Inhibitory effects of Indigofera aspalathoides on 20-methylcholanthrene-induced chemical carcinogenesis in rats

S Selva Kumar1, CM Karrunakaran1, M. R. K. Rao1, MP Balasubramanian2
1 Department of Industrial Biotechnology, Bharath University, Chennai 600 073, India
2 Department of Pharmacology and Environmental Toxicology, Dr. A. L. M. Post graduate Institute of Basic Medical Sciences, University of Madras, Taramani, Chennai 600 113, India

Date of Submission 07-Apr-2010
Date of Acceptance 09-Dec-2010
Date of Web Publication 11-Jan-2011

Correspondence Address:
S Selva Kumar
Department of Industrial Biotechnology, Bharath University, Chennai 600 073
India

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1477-3163.75458

Abstract

Background: The anticancer and antioxidant effects of the aqueous extract of Indigofera aspalathoides on 20-methylcholanthrene (20-MCA) induced fibrosarcoma were investigated in male albino rats. Materials and Methods: The rats were divided into four different groups, each group consisting of six animals. Group I animals were served as normal control, Group II animals were fibrosarcoma-bearing animals after the incubation period, Group III animals were fibrosarcoma-bearing animals, treated with aqueous extract of I. aspalathoides intraperitoneally at a dose of 250 mg/kg b.w. for 30 days and Group IV animals were administered with the aqueous extract of I. aspalathoides alone, at a dose of 250 mg/kg b.w. for 30 days, served as drug control animals. After the experimental period, all the rats were weighed and killed by cervical decapitation. The serum was separated from the blood for analysis. The weights of the liver and the kidneys were noted. The fibrosarcoma was proved by pathological examinations. The liver and kidney tissues were excised and then homogenized in an ice-cold buffer. These tissues were used for biochemical analysis. Results: The activities of antioxidant enzymes, e.g. catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD), in blood serum, liver, and kidney of control and experimental animals, respectively, have been reported. Conclusion: The present observations suggested that the aqueous extract of I. aspalathoides treatment enhanced the recovery from 20-MCA-induced fibrosarcoma due to its antioxidants and antineoplastic properties.

Keywords: Antioxidant enzymes, chemoprevention, fibrosarcoma, Indigofera aspalathoides, 20-MCA

How to cite this article:
Kumar S S, Karrunakaran C M, Rao M, Balasubramanian M P. Inhibitory effects of Indigofera aspalathoides on 20-methylcholanthrene-induced chemical carcinogenesis in rats. J Carcinog 2011;10:1

How to cite this URL:
Kumar S S, Karrunakaran C M, Rao M, Balasubramanian M P. Inhibitory effects of Indigofera aspalathoides on 20-methylcholanthrene-induced chemical carcinogenesis in rats. J Carcinog [serial online] 2011 [cited 2021 Oct 15];10:1. Available from: https://carcinogenesis.com/text.asp?2011/10/1/1/75458

Introduction

Cancer remains a major public health problem in the world.[1] This disease is responsible for approximately several million deaths annually, mainly in the under developed and the developing countries. [2] In the United States alone, as the leading cause of death, cancer accounts for more than 25% of all the deaths in human beings. It is considered as an advisory of modernization and advanced pattern of sociocultural life, dominated by the western medicine. Multidisciplinary scientific investigations are making best efforts to control this disease, but, sure shot and perfect cure is yet to be brought into the scenario of world medicine. Many of the carcinogens appear to act solely by increasing proliferation. Molecular genetics of cancer reveal that cell division is essential in the complex process of genesis of human cancer. Cell division per second increases the risk of various kinds of cancer. [3] Cell division is necessary for conversion of DNA adducts or other damages to single-strand DNA to gaps or mutations. The development of a fully malignant tumor is to involve the activation or altered expression of protooncogene to oncogene and the loss or inactivation of tumor suppressor genes, the function of which is to control normal cellular activity. [4] Epidemiologic evidence indicates that increased cell division induced by exogenous or endogenous stimulation is a common denominator in the cancers.

