the effect of the aqueous extract of the roots of asparagus racemosus on hepatocarcinogenesis...

ASPARAGUS RACEMOSUS ON HEPATOCARCINOGENESIS 1175

Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 22, 1175–1182 (2008)DOI: 10.1002/ptr

Copyright © 2008 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 22, 1175–1182 (2008)Published online in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2391

The Effect of the Aqueous Extract of theRoots of Asparagus racemosus onHepatocarcinogenesis initiated byDiethylnitrosamine

Alka Agrawal1, Meenakshi Sharma1, Santosh Kumar Rai1, Bharat Singh2, Manisha Tiwari1*and Ramesh Chandra1

1Dr B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110 007, India2Department of Pathology, University College of Medical Sciences, Delhi, India

Histopathological studies of the hepatic tissues of Wistar rats treated with diethylnitrosamine (DEN) (200 mg/kg b wt, i.p.) once a week for 2 weeks, followed by treatment with DDT, a tumor promoter (0.05% in diet)for 2 weeks and kept under observation for another 18 weeks, demonstrated the development of malignancy.Pretreatment of Wistar rats with the aqueous extract of the roots of Asparagus racemosus prevented theincidence of hepatocarcinogenesis. Immunohistochemical staining of the hepatic tissues of rats treated withDEN showed the presence of p53+++++ foci (clusters of cells expressing the mutated p53 protein), whereasan absence of p53+++++ foci was observed in Wistar rats pretreated with the aqueous extract of the roots ofAsparagus racemosus. The microsections of the hepatic tissue of rats treated with DEN followed by treat-ment with the aqueous extract of Asparagus racemosus showed an absence of p53+++++ foci. The results of thebiochemical determinations also show that pretreatment of Wistar rats with the aqueous extract of Asparagusracemosus leads to the amelioration of oxidative stress and hepatotoxicity brought about by treatment withDEN. These results prove that the aqueous extract of the roots of Asparagus racemosus has the potentialto act as an effective formulation to prevent hepatocarcinogenesis induced by treatment with DEN.Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: hepatocarcinogenesis; Asparagus racemosus; p53; diethylnitrosamine; histopathology.

INTRODUCTION

Hepatocarcinogenesis or liver cancer is one of the lead-ing causes of cancer related deaths worldwide and isthe major malignancy complicating chronic liver dis-ease (Olynyk et al., 2006; Daniel et al., 2006). Preven-tive approaches to therapy are limited and treatmentby chemotherapy is expensive with high recurrencerates. The commonly used drugs include doxorubicin(adriamycin) which is an anthracycline antibiotic derivedfrom Streptomyces peucetius. Doxorubicin is a DNAintercalating agent that interferes with DNA and RNAsynthesis. Doxorubicin is known to lead to significantnephrotoxicity and ototoxicity. Other primary toxiceffects include nausea, myelosuppression, peripheralneuropathy and hepatotoxicity (Codde et al., 1990;Griffen et al., 1988; Kunitomo et al., 1985; Bechteret al., 1987; Raiczyk and Pinto, 1988). Another drugused for the treatment of hepatocarcinogenesis is 5-fluorouracil (5FU). This drug inhibits thymidine synthesisand is incorporated into RNA and DNA. Dose limitingtoxic effects of this drug include leukopenia and throm-

bocytopenia, stomatitis, dysphagia and immunosuppres-sion (Hoshino et al., 2005).

There is therefore, a need for a drug that can curethe disease and offer minimal toxic effects. Naturalcompounds may have an advantage over these drugsbecause, being constituents of living systems, they maybe less toxic and hence more acceptable for humanapplication. Ayurveda, the Indian system of medicinecontains many herbal remedies thought to be activeagainst a variety of diseases including cancer (Dahanukarand Thatte, 1997; Patwardhan et al., 1990). The presentwork was undertaken to find a herbal curative againstdiethylnitrosamine (DEN) induced hepatocarcinogenesis.

