110, 2006 ISSN 1811-7775 2006 Asian Network for Scientific Information Protective Effect of Jasminum grandiflorum Linn. On DMBA-induced Chromosomal Aberrations in Bone Marrow of Wistar Rats 'Shanmugam Manoharan, 'Kuppusamy Panjamurthy, Krishnamurthy Vasudevan, Dhanarasu Sasikumar and 'Kalivaperumal Kolanjiappan Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar- 608002, Tamil Nadu, India Department of Zoology, Faculty of Science, Annamalai University, Annamalai Nagar-608002, Tamil Nadu, India Department of Sidda Medicine, Tamil University, Thanjavur, Tamil Nadu, India Abstract: The present study has investigated the protective effects of Jasminum grandiflorum flowers and leaves in 7,12-dimethylbenz(a)anthracene (DMBA) induced chromosomal abnormalities in bone marrow of female wistar rats. Micronuclei quantification and detection of chromosomal aberrations were performed to detect and quantify the genotoxic action of DMBA. Oral pretreatment of J. grandiflorian [lower and leaf extracts to DMBA treated rats significantly reduced the frequency of micronucleated polychromatic erythrocytes (MnPCEs) the in rat bone marrow. Also, the plant extracts significantly decreased the percentage of aberrant cells; the number of chromatic and chromosomal breaks in DMBA treated rals. Presenil results thus demonstrate that the plant products of J. grandiflorum (flowers and leaves) have potent protective effects in DMBA-induced chromosomal aberrations in female wistar rats. Key words: DMBA, chromosomal aberrations, micronuclei, Jasminum grandiflorum INTRODUCTION Several studies have consistently shown that the development of cancer is related to genetic damage rate in somatic cells. Agents that can alter an organism's genome by causing toxic effect on cellular genetic materials are referred to as mutagenic. Such substances increase the eggog rate in the reduplication of the genome and induce mutations by damaging the organism's DNA (Schmid, 1973; Agarwal et al., 1994). An abnormality in chromosomal structure (translocation, deletion. duplication) and chromosomal number (aneuploidy and polyploidy) due to exposure to chemical or physical satelietatormulagern is referred to as chromosomal aberrations. Cytogenetic studies have shown chromosomal aberrations in cell populations of both human and experimental cancer (Agarwal et al., 1994; Gaizev et al., 1996). An increase in chromosomal breakage and chromosome loss is associated with increased risk of cancer and in the progression of neoplastic transformation (Guerin et al., 1978). Bone marrow micronucleus Test and detection of chromosomal to aberrations have been widely used as a tool to indicate carcinogen-induced DNA damage as well as the antimutagenic effect of natural and synthetic chemopreventive agents (Hagmar et al. 2001; Bhuvaneswari et al., 2004). assess Carcinogen induced mutations in somatic cells have been implicated in cell death and pathogenesis of several diseases including cancer. The mutagenic and carcinogenic action of mutagenic substances involves overproduction of DNA-attacking reactive oxygen species (Ames et al., 1973). 7,12-dimethylbenz (a) anthracene (DMBA), the polycyclic aromatic hydrocarbon, is an immunosuppressor and potent organspecific carcinogen. It has been reported that DMBA exposure result in a marked increase in tumor burden and tumor volume in rodent models and pronounced mutagenic response in several in vivo and in vitro mutation assay systems (Chang et al., 1996). Studies have demonstrated the DNA damaging and mutagenic effects of DMBA in experimental models (Chandra et al., 2003). Both in vivo and in vitro studies have demonstrated polyploidy and sister chromatic exchanges in DMBACorresponding Author: Dr. Shanmugam Manoharan, Department of Biochemistry and Biotechnology, Faculty of Science, Amamalai University, Amanalainagar-608 002, Tamil Nadu, India, Tel: 91 04144 238343 Fax: 91 04144 238145 406