Fibrosarcoma is a tumor composed of collagen fibers forming mesenchymal cells of the fibroblasts, and they arise from subcutaneous fibrous tissues. Fibrosarcoma causes no characteristic symptoms and is difficult to diagnose clinically. 20-Methylcholanthrene (20-MCA) causes a wide range of tumors in all animal species. This compound belongs to polycyclic aromatic hydrocarbons (PAH). PAHs are ubiquitous environmental agents commonly believed to significantly contribute to human as well as animal cancers. These chemicals are formed in the process of incomplete combustion of organic materials and are formed widely in the environment, for example, in engine exhaust, cigarette smoke, soil, water, and food. The human exposure to PAHs is therefore unavoidable. Like many other carcinogens, polycyclic aromatic hydrocarbons (20-MCA) are metabolized enzymically to various metabolites, of which some are reactive. In the large group of enzymes involved in carcinogenic metabolism, 20-MCA has been used as an effective experimental model in the field of carcinogenesis and chemoprevention. Cancer is the leading cause of mortality worldwide, and failure of conventional chemotherapy to effect major reduction in the mortality indicates that new approaches are critically needed. [5] An extremely promising strategy for cancer prevention today is chemoprevention, which is defined as the use of synthetic or natural agents to block the development of cancer in humans.[6] A variety of bioactive compounds and their derivatives have been shown to inhibit carcinogenesis in a number of experimental systems involving initiation, promotion, and progression. [7] Plant vegetables and herbal plants used as folk, and traditional medicines have been accepted currently as one of the main sources of cancer chemoprevention, drug discovery, and development. [8] Antitumor activities have been reported in several plant species. [9] However till now, few research have been reported in several plant species in recognition. Natural products have attracted recent attention as chemopreventive agents because of its availability and lack of toxicity and side effects. Hence, this study focuses on the promising anticancer efficacy of the aqueous extract of Indigofera aspalathoides on 20-MCA-induced fibrosarcoma in rats. I. aspalathoides is a shrub and this plant is widely present in India and Sri Lanka. It is thought to be an important medicinal plant in traditional system of Indian Medicine. This is one of the important ingredients of the specific oil for syphilitic and other skin diseases.

Materials and Methods

Animals

Wister strain male albino rats, weighing 100-120 g, were obtained from TANUVAS-LAMU, Madhavaram, Chennai, India. The animals were fed with normal pellet diet (rat chew) and water ad libitum. The study protocol, approved by the Ministry of Social Justice and Empowerment, Government of India, was followed [Institutional Animal Ethics Committee (IAEC) number 07/15/02].

Chemicals

All the chemicals and reagents used were of analytical grade and were purchased from M/S Sigma Chemicals, USA.

Plant materials

Fresh aerial parts (leaves, stems, and seeds) of the plant of I. aspalathoides were obtained and authenticated by the Chief Botanist, Tamil Nadu Aromatic and Medicinal Plants Corporation Limited (TAMPCOL) and Government Siddha Medical College campus, Arumbakkam, Chennai, India.

Preparation of plant extract

One kilogram of the shade dried and coarsely powdered aerial parts of the plant I. aspalathoides was charged in an aspiration bottle and allowed to soak in double distilled water for 2 days at room temperature. The extract was filtered and concentrated on a water bath. The inorganic material was precipitated and filtered off. The filtrate was again concentrated in a china dish and dried in vacuum. The yield of the extract was 10% w/w of the powdered aqueous extract. This was stored in a refrigerator for further and future use. The dose of the aqueous extract of I. aspalathoides was selected on the basis of acute toxicity study, and the LD 50 of the extract was found to be 2500 mg/kg b.w. The plant extract administration did not produce any abnormalities, e.g. atoxic, circling, lacrimation, and labored breathing in the animals during the experimental period. The dose level selected for this study was nontoxic and safe.

Acute toxicity studies

A cute toxicity study of AEIA was done as per OECD guideline 425 using albino male rats. The animals were kept fasting for overnight providing only water, after which the extract was administered orally for one animal at the limit dose of 2500 mg kg -1 and observed for 14 days (special attention for the first 4 h of administration followed by the next 20 h). If the animal dies, the limit test was terminated and main test was conducted. If the animal survives, four additional animals were dosed sequentially so that five animals were tested. However, if three animals died, the limit test was terminated and the main test was performed. The LD 50 is greater than 2500 mg kg -1 if three are more animals survived. If an animal unexpectedly dies during the study and there are other survivors, it is advisable to stop dosing and observing all animals to see if other animals will also die during a similar observation period.

