Role of Prosopis cineraria against N-nitrosodiethylamine-induced liver tumor in rats with reference to marker enzymes and nucleic acid contents

  • Naina Mohamed Pakkir Maideen School of Pharmacy, Vels Institute of Science, Technology and Advanced Studies, Vels University, Chennai 600 117, India.
  • Ravichandiran Velayutham School of Pharmacy, Vels Institute of Science, Technology and Advanced Studies, Vels University, Chennai 600 117, India.
  • Gobinath Manavalan Ratnam Institute of Pharmacy, Pidathapolur, Muthukur, Nellore 524 346, Andhrapradesh, India.
Keywords: Liver tumor, N-nitrosodiethylamine, Marker enzymes, Nucleic acid content, Prosopis cineraria
DOI: 10.3329/bjp.v6i2.9027

Abstract

The effect of methanol extract of Prosopis cineraria against experimental liver tumor in rats was studied. Liver tumor was induced by the administration of N-nitrosodiethylamine (200 mg/kg) and it was promoted by phenobarbital administration. Methanol extract (200 and 400 mg/kg) was administered to determine the protective activity. Administration of methanol extract suppressed the liver tumor effectively as revealed by  the decrease in elevated levels of aryl hydrocarbon hydroxylase, lactate dehydrogenase, g-glutamyl transpeptidase (g-GTP), 5’nucleotidase, deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). We found that methanol extract may extend its protective role by modifying the levels of  marker enzymes and nucleic acid contents.

Introduction

Hepatocellular carcinoma (HCC) is one of the ten most common human cancers, with a worldwide incidence of over one million cases every year (Arya et al., 1988). A large number of agents including natural and synthetic compounds have been identified as having some potential cancer chemopreventive value. Plants and plant products have been shown to play an important role in the management of various liver disorders.

Prosopis cineraria Linn (Leguminosae) is a small tree found in dry and arid regions of Arabia and in regions of India mainly Rajasthan, Haryana, Punjab, Gujarat, Western Uttar Pradesh and drier parts of Deccan and extends as far as South in Tuticorin.

This plant is used for the treatment of several ailments, including safeguard against miscarriage and inflammation. The literature survey has shown that there is no work being done on the protective effect of P. cineraria against liver tumor. Hence, our present study is aimed to evaluate the protective activity of methanol extract against n-nitroso diethylamine (DEN)-induced phenobarbital promoted liver tumor by regulating the levels of marker enzymes and nucleic acid contents in rats.

Materials and Methods

Collection of the plant material

P. cineraria (Leguminosae) collected in the month of November 2009 from kolli hills, Tamilnadu, India and identified by Botanical Survey of India, Coimbatore, and Tamilnadu, India. A voucher specimen has been kept in our laboratory for future reference.

Preparation of extract

The leaves of P. cineraria were dried under shade and then powdered with a mechanical grinder. The powder was passed through sieve No. 40 and treated with petroleum ether for dewaxing as well as to remove chlorophyll and it was later packed into soxhlet apparatus with methanol and subjected to hot continuous percolation using Soxhlet apparatus. After the completion of extraction, it was filtered and the solvent was removed by distillation under reduced pressure. The extract was stored in desiccator.

Phytochemical screening

The methanol extract was subjected to preliminary phytochemical investigations (Kokate et al., 1991) and was found with the presence of various constituents like alkaloids, carbohydrates, glycosides, phenolic compounds, tannins and flavanoids.

Animals

Healthy male Wistar albino rats (6-8 weeks old) were used throughout the study. The animals were purchased from King Institute of Preventive Medicine, Chennai and maintained in a controlled environmental condition of temperature (23 ± 2°C) and relative humidity (50-70%) on alternatively 12 hours light/dark cycles. All animals were fed standard pellet diet and water ad libitum

Acute toxicity studies (LD50)

Mice received methanol extract at various doses (500-2,000 mg/kg) orally by gavage. They were observed for toxic symptoms continuously for the first 4 hours after dosing. Finally, the number of survivors was noticed after 24 hours. In the toxicity study, no mortality occurred within 24 hours under the tested doses of methanol extract.

Sources of chemicals

DEN, bovine serum albumin and 2,4,6-trinitrobenzene sulfonate, was obtained from Sigma Chemical Company, St. Louis, MO, USA. All other chemicals used were of analytical grade obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai and Glaxo Laboratories, Mumbai.