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Intl. J. Pharmacol., 2 (4): 406-410, 2006 induced genotoxicity (Guerin et al., 1978; Bhuvaneswari et al., 2001). It has been suggested that the N-ras mutation is an earliest event in DMBA-induced leukemogenesis (Osaka et al., 1996). Medicinal plants exert their chemopreventive potential by interfering with covalent interaction of a carcinogen with DNA, modifying DNA repair process. antioxidant properties and preventing cellular proliferation. Profound evidence has revealed that. medicinal plants can reduce genetic damages induced by mutagens and carcinogens (Premkumar et al., 2004; Balasenthil et al., 2000). Although the protective roles of some medicinal plants were reported in carcinogeninduced genotoxicity, several other medicinal plants remain to be investigated. Jasminum grandiflorum is a well-known glabrous twining shrub widely grown in gardens throughoul. India. J. grandiflorum flowers and leaves are largely used in folk medicine to prevent and treat breast cancer. J. grandiflorum flowers are traditionally used to women when brewed as a tonic as it aids in preventing breast cancer and stopping uterine bleeding (Joshi, 2000). We recently demonstrated the anticarcinogenic and antilipidperoxidative effects of J. grandiflorum in DMBA-induced mammary carcinogenesis (Kolanjiappan et al., 2005). However, no reports are available on the protective action of J. grandiflorum in DMBA induced chromosomal aberrations in wistar rats. The present study was thus designed to evaluate the protective effect of J. grandiflorum flowers and leaves against. DMBAinduced chromosomal aberrations in bone marrow of female wistar rats. MATERIALS AND METHODS Animals: Female Wistar albino rats, 7-8 weeks old, weighing 130-140 g were used for the study. The animals were obtained from central animal house, Rajah Muthiah Institute of Health Science, Annamalai University, Annamalainagar, India. The rats were housed In polypropylene cages and were maintained in controlled atmosphere (temperature of 22+2°C and 50-70% humidity) with a 12h light: dark (LD) cycles in an experimental room. The local institutional animal ethics committee, Annamalai University, Annamalainagar, India, approved the experimental design. Chemicals: The carcinogen, 7, 12-dimethylbenz (a) anthracene (DMBA), colchicine, Giemsa, May-Grunwald's stain were obtained from Sigma-Aldrich Chemicals Pvt. Lid., Bangalore, India. All other chemicals used were of analytical grade. Preparation of the plant extract: .. grandiflorum flowers and leaves were obtained from the gardens around Chidambaram, Tamil Nadu, India, and identified by the Botanist, Department of Botany. Annamalai University. A voucher specimen was deposited, in the Department of Botany, Ammamalai University. The aqueous and ethanolic extracts of J. grandiflorum flowers and leaves were prepared according to the method of Hossain et al (Hossain et al.. 1992). Five hundred gram of fresh flowers or leaves of J. grandiflorum were dried, powdered and then soaked in 1500 mL of 95% ethanol overnight. After filtration the residue obtained was again resuspended in equal volume of 95% ethanol for 48 h and filtered again. The above two filtrates were mixed and the solvent was evaporated in a rotavapour at 40-50°C under reduced pressure. An 11% semisolid light yellow material of J. grandiflorum flowers and 12% semisolid light greenish yellow material of leaves were obtained. Hundred gram of dried fine powder of J. grandiflorum leaves or flowers were suspended in 250 ml of water for 2 h and then heated at 60-65°C for 30 min. The extract was collected and the processes were repeated three times with the residual powder and the collected extract was pooled and passed through line cotton cloth. The filtrates