Acute toxicity test

The AEIA has not shown any mortality at the limit dose of 2500 mg kg -1 b.w. AEIA was found to be safe even at a higher concentration. Based on this, the dose for the chemoprevention activity was decided.

Induction of fibrosarcoma

Fibrosarcoma was induced in Wister strain of male albino rats by subcutaneous implantation of the Millipore filter disc, impregnated with 5% suspension of 20-MCA in paraffin oil. [10] Tumors which appeared in about 4 weeks after implantation were highly localized and were maintained by serial transplantation. The tumor was minced and suspended in normal saline. A suspension of about 1 × 10 6 cells in 0.5 ml of saline was injected, subcutaneously, into the thigh. The transplanted tumor became palpable in 4-6 days time.

Experimental design

The rats were divided into four different groups, each group consisting of six animals. Group I animals were served as normal control, Group II animals were fibrosarcoma-bearing animals after the incubation period, Group III animals were fibrosarcoma-bearing animals, treated with the aqueous extract of I. aspalathoides intraperitoneally at a dose of 250 mg/kg b.w. for 30 days and Group IV animals were administered with the aqueous extract of I. aspalathoides alone, at a dose of 250 mg/kg b.w. for 30 days, served as drug control animals. After the experimental period, all the rats were weighed and killed by cervical decapitation. The weights of the liver and the kidneys were noted. The fibrosarcoma was confirmed by the pathologist, Dr. Vijayalakshmi, Professor of Pathology, Madras Medical College, Chennai, India, as shown in the photographs of sections of liver and kidney histopathology. The blood was collected and centrifuged to separate the serum. The liver and kidney tissues were excised and then homogenized in an ice-cold buffer. These serum and tissues were used for biochemical analysis. Antioxidant enzymes such as catalase (CAT) was assayed by the method of Sinha, [11] the level of superoxide dismutase (SOD) was determined using the method of Marklund and Marklund [12] and that of glutathione peroxidase (GPx) was estimated by the method of Rotruc et al[13] in the liver and kidney tissues. The tissue protein was estimated by the method of Lowery et al[14] using bovine serum albumin as standard.

Statistical analysis

One-way analysis of variance (ANOVA), using SPSS 7.5 Student Version, was used for statistical significance between the groups.

Results and Discussion

In the recent times, focus on plant research has increased all over the world and a large body of evidence has gathered to show the increased potential of medicinal plants used in various traditional systems. The following results are ample proof from in one such study that we have conducted.

[Figure 1][Table 1] and [Table 2] show the activities of antioxidant enzymes, e.g. CAT, GPx and SOD in the serum, liver and kidney, respectively, of control and experimental animals. In fibrosarcoma-induced (Group II) animals, the activity of antioxidant enzymes was decreased significantly (P < 0.001), when compared to normal control (Group I) animals. The antioxidant enzyme activities were reverted to normalcy in fibrosarcoma-bearing, drug-treated Group III animals when compared to Group II animals. No significant changes were observed in (Group IV) rats, when compared to the normal control (Group I) animals.

Figure 1: The graphs for serum catalase, superoxide dismutase, and glutathione peroxidase levels. The letter “a” represents Group II, III and IV compared with Group I; b represents Group III compared with Group II. Values are mean ± SD; n = 6. *P < 0.001, #P < 0.01, @P < 0.05; NS, not significant

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Table 1: The activities of antioxidant enzymes in liver of control and experimental animals

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Table 2: The activities of antioxidant enzymes in kidney of control and experimental animals