Experimental protocol

The rats were divided into four groups, each group consisting of six animals. Group 1 served as control animals and were treated with distilled water orally for 20 weeks.  Liver tumor was induced in Group 2, 3, and 4 using single intraperitoneal injection of DEN at a dose of 200 mg/kg body weight in saline. Two weeks after the DEN administration, the carcinogenic effect was promoted by 0.05% phenobarbital, which was supplemented to the experimental animals through drinking water for up to 20 successive weeks (Yoshiji et al., 1991). Whereas Group 2 animals receive DEN alone, Group 3 animals were treated with methanol extract (200 mg/kg, dissolved in 0.3% carboxymethyl celluose)   simultaneously for 20 weeks from the first dose of DEN and Group 4 animals treated with methanol extract (400 mg/kg, dissolved in 0.3% carboxymethyl celluose) simultaneously for 20 weeks from the first dose of DEN. At the end of experiments, animals were fasted overnight and were killed by cervical decapitation. Blood was collected and serum separated out. The liver were immediately removed and suspended in ice cold saline. At the end of experimental period of 20 weeks biochemical parameters were analyzed.

Measurement of marker enzymes

The aryl hydrocarbon hydroxylase assayed by the modified method of Cantrell et al. (1973). The activity of lactate dehydrogenase was assayed by the method of King (1965). The activity of g-glutamyl transpeptidase (g-GTP) was evaluated by using the method of Orlowski and Meister (1965). 5¢-Nucleotidase was estimated by the method of Belfield and Goldberg (1969).

Measurement of nucleic acid contents

The nucleic acids were extracted by the method of Schneider (1957). Deoxy ribonucleic acid (DNA) was estimated by the method of Burton (1956) and Ribonucleic acid (RNA) was estimated by the method of Rawal et al. (1977).

Statistical analysis

The values were expressed as mean ± SEM. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey-Kramer multiple comparisons test. P values <0.05 were considered as significant.

Results

The activities of the marker enzymes (arylhydrocarbon hydroxylase, lactate dehydrogenase, g-GTP and 5’-nucleotidase) in the liver of control and experimental animals were found to be a significant (p<0.001) rise in the enzyme activities in tumor bearing animals when compared with control (Table I). The rise (p<0.001) in the activities of these marker enzymes found in Group 2 tumor bearing animals was significantly decreased in Group 3 and 4 methanol extract-treated animals respectively on dose-dependent manner when compared with tumor bearing group.

Table I: Effect of methanol extract of Prosopis cineraria on the activities of some marker enzymes in the liver of control and experimental rats

Group Treatment Arylhydrocarbon hydroxylase Lactate dehydrogenasea γ-Glutamyl transpeptidasea 5'nuleotidasea
1 Control 0.7 ± 0.0 1.4 ± 0.0 6.9 ± 0.2 1.9 ± 0.1
2 Tumor bearing 1.1 ± 0.0a 2.6 ± 0.1a 12.8 ± 0.2a 3.7 ± 0.3a
3 Methanol extract 200 mg/kg 0.9 ± 0.0a,b 2.3 ± 0.1a,b 10.5 ± 0.3a,b 3.0 ± 0.1a,c
4 Methanol extract 400 mg/kg 0.9 ± 0.0a,b 1.8 ± 0.0a,b 8.7 ± 0.2a,b 2.2 ± 0.1b
n = 6 animals in each group; Each value is expressed as mean ± SEM; ap<0.001 Vs control; bp<0.001; cp<0.01 Vs tumor bearing animals; Data were analyzed by one-way ANOVA followed by Tukey-Kramer multiple comparison test; aUnits: Aryl hydrocarbon hydroxylase- mmoles of fluorescent phenolic metabolites formed/min/mg/protein, Lactate dehydrogenase- mmoles of pyruvate liberated/min/mg/protein, g-GTP- nmoles of p-nitroaniline formed/min/mg/protein, 5'nuleotidase-nmoles of Pi liberated/min/mg/protein

Tumor bearing animals showed a significantly increased nucleic acid contents (DNA and RNA) in liver tissues (p<0.001; Figure 1). Methanol extract (200 and 400 mg/kg) treatment resulted in a significant  decrease in the levels of nucleic acid contents in Group 3 and Group 4 animals. Methanol extract-treated Group 4 shows more restoration than treated Group 3.