were evaporated at 40-50°C in a rotavapour. J. grandiflorum leaves yielded 13% greenish brown semisolid and flowers yielded 12.5% brownish semisolid. All the extracts were stored at 0-4°C until used. A known amount of the residual extract. (300 mg kg bw (ethanolic), 500 mg kg¯¯ bw (aqueous)) was suspended in distilled water and was orally administered to the animals by gastric incubations using force-feeding needle during the experimental period. A total number of 60 animals were divided into 10 groups and each group contained 6 animals. Groups 2 to 5 animals were pretreated with aqueous extract of J. grandiflorum flowers (500 mg kg bw), ethanolic extract of J. grandiflorum flowers (300 mg kg bw), aqueous extract of J. grandiflorum leaves (500 mg kg bw) and ethanolic extract of J. grandiflorum leaves (300 mg kg bw), respectively for five days. At the end of the 5th day, groups 1 to 5 animals were intraperitoneally injected with DMBA (30 mg kg bw, single dose) after 2 h of administration of the plant extracts. Groups 6 to 9 animals were received aqueous extract of J. grandiflorum flowers (500 mg kg bw), ethenolic exitacl of J. grandiflorum Plowers (300 mg kg bw), aqueous extract of J. grandiflorum leaves (500 mg kg bw) and ethenolic extract of J. grandiflorum leaves (300 mg kg bw) alone respectively and were not received DMBA. Groups 10 407

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Intl. J. Pharmacol., 2 (4): 406-410, 2006 served as control and all animals were provided standard pellets and water ad libitum. All the animals were sacrificed at the 6th day by cervical dislocation for the assessment of micronucleus and chromosomal aberrations. Assessment of chromosomal aberrations in bone marrow was carried out according to the procedure of (Kilian et al., 1977). The femur bones were removed from animals injected intraperitoneally with 0.1% colchicines, (1 mL/100 g body weight 90 min before sacrificing the animals. The bone marrow contents were flushed into 5 mL of physiological saline and centrifuged at 500 g for 5 min. The sediment obtained were resuspended in 6 mL of hypotonic KCI (0.075 M) and incubated at 37°C for 25 min. The pellets were then fixed using methanol: acetic acid (3:1) fixative and stained with Giemsa stain. One hundred well spread metaphase cells were scored for each animal and structural chromosomal aberrations were observed and recorded. Bone marrow micronucleus test was carried out according to the method of Schimid (1975). The femur bones removed from the Wistar rats were cleaned and the content was flushed into tube containing 1 mL of calf scrum and was centrifuged at 500 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-Grunwald stain followed by Giemsa stain. The frequency of MnPCDs in each group was calculated by scoring polychromatic erythrocytes (PCEs) per animal. 2500 Statistical analysis: The values are expressed as mean SD. The statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Duncan's multiple range test. (DMRT). p-values less than 0.05 were considered statically significant. RESULTS The frequency of MnPCEs and chromosomal aberrations in control and experimental animals in each group are given in Table 1 and 2, respectively. All groups (groups 2-5) Treated with DMBA showed a high frequency of MnPCLS and chromosomal aberrations (structural aberrations: chromatic gap, chromosomal gap. chromatic break, chromosomal break, fragment, minute) as compared to control animals. IIowever, rats treated with DMBA alone (group 1) showed highest frequency of MnPCEs and chromosomal aberrations as compared to control rats. The frequency of MnPCEs and chromosomal abnormalities were significantly reduced in DMBA treated animals pretreated with oral Table 1: Effect of J. grandiflorum on DMBA-induced bone marrow micronuclei formation Group Parameters DMDA PCE&NCEs 0.8110.0 