The plant Asparagus racemosus Wild (family Liliaceae)is also known as Shatavari. Asparagus racemosus isrecommended in Ayurvedic texts for the preventionand treatment of gastric ulcers, dyspepsia and as agalactogogue. Asparagus racemosus has also been usedby Ayurvedic practitioners for the treatment of inflam-mation and liver diseases (Goyal et al., 2003; Sairamet al., 2003). The alcohol extract of root of Asparagusracemosus has been shown to reduce significantly theenhanced levels of alanine transaminase, aspartatetransaminase and alkaline phosphatase in CCl4-inducedhepatic damage in rats (Goyal et al., 2003).

In the present study an attempt was made to find aherbal curative for hepatic pathologies induced by DEN,an initiator of hepatocarcinogenesis and DDT, a tumorpromoter. The effect of the aqueous extract of Asparagus

Received 18 September 2006Revised 17 August 2007

Accepted 4 September 2007

* Correspondence to: M. Tiwari, Dr B. R. Ambedkar Center for BiomedicalResearch, University of Delhi, Delhi 110 007, India.E-mail: [email protected], [email protected]/grant sponsor: Defence Research and Development Organization(DRDO), India.Contract/grant sponsor: Council of Scientific and Industrial Research.


1176 A. AGRAWAL ET AL.

racemosus (200 mg/kg b wt) was observed on histopatho-logical changes in rats administered DEN and rats ad-ministered DEN and DDT and kept under observationfor a total of 22 weeks.

p53 is known to be a potent tumor suppressor andthe p53 gene is the most common target for geneticalterations in human cancers. The p53 protein sup-presses abnormal cell proliferation and represents animportant mechanism for protection against cancer.Normal cells express wild type p53 protein, however,cancerous cells produce mutant forms of p53 (Rotteret al., 1993; Sluss and Jones, 2003). Clusters of cellsexpressing a mutant conformation of the p53 proteincan be detected by immunohistochemical staining(Rebel et al., 2001).

This study determined the effect of the aqueousextract of the roots of Asparagus racemosus on p53protein expression in rats treated with DEN and treatedwith a combined dosing regimen of DEN and DDT.p53+ foci (clusters of cells expressing mutant p53 pro-tein) were immunohistochemically stained in sectionsof rat liver using Pab 240, an antibody specific for p53mutant conformation.

MATERIAL AND METHODS

Chemicals. All the chemicals and reagents used in thepresent studies were of an analytical grade or equiva-lent and were procured from M/S Sigma Chemical Co.,St Louis, MO, USA, E. Merck, Germany and M/S FineChemicals, India. Organic solvents were freshly distilledbefore use.

Experimental Wistar rats and diet. Adult male Wistarrats (maintained in the animal house facility, Dr B. R.Ambedkar Center for Biomedical Research, Univer-sity of Delhi), weighing approximately 100–125 g weredivided into groups of six Wistar rats each. The Wistarrats were reared on laboratory chow, fed ad libitumand had free access to water at all times. The room wasmaintained at 25 °C ± 2 °C with natural daytime and nolight after 1900 h, until morning. All the experimentswere performed in the morning according to currentguidelines for the care of the laboratory Wistar ratsand the ethical guidelines for the investigation of experi-mental pain in conscious Wistar rats (Zimmerman,1983).

Preparation of plant extracts. The dried roots of Asparagusracemosus were obtained from the laboratory of DrK. K. Srivastava at the Defence Institute of Physiologyand Allied Science (DIPAS), Delhi and identified byDr Kshivsagar S. R., Aravali Biodiversity Park, Centrefor Environmental Management and Degraded Eco-system (CEMDE), University of Delhi, Delhi. Roots (50 g)of Asparagus racemosus were dried and crushed to acoarse powder. The powdered roots were soaked in waterfor 24 h. The aqueous suspension was then filtered,rotary evaporated and lyophilized. A yield of 5.9 g(11.8% w/w) was obtained from the aqueous extract.