Figure 1: Effect of methanol extract of P. cineraria on activities of DNA and RNA in liver tissues against DEN-induced liver cancer in rats n = 6; Each value is expressed as mean ± SEM; Group 1: control animals, Group 2: liver tumor bearing animals, Group 3 and 4: methanol extract 200 and 400 mg/kg treated. ap<0.001; bp<0.01 Vs control; dp<0.001 Vs tumor bearing animals; Data were analysed by one-way ANOVA followed by Tukey-Kramer multiple comparison test   

Discussion

Change in metabolism that occurs during malignancy (Stefanini, 1985) reflected by the abnormal variation in the marker enzymes. Cancer marker enzymes functioning as an indicator of cancer response to therapy. The marker enzymes such as aryl hydrocarbon hydroxylase, g-GTP, 5'-nucleotidase and lactate dehydrogenase are specific indicators of tissue damage (Durak et al; 1993 and Ferringo et al; 1994). The increase in the activities of these enzymes may be due to the increased tumor incidence. Aryl hydrocarbon hydroxylase is a large group of cytochrome P450 mono-oxygenases that complex with NAD (P) H-flavin oxidoreductase in numerous mixed-function oxidations of aromatic compounds. They are functioning as catalysts in hydroxylation of a broad spectrum of substrates and are important in the metabolism of steroids, drugs, and toxins such as phenobarbital, carcinogens.

g-GTP is a transferase enzyme which catalyses the transfer of gamma glutamyl groups from a large variety of peptide donors to a wide range of aminoacids and peptide receptors (Valentich and Moris, 1992). g-GTP activities were increased in cancer conditions. Chemical carcinogens   entering the liver   initiate some systematic effects which in turn induce g-GTP synthesis. These elevations show the progress of carcinogenic process, since its ability is correlated with growth rate, histological differentiation and survival time of the host. An increased level of g-GTP was observed in cancerous cells (Ngo and Nutler, 1994). This rise may indicate the presence of tumor and the reports show that g-GTP activity in liver was significantly increased in tumor bearing rats than in control group.

Patients with solid tumors, reported to have altered 5'-nucleotidase. And the 5'-nucleotidase has been described as an important marker for differentiation of B-lymphocytes. In our study, 5'-nucleotidase has been increased significantly in the hepatoma bearing rats. It has been demonstrated that increased activity of 5'-nucleotidase in carcinoma of liver, gastrointestinal tract and pancreas (Rosi et al., 1998) also observed an increased activity of 5'-nucleotidase in leukemia patients. During methanol extract treatment 5'-Nucleotidase activity got reduced significantly on dose-dependent manner.

Lactate dehydrogenase is a cytoplasmic enzyme which is involved as a catalyst in the oxidation of lactate to pyruvate and vice versa. It is a marker for membrane integrity and is a regulator of many biochemical reactions in the body tissues and fluids. It has been reported that an excessive activity of lactate dehydrogenase  found in malignant cells which spreads through the organs of tumor bearing rats. Changes in permeability of cell membranes and the leakage to soluble enzymes caused by increased enzyme activity in the serum of patients with lung and ovarian cancer (Bose and Mukherjee, 1994). Enhanced glycolysis using the growth of tumor caused by elevated activity of Lactate dehydrogenase in tumor bearing rats. The treatment with methanol extract (200 and 400 mg/kg) causes controlled glycolysis which has reduced lactate dehydrogenase activity and protected the membrane integrity. In methanol extract-treated groups, these enzymes level were reverted to near normal level, attributed to the antimutagenic and anticarcinogenic activity of methanol extract.

Nucleic acid content of tumor is found to be an important indicator of prognosis, because it is well correlated with the size of the tumor in the cancerous condition (Gallagher, 1986). In diseased state, the degree of malignancy increases with the defective abnormalities in DNA. Reports reveal  that  abnormal  amount  of  DNA  was  observed  in  various  cancers including breast carcinoma, endometrial carcinoma and lung carcinoma (Ellis et al., 1991). In the present study, an increased activity was observed in DEN induced liver cancer animals and this may be due to the over expression of many enzymes which are necessary for DNA synthesis in tumor cells.

RNA levels were found to be increased in the cancerous condition as DNA and RNA are directly related to each other, an abnormally increased content of DNA may lead to an increased transcription, which in turn increased RNA content in tumor cells. The mechanisms by which tea polyphenols may act includes the inhibition of promutagen activation, the inactivation of mutagens and carcinogens, blocking and scavenging of reactive molecules, modulation of DNA replication or repair, inhibition of promotion, and inhibition of invasion and metastasis of tumor cells. These mechanisms are currently being progressively clarified. Most of the reports on mechanisms, however, still remain as suggestive or speculative (Kurado and Hara, 1999). In methanol extract- (200 and 400 mg/kg) treated animals, the nucleic acid levels were decreased due to its inhibition of mutagenesis process.