1 2 3 .1 S g Tlower (Aqueous Ext) DMBA g Flower (Alcoholic Ext) DMMA Jg Leaf (Aqueous Ext.) - DMBA g Leaf (Alcoholic Text) DMBA J & Flower (Aqueous Text.) 7 Jg Flower (Alcoholic Ext) 8 Jg Leaf (Aqueous Ext) h Jg Leaf (Alcoholic Ext) 10 Contral MnPCEs 2500PCEs PC'Es* (%) 67.591.21 39.3112.526 11.80 0.9110.05 18.10 26.3212.83 0.91 10.08 17.50 11.1.512.95d 0.95 10.05 18.60 31.9113.626 0.9210.07 17.90 6.8610.92 5.1110.580 7.130.57 1.040.00 51.10 5.93 10.52 5.76 10.3.1° 1.020.07 1.010.08 1.01 10.08 50.00 51.00 19.90 1.01 10.07 50.10 *Values are expressed as can+SD (n 6; 2500 PCEs were scored per animal). Values not sharing a common superscript significantly differ al p<0.05. (DMRT) Perventage of polychromatic cryocytes was calculated as follows: [PCEs (PCEs+NCEs)] x100 Table 2: Mitotic index and frequencies of chromosomal abnormalities in experimental and control animals Chromosomal aberrations rat' Group 1 Parameters DMBA 2 Jg Flower (Aqueous ext) - DMBA Mitotic index (%) 1.76=0.06 2.31 0.83 Total aberrations Abnormal 3 J. Flower (Alcoholic Fxt) DMHA 2.48 0.91 G* 12.92=0.18 7.51-0.08 5.17=0.07 B' B" M rat metaphase rat 4 Leaf (Aqueous Ext) DMHA 2.43 0.76 5 g Leaf (Alcoholic Fxt) DMHA 2.12=0.92 6.93=0.07 5.16=0.06 6 Flower (Aqueous ext) 4.09=1.02 7 J. Mower (Alcoholic Ext) 4.27-1.04 X Leaf (Aqueons Ext) 4.32 1.07 ri Leaf (Alcoholic Ext) 10 Control 4.14=1.03 4.72=1.07 [1.46=0.01 0.44 0.03 0.46 0.01 0.43=0.02 0.48=0.02 1.01 0.06 1.50 0.08 1.33=0.00 0.33 0.01 0.31 0.01 9.14±1.47" 6.92±1.07 2.16±0.46' 2.43+0.35 2. 25±0.37 2.4310.20 2. 57±0.64 1.6+0.10 Values are expressed as mean±SD); n-6. Values not sharing a common superscript significantly differ at p<0.05. (DMRT) (7- Cap, B'- Chromatic Break, P. Tsochromatid Break, F- Fragment, M-minute. A-Mitotic index has been calculated by analyzing 1000 cells/animal (for a total of 6000 cells/treatment) and percentage of the mitotic cells calculated for each treatment group. B- Frequency per 100 cells. Fach chromosomal aberration has been counted by analyzing 100 cells/animal (6 animals/group, for a total of 600 cells/treatment] and mean±SD) were calculated per treatment group. Chaps were not included in total chromosomal aberrations 7.54±0.24 4.9±0.17 3.48±0.10 5.14±0.07 3.61±0.08 1.30.01 1.1±0.01 1.33±0.02 1.16±0.02 2.4.5±0.192 1.17±0.06 1.16±0.05 1.0005 1.16±0.06 0281020 0.16±0.01 03310102 0.16±0.01 7.10-0.76 2.36=0.1.5 18.63±1.54 1.5.1.5±1.85 5.16 0.50 3.33 0.54 1.17 0.06 12.12±1.62" 10.31±1.61 1.1 (0.011 11 5.60=0.52 3.16=0.51 1.8.4-0.05 1.65=0.04 1.84 0.05 1.67-0.05 1.33=0.02 9.21±1.01 11.26±1.81" 6.27±1.59 9.35±1.65" 0.28=0.02 0.33=0.02 0.32=0.02 1.42±0.06' 1.46±0.67' 1.57±0.09 1.57±0.08 408

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Intl. J. Pharmacol., 2 (4): 406-410, 2006 administration of J. grandiflorum flower and leaf extracts. Of these extracts, the ethanolic extract of J. grandiflorum flowers showed better effect in DMBA induced genotoxicity. Oral administration of J. grandiflorum plant extracts alone displayed no significant differences in MnPCEs and chromosomal abnormalities as compared to control rats. DISCUSSION The aim of the present study was to demonstrate the protective effects of J. grandiflorum flowers and leaves in DMBA induced bone marrow chromosomal abnormalities in female wistar rats. Previous studies from our laboratory demonstrated that. J. grandiflorum flowers significantly reduced the incidence of tumor formation and tumor volume as well as reduced the levels of lipid peroxides and improved the antioxidant defense mechanism in DMBA induced mammary carcinogenesis (Kolanjiappan et al., 2005). The rodent. bone marrow and peripheral blood micronucleus test have been used as tool to evaluate the mutagenicity of environmental mulagens and carcinogens (Ilagmar et al., 2001; Ilayashi et al., 2000). The purpose of the micronucleus