Phytochemical analysis. The chemical constituents ofthe extract were identified by qualitative chemical testsusing the procedures of Kokate et al. (1996).

DEN induced hepatocarcinogenesis. Rats were dividedinto seven experimental groups with six Wistar rats ineach group.

Group I (Control): Wistar rats were administered asingle daily dose of saline (0.9%) intraperitoneally (i.p.)for 22 weeks.

Group II: The Wistar rats received DEN (200 mg/kgbwt, i.p.) once a week for 2 weeks. The Wistar ratswere observed for another 20 weeks.

Group III: The Wistar rats were administered DEN(200 mg/kg bwt, i.p.) once a week for 2 weeks followedby treatment with DDT, a tumor promoter (0.05%,powdered and mixed in diet) for 2 weeks. The Wistarrats were observed for another 18 weeks.

Group IV: Wistar rats were orally administered anaqueous extract of Asparagus racemosus (200 mg/kgbwt, orally) for 20 days, followed by administration ofDEN (200 mg/kg bwt, i.p.) once a week for 2 weeks.The Wistar rats were kept under observation for an-other 17 weeks.

Group V: Wistar rats were administered DEN(200 mg/kg bwt, i.p.), once a week for 2 weeks followedby the oral administration of 200 mg/kg bwt, of theaqueous extract of Asparagus racemosus for 20 days.The Wistar rats were observed for another 17 weeks.

Group VI: The Wistar rats were orally administeredaqueous extract of Asparagus racemosus (200 mg/kgbwt) for 20 days and then treated with DEN (200 mg/kg bwt, i.p.) for 2 weeks, followed by treatment with0.05% DDT (powdered and mixed in diet) for another2 weeks. The Wistar rats were observed for another15 weeks.

Group VI: The Wistar rats were administered DEN(200 mg/kg bwt) once a week for 2 weeks, followed bytreatment with DDT (0.05%, powdered and mixed indiet). The Wistar rats were then orally administeredwith the aqueous extract of Asparagus racemosus(200 mg/kg bwt) for 20 days. The Wistar rats wereobserved another 15 weeks.

Although both DEN and DDT have differentmechanisms of action, in this study DEN (a genotoxiccompound) was used as an initiator and DDT (a non-genotoxic compound) as a promoter of hepatocarcino-genesis in the rat model. Earlier studies in variouslaboratories showed that a single i.p. administration ofDEN for 2 weeks can initiate liver cancer in 22 weeks.DDT was given after DEN to promote the processof hepatocarcinogenesis. It may be added that the bestroute for administration for DEN is intraperitone-ally and orally for DDT, as already reported in theliterature. The extract of Asparagus racemosus wasadministered orally because it is the simplest andmost convenient route for administering the extract. Achemical/drug administered orally passes through theintestinal wall and liver before it is transported via thebloodstream to the target site. Since this was a study ofthe prevention of hepatocarcinogenesis, an oral routeof administration was chosen for the plant extract.

Biochemical determinations. After a total of 22 weeks,the Wistar rats were killed using ether as an anaes-thetic. Blood was drawn from retroorbital sinus usingcapillary tubes, into dried test tubes. The serum wasseparated for biochemical estimations. The liver wasquickly removed in ice-cold saline, perfused with 0.9%saline, cleaned, wiped dry and weighed and processed



for histopathology. The 20% homogenate of liver in0.1 M potassium phosphate buffer (pH 7.4) with 0.25 M

sucrose, was centrifuged at 800 × g for 5 min at 4 °Cin an IEC-20 refrigerated centrifuge (Rotor No894) toseparate the nuclear debris. The supernatant obtainedwas centrifuged at 10 500 × g for 20 min at 4 °C toobtain the post mitochondrial supernatant (PMS) whichwas used as a source for estimation of glutathione S-transferase, catalase, glutathione reductase, reducedglutathione and protein.