Conclusion

The antitumor properties of the methanol extract may be due to the presence of flavanoids and all these observations clearly indicate a significant protective activity of methanol extract of P. cineraria.

Ethical Issue

The research has followed the national ethical standards for the care and use of laboratory animals and it was approved by the Institutional Animal Ethics Committee constituted for the purpose. The oral acute toxicity study of the extract was carried out in Swiss albino mice using up and down procedure as per OECD guidelines (OECD, 2001). 

References

Arya S, Asharaf S, Parande C. Hepatitis B and delta markers in primary hepatocellular carcinoma patients in the Gizan area of Saudi Arabia. APMIS (Suppl.). 1988; 3: 30-34.

Belfield A, Goldberg DM. Application of a continuous spectrophotometric assay for 5’-nucleotidase activity in normal subjects and patients with liver and bone disease. Clin Chem. 1969; 15: 931-39.

Bose CH, Mukherjee M. Enzymatic tumor markers in ovarian cancer: A multiparametric study. Cancer Lett. 1994; 77: 39-43.

Cantrell E, Abreu M, Busbee D. A simple assay of aryl hydrocarbon hydroxylase in cultured human lymphocytes. Biochem Biophys Res Commun. 1976; 70: 474-79.

Durak I, Umitisik CA, Canbolt O, Akyol O, Kavutul M. Adenosine deaminase, 5’-nucleotidase xanthine oxidase, SOD, CAT activities in cancerous and noncancerous human laryngeal tissues. Free Radical Biol Med. 1993; 15: 681-84.

Ellis CN, Burnette JJ, Sedlack R, Dyas C, Balkemore WS. Prognostic applications of DNA analysis in solid malignant lesions in humans. Surgery 1991; 173: 329-42.

Ferringo D, Buccheri G, Biggi A. Serum tumor markers in lung cancer: History, biology and clinical application. Eur Respir. 1994; 7: 186-97.

Gallagher RE. Biochemistry of neoplasia. In: Comprehensive textbook of oncology. Moosa AR, Robson MC, Schimpff SC (eds). Baltimore, USA, Williams and Wilkins, 1986, pp 36-45.

King J. In: Practical clinical enzymology. London, D. van. Nostrand Co., 1965, pp 83-93.

Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 1st ed. Pune, Nirali Prakasan, 1990, p 123.

Kuroda Y, Hara Y. Antimutagenic and anticarcinogenic activity of tea polyphenols. Mutation Res. 1999; 436: 69-97.

Ngo EO, Nutler LM. Status of glutathione and glutathione metablolising enzymes in mendione resistance human cancer cells. Biochem Pharmacol. 1994; 47: 421-24.

Orlowski K, Meister A. Isolation of y-glutamyl transpeptidase from hog kidney. J Biol Chem. 1965; 240: 338-47.

Rawal UM, Patel US, Rao GN, Desai RR. Clinical and biochemical studies on cateractous human lens III. Quantitative study of protein, RNA and DNA. Arogya J Health Sci. 1977; 3: 69-72.

Rosi F, Tabucchi A, Carluci F, Galieni P, Laukia F, et al. 5-nucleotidase activity in lymphocytes from patients affected by β-cell chronic lymphocytic leukemia. Clin Biochem. 1998; 31: 269-72.

Schneider WC. In: Methods in enzymology. Colowick SP, Kaplan NO. (eds). Vol. III. New York, Academic Press, 1957, p 680.

Stefanini M. Enzymes, isoenzymes and enzyme variants in the diagnosis of cancer. Cancer 1985; 55: 1931-36.

The Organization of Economic Cooperation and Development (OECD). The OECD guideline for testing of chemicals: 420 Acute oral toxicity. Paris, OECD, 2001, pp 1-14.

Valentich AM, Moris B. Effects of essential fatty acid deficiency on GGT activity of rat pancreas. J Nutri Biochem. 1992; 3: 67-70.

Yoshiji H, Nakae D, Kinugasa T, Matsuzaki M, Denda A, Tsujii T, Konishi Y. Inhibitory effect of dietary iron deficiency on the induction of Putative preneoplastic foci initiated with diethylnitrosamine and promoted by phenobarbital. Br J Cancer. 1991; 64: 839-42.

Published
2012-01-02

Apply citation style format of Bangladesh Journal of Pharmacology

Section
Research Articles
Financial Support
Self-funded
Conflict of Interest
Authors declare no conflict of interest