assay is to identify a mutagenic substance that causes cytogenetic damages, which results in the formation of micronucleus containing lagging chromosome fragments or whole chromosomes. Measurement of micronuclei polychromatic crythrocytes is suitable due to the reasons that their macronucleus is extruded in the processes of their formation from erythroblasts (Gaizev et al., 1996; Hayashi et al., 2000). Chromosomal aberrations are well detected in the cells in the metaphase of mitosis. due to the fact that the chromosomes are compact in shape and their structure is easy to examine at this stage (Hagmar et al., 2001). in can cause DMBA is present in the environment as a product of incomplete combustion of complex hydrocarbons. DMBA. being an indirect carcinogen, requires further metabolic activation to become an ultimate carcinogen. The thiol epoxides and other toxic reactive oxygen species formed during metabolic activation of DMBA chromosomal damage by binding with adenine residues of DNA. It has been suggested that DMBA is highly mutagenic to lac I in mammary tissues and that adducts with both GC and A:T base pairs participate in forming mutations in DMBA-treated Big Blue rats (Manjanatha et al., 1998). Ha-ras mutations occur in the very early pre-dysplastic phase and amplification and over expression of C-erbB and its product, the epidermal growth factor receptors occur in the late phases of DMBA Treatment in DMBA induced hamster buccal pouch carcinogenesis (Husain et al., 1989). Quintanilla et al. (1991) demonstrated that DMBA caused predominantly A to T transversions in Ha-ras codon 61 in mouse skin and hamster buccal pouch tumors, which is consistent with the ability of DMBA to form bulky adducts with adenosine. An increase in micronucleus frequency and chromosomal aberrations in bone marrow of DMBA painted or injected rexdents have been reported (Guerin et al., 1978; Bhuvaneswari et al., 2004). was Substances, which reduce genetic instability in somatic cells, are said to be antimutagenicity agents. Medicinal plants with bioactive chemopreventive principles can minimize the deleterious effects of mutagens and carcinogens. The protective role of J. grandiflorum on micronuclei frequency and chromosomal aberrations induced by DMBA investigated in Wistar rals. DMBA elevated MnPCEs frequency in the bone marrow of DMBA injected animals, was suppressed by J. grandiflorum flower and leaf extracts. Oral administration of J. grandiflorum lower and leaf extracts significantly decreased the percentage of aberrant cells and the number of chromatic as well as chromosomal breaks in DMBA-injected animals. Although the exact mechanism of protective role of J. grandiflorum is unclear, the possible mechanisms include an enzymatic induction and increase enzymatic degradation of DMBA by liver, modification of biodistribution of DMBA modification of erythropoiesis and/or enhance the antioxidant defense mechanisms to neutralize the toxic effects of reactive oxygen species generated by DMBA. The present study thus demonstrates the protective role of J. grandiflorum in DMBA-induced chromosomal aberrations in female Wistar rats. ACKNOWLEDGEMENTS The authors are grateful to Dr. A. Ramesh, Professor and Ilead, Dr. S.T. Santhiya, Professor and Mrs. A. Saraswathi Chitra Devi, Research Scholar, Department. of Genetics, Dr. ALM Postgraduate Institute of Basic Medical Sciences, Tharamam, Chennai, India, and Dr. C.R. Ramachandran, Dean. Faculty of Dentishy, Annamalai University, Mr. G. Gunasekaran, Lab technician, Department of Biochemistry, Annamalai University, for their kind help and support in carrying out. This sturly. Financial support from Indian Council of Medical Research (ICMR), New Delhi, to Mr. K. Kolanjiappan, in the fon of TCMR-SRF is gratefully acknowledged 409

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