The biochemical parameters serum glutamic oxalo-acetic transaminase (SGOT), serum glutamate pyruvatetransaminase (SGPT) and serum alkaline phosphatase(SALP) were assayed according to standard methodsof Bergmeyer and Bernt (1974) and Reitman andFrankel (1957), respectively, using an assay kit. Thecontents of reduced glutathione and malondialdehyde(MDA) were determined by the methods of Jollowet al. (1974) and Yagi et al. (1979), respectively. Theactivities of glutathione-S-transferase (GST), glutathionereductase (GR) and catalase were determined by themethods of Habig et al. (1974), Carlberg and Mannervik(1975) and Cohen et al. (1970), respectively. The pro-tein content was measured by the methods of Lowryet al. (1951) using bovine serum albumin as standard.

Liver histopathological assessment. Formalin (10%) wasused as a fixative. After paraffin embedding, liver sec-tions were cut at 3 μm thickness, using a microtome,with disposable blades. The sections were stained inMayer’s hematoxylin and eosin and mounted in DPXmountant. Six slides were checked in each group.Photomicrographs were obtained using an Olympusmicroscope-BX-60/PM 20.

Liver immunohistochemistry. The liver sections cut at3 μm thickness using a microtome were rehydrated andtreated with H2O2 to eliminate endogenous peroxidaseactivity and then nonspecific binding was blocked bydiluting Pab 240 1:25 in PBS, containing 5% normalrabbit serum, 0.2% BSA and 0.1% saponin, containing5% normal rabbit serum. The tissues were then incu-bated sequentially with:

(a) Primary antibody. To stain the p53 protein in mutantconformation, Pab240 was used as a primary anti-body (Biosource International). Pab240 was diluted1:25 in PBS, containing 5% normal rabbit serum,0.2% BSA and 0.1% saponin, and incubated over-night with sections at 4 °C. The liver sections wereprepared from the paraffin block prepared for thehistopathological studies. However, in this case liversections of 3 μm were cut and section mounted basalside up. Unbound antibody was removed in a triplewash of 5 min in PBS/Tween 0.5%.

(b) Secondary antibody. The secondary antibody, rabbitantimouse (IgG1)-biotinylated, diluted 1:50 in PBS,containing 0.2% BSA and 0.1% saponin, was usedto incubate the liver section for 1 h at room tem-perature. Excess of this antibody was removed in atriple wash of 5 min in PBS. The liver sections wereincubated for 45 min in streptavidin-biotin-peroxidasecomplex. After a triple wash of 5 min in PBS, thesections were stained for 3 min in 50 mL of substratesolution, containing 40 mg of 3,3′-diaminobenzidineand 100 μL of 30% H2O2. The peroxidase reaction

was stopped by a triple wash of 5 min in PBS. Theliver sections were mounted basal side up in 7.0%gelatin and 50% glycerol, in distilled water.

(c) Substrate/Chromogen preparation. To prepare 5 mLof DAB (3,3′-diminobenzidine) chromogen: 4 dropsof DAB chromogen concentrate was added to avial of substrate buffer (5 mL) and mixed.

Development: The mixture was applied to liver sec-tions within 20 min of preparation.

Incubated specimens with substrate/chromogen untildesired staining intensity is achieved. Photomicrographswere obtained using an Olympus microscope-BX-60/PM 20.

The p53 protein was detected using the UltratechHRP (DAB streptavidin-Biotin) Universal DetectionSystem catalogue #2765.

Statistical analysis. Student’s t-test was used to com-pare the control rats with DEN treated rats and controlrats with rats treated with DEN and DDT. Differencesbetween treated rats were assessed by the analysis ofvariance (ANOVA) followed by Tukey-Kramer multi-ple comparisons test to judge whether the differencesamong several treatments was significant or was just amatter of sampling fluctuations. Data are expressed asmean ± SEM.

RESULTS

Phytochemical screening

The results of the phytochemical screening of the aque-ous extract of Asparagus racemosus are summarized.The screening tests of the aqueous extract of Asparagusracemosus were positive for steroids, phytosterols,carbohydrates, tannins, anthraquinones, saponins, glyco-sides and flavonoids and negative for terpenoids, aminoacids and alkaloids.