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Enzymatic antioxidants provide a major intracellular antioxidant protection by removing the superoxide radicals and H 2 O 2 . Superoxide radicals may be reduced by the enzyme SOD to form H 2 O 2. CAT converts H 2 O 2 into neutral products-O 2 and H 2 O . GPx catalyses the destruction of H 2 O 2 and other lipid hydrogen peroxides by using glutathione as an electron donor. Cytoprotective enzymes, which are located within both hydrophilic and hydrophobic compartments of the antioxidant in intra- and extracellular fluids, are involved in the scavenging of free radicals. Elevation in MDA production, in neoplastic conditions, may in part be attributed to the effective inhibition of free-radical scavenging enzymes. Decreased activity of NADPH-dependent glutathione reductase for the conversion of GSSG-GSH can be ascertained by the decreased level of GSH. Under conditions of oxidative stress, the NADP/NADPH ratio will switch in favor of NADP, indicating decreased glucose-6-phosphate dehydrogenase activity. The paucity of NADPH production will, in turn, decrease the CAT activity. [15] Superoxide radicals play an important role in cell physiology. SOD is widely distributed in cells with high oxidative metabolism and has been proposed to protect such cells against the deleterious effects of superoxide anions. GPx is considered to be the most important H 2 O 2 -removing enzyme in mammalian cells and is more important than CAT in removing H 2 O 2 . [16] The activity of GPx is dependent on the availability of GSH, which in turn, is maintained by the de novo synthesis. Decreased level of GSH can be ascribed to depress the activity of NADPH-dependent GR, which is required for the conversion of GSSH to GSH. Therefore, a decrease in NADP production is due to glucose-6-phosphate dehydrogenase inhibition and a decrease in the GSH level may be responsible for the impaired functioning of GPx in neoplastic tissues. Decreased GPx activity was also observed in red blood cells of untreated patients with malignant lymphoma. [17]

Our findings agree well with this and the activity of GPx in the liver was significantly decreased in fibrosarcoma-bearing animals. The levels of antioxidant enzymes, e.g. CAT, SOD and GPx, were corrected to near normalcy in fibrosarcoma-bearing animals by treating with aqueous extract of I. aspalathoides.

Antioxidants have also been advocated to impart anticancer activities by several other mechanisms, [18] e.g., trapping the ultimate carcinogen, blocking the metabolic activation of carcinogen, modulating xenobiotic metabolizing enzymes, scavenging of free radicals, inhibiting generation of free radicals, inhibiting promotion stage of carcinogens by inhibiting cell proliferation through blocking of the lipoxygenase/cycloxygenase pathway or by lowering ornithine decarboxylase activity and by decreasing the bioavailability of ultimate carcinogen. Treatment with I. aspalathoides showed protective action against reactive oxygen species (ROS), induced by malignant tumor, possibly through its ability as an antioxidant in quenching the superoxide anions or free radicals.

These observations clearly indicate a chemopreventive function of the extract. Previous studies conducted showed that the extract has antitumor activity, but has no subacute toxicity. [19] The preliminary phytochemical studies have shown the presence of alkaloids and flavanoids in the aqueous extract of I. aspalathoides. Flavanoids are known to possess antimutagenic and anticarcinogenic effects. Moreover, flavanoids have a chemopreventive role in cancer, through the induction of enzymes affecting carcinogen metabolism and inhibit various activities of tumor promoters, which are involved in the process of carcinogenesis.

Chemopreventive effect of the aqueous extract of I. aspalathoides may be due to the presence of these compounds. Our results clearly indicate a significant chemotherapeutic effect of the aqueous extract of I. aspalathoides. Further studies to characterize the active principles and to elucidate the mechanism of action of the aqueous extract of I. aspalathoides are in progress.

Conclusion

The present observations suggested that the aqueous extract of I. aspalathoides treatment enhanced the recovery from 20-MCA-induced fibrosarcoma due to its antioxidants and antineoplastic properties.

Acknowledgements

The author is grateful to Prof. Dr. B. Nagarajan, Cancer institute, Chennai, for his help and advice.

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Authors

Dr. Sivagnanam Selva Kumar, EDUCATION Ph.D., (2000-2005) Molecular Biology- Pharmacology & Environmental Toxicology University of Madras, Chennai. M.Sc., MOLECULAR BIOLOGY, (1997-1999) Department of Biochemistry and Molecular Biology University of Madras, Chennai. EXPERIENCE Employed as a lecturer in Bio-Technology in Bharath College of Science & Management, Thanjavur since 2005-2006. Presently employed with Bharath University, Selaiyur, Chennai as lecturer in the Department of Bio-Technology. (July’2006 to Till date) AWARD/SCHOLARSHIP “Disabled person with higher achievement” national level Professional Scholarship (2002-2003). National center for promotion and Employment for Disabled people (NCPEDP) New Delhi.

Figures

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 Tables

  [Table 1][Table 2]