Histopathological observation

The liver microsections of control group Wistar ratsshowed normal hepatic cells with well-preserved cyto-plasm, prominent nucleus and nucleolus and prominentcentral vein (Fig. 1).

Liver microsections of Wistar rats treated with DENshowed focal areas of mild dysplastic change. Theintervening area showed normal hepatocytes. The livermicrosection confirmed that the cells were premalignant(Fig. 2).

The liver microsections of the Wistar rats treatedwith DEN and DDT showed a well maintained lobularand cord pattern. The hepatocytes showed markednuclear changes. The nuclei were enlarged with clumpedchromatin and macronuclei. There was evidence ofliver cell dysplasia and cells were premalignant. Thesehistopathological studies confirm the induction ofhepatocarcinogenesis by DEN, in a rat model (Fig. 3).

The liver microsections of Wistar rats treated withthe aqueous extract (200 mg/kg bwt) of Asparagusracemosus followed by treatment with DEN showeda well maintained lobular and cord pattern. The



The liver microsections of the group treated withDEN and DDT followed by treatment of the aqueousextract of Asparagus racemosus showed mild dysplasiaof hepatocytes with steatosis and mild chronic portaltriaditis.

It may be noted that the same dose was administered(200 mg/kg b wt) irrespective of the carcinogen (DEN/DDT) as a dose dependent study (50, 100, 200 and300 mg/kg b wt) showed that a dose of 200 mg/kg b wtwas least toxic to the liver and most effective for pre-venting hepatocarcinogenesis (data not shown). In addi-tion, keeping the dose the same in all treatment groupswill give a better idea of the effect of the extract of theplant in all types of conditions/chemical treatments. Thedose of the aqueous extract of Asparagus racemosuswas made in water and administered orally.

Figure 1. Group 1: Liver section of normal control rat showing(at 1000× magnification) normal hepatic cells with well-preservedcytoplasm, prominent nucleus and nucleolus.

Figure 2. Group 2: Liver section of DEN (200 mg/kg b wt)treated rat showing (at 1000× magnification) focal areas ofmild dysplastic changes. Microsection shows the presence ofpremalignant cells.

Figure 3. Group 3: Liver section of DEN (200 mg/kg b wt) andDDT (0.05%, in diet) treated rat showing (at 1000× magnifica-tion) enlarged nuclei with clumped chromatin and macronuclei.Sheets of malignant hepatocytes are observed, showing mito-tic figure and prominent nucleoli and hyperchromatic nuclei.

hepatocytes appeared normal. No dysplasia or inflam-mation was noted. No nuclear changes were seen, thusthere was no evidence of malignancy (Fig. 4).

The liver microsections of Wistar rats treated withDEN (200 mg/kg bwt) followed by treatment with theaqueous extract of Asparagus racemosus (200 mg/kgbwt) showed marked fatty change in the liver tissue.No inflammation or malignancy was noted in these liversections. However, the histopathological changes indi-cate diffuse steatosis (Figure not included).

The liver microsections of the group of Wistar ratspretreated with the aqueous extract of Asparagusracemosus followed by treatment with DEN and DDT,showed large areas of fatty change with foci of fibrosis.The preserved hepatocytes showed degenerativechanges. Periportal chronic inflammation was noted. Nomalignancy was seen in the liver sections. This livermicrosection showed marked steatosis with cytotoxicperiportal hepatitis and fibrosis.

Figure 4. Group 4: Liver section of animal treated with aqueousextract (200 mg/kg b wt) of Asparagus racemosus followed bytreatment of DEN (200 mg/kg b wt) showing (at 1000× magnifi-cation) a well maintained lobular and cord pattern. No dysplasia,inflammation or nuclear changes are seen and there is no evi-dence of malignancy.



Immunohistochemical studies

p53+ foci were immunohistochemically stained in liversections of treated rats using Pab240, an antibodyspecific for p53 mutant conformation. The density ofp53+ foci is a direct measure of tumor risk. The liversections of Wistar rats, which were taken as control,showed an absence of p53+ foci (Fig. 5).

The liver sections of the Wistar rats treated with DENalone and a combination of DEN and DDT, showeddistinct clusters of cells with p53+ foci (Fig. 6).

Immunohistochemical studies showed an absence ofp53+ foci in the liver sections of Wistar rats, pretreatedwith the aqueous extract of Asparagus racemosus,followed by treatment with DEN (Fig. 7).

There was an absence of p53+ foci in liver sectionstreated with the aqueous extract of Asparagus racemosusfollowed by treatment with DEN and DDT. No p53+foci were seen in the liver sections of Wistar rats treatedwith DEN followed by treatment with the aqueousextract of Asparagus racemosus. Wistar rats treated withDEN and DDT followed by the administration of theaqueous extract of Asparagus racemosus showed noclusters of p53+ foci in hepatocytes.

Figure 5. Liver section of normal control rat. Immunohisto-chemical staining carried out after 22 weeks. Section shows (at40× magnification) no p53+ foci are observed.

Figure 6. Liver section of DEN (200 mg/kg b wt) treated rat.Immunohistochemical staining carried out after 22 weeks. Sec-tion shows (at 400× magnification) the presence of p53+ foci.

Figure 7. Liver section of aqueous extract (200 mg/kg b wt) ofAsparagus racemosus followed by treated with DEN (200 mg/kg b wt). Section shows (at 400× magnification) no p53+ foci.

Table 1. Effect of aqueous extract of Asparagus racemosus onMDA levels and reduced glutathione in serum of control andexperimental Wistar rats

GSHa MDA levelb

Control 74.76 ± 0.47 1.34 ± 0.34DEN 54.33 ± 0.37••• 4.5 ± 0.22•

DEN + DDT 52.56 ± 0.43••• 5.35 ± 0.25•

PM + DEN 72.07 ± 0.48†† 2.2 ± 0.16††

PM + DEN + DDT 70.2 ± 0.4** 2.15 ± 0.30**

DEN + PM 73.54 ± 0.22††† 2.20 ± 0.11††

DEN + DDT + PM 63.16 ± 0.5*** 2.9 ± 0.30***

Values are expressed as mean ± SD (n = 6).b nmol of MDA conjugate formed/mL of serum.a nmol of GSH/mg protein.• p < 0.001 compared with control. ••• p < 0.01 compared withcontrol. †† p < 0.005 compared with DEN treated rats. ††† p < 0.01compared with DEN treated rats. ** p < 0.005 compared withDEN + DDT treated rats. *** p < 0.01 compared with DEN +DDT treated rats.

Effect of aqueous extract on MDA and reducedglutathione (GSH) levels

The production of MDA increased significantly inWistar rats treated with DEN and Wistar rats treatedwith a combination of DEN and DDT, compared withsaline treated rats (DEN: p < 0.001; DEN + DDT: p <0.001) (Table 1). Pretreatment of Wistar rats with theaqueous extract of Asparagus racemosus reduced theDEN induced MDA production (p < 0.005). Treatmentof Wistar rats with DEN followed by treatment withaqueous extract decreased the elevated MDA levels(p < 0.005).

A significant decline in MDA levels was noted inWistar rats pretreated with the aqueous extract ofAsparagus racemosus followed by treatment with DENand DDT (p < 0.005).

DEN treatment decreased hepatic GSH levels(p < 0.01). Pretreatment with the aqueous extractof Asparagus racemosus followed by treatment withDEN, increased GSH levels (p < 0.005). There was a



significant increase in GSH levels in the Wistar ratspretreated with the aqueous extract of Asparagusracemosus followed by treatment with a combinationof DEN and DDT (p < 0.005) (Table 1).

Effect of aqueous extract on glutathione-S-transferase(GST), glutathione reductase (GR) and catalase(CAT)

The activities of GST, GR and catalase decreased sig-nificantly in DEN treated Wistar rats (p < 0.001, p <0.001, p < 0.001) when compared with the control group.Pretreatment with the aqueous extract of Asparagusracemosus, in DEN treated Wistar rats reversed thedecline in enzyme activity brought about by DEN treat-ment (GST: p < 0.005, GR: p < 0.01, CAT: p < 0.05).Treatment with DEN followed by administration of theaqueous extract of Asparagus racemosus caused a sig-nificant increase in GST activity (p < 0.005). There wasa significant increase in the activities of glutathione

reductase and catalase after treatment with aqueousextract following treatment with DEN (GR p < 0.001,CAT p < 0.001) (Table 2).

Hepatotoxicity: Effect of aqueous extract on alaninetransaminase (ALT), aspartate transaminase (AST)and alkaline phosphatase (ALP)

The level of ALT, AST and ALP were high in DENtreated Wistar rats compared with the control group.Pre-administration of aqueous extract of Asparagusracemosus to DEN treated rats reversed the increasein liver marker enzymes brought about by DEN treat-ment (ALT: p < 0.005, AST: p < 0.01, ALP: p < 0.01).

DEN and DDT significantly increased the levels ofliver marker enzymes (p < 0.001). Pretreatment withthe aqueous extract of Asparagus racemosus significantlyreduced the elevated levels of liver marker enzymes(ALT: p < 0.001, AST: p < 0.001, ALP: p < 0.001)(Table 3).

Table 3. Effect of aqueous extract of Asparagus racemosus on transaminase and alkalinephosphatase levels in serum of control and experimental Wistar rats

Group SGOTa SGPTb ALPc

Control 6.8 ± 0.22 9.04 ± 0.44 6.23 ± 0.17DEN 14.7 ± 0.32• 15.13 ± 0.19• 10.38 ± 0.26•

DEN + DDT 15.85 ± 0.16• 15.14 ± 0.43• 14.6 ± 0.34•

PM + DEN 7.0 ± 0.15†† 7.99 ± 0.11†† 6.08 ± 0.15†††

PM + DEN + DDT 7.37 ± 0.24* 8.27 ± 0.12* 5.97 ± 0.21*

DEN + PM 9.0 ± 0.11††† 9.97 ± 0.10††† 9.98 ± 0.13DEN + DDT + PM 10.3 ± 0.15*** 10.3 ± 0.05*** 8.07 ± 0.08**

Values are expressed as mean ± SD (n = 6).a SGPT level given in IU.b SGOT level given in IU.c Alkaline phosphatase KA units.• p < 0.001 compared with control. †† p < 0.005 compared with DEN treated rats. ††† p < 0.01compared with DEN treated rats. * p < 0.001 compared with DEN+DDT treated rats. ** p < 0.005compared with DEN+DDT treated rats. *** p < 0.01 compared with DEN+DDT treated rats.

Table 2. Effect of aqueous extract of Asparagus racemosus on the activities of glutathione-S-transferase (GST), glutathione reductase (GR) and catalase (CAT)

GSTa Glutathione reductaseb Catalasec

Control 15.07 ± 0.30 16.53 ± 0.42 12.13 ± 0.24DEN 4.58 ± 0.08• 12.01 ± 0.19••• 4.58 ± 0.18•

DEN + DDT 3.59 ± 0.23• 4.54 ± 0.29•• 3.97 ± 0.28•

PM + DEN 11.03 ± 0.31†† 15.61 ± 0.33††† 10.87 ± 0.22†††

PM + DEN + DDT 10.23 ± 0.15* 8.30 ± 0.19** 10.90 ± 0.10**

DEN + PM 12.63 ± 0.39†† 16.58 ± 0.28††† 10.33 ± 0.28†††

DEN + DDT + PM 4.13 ± 0.46 14.98 ± 0.11* 9.5 ± 0.15***

Values are expressed as mean ± SD (n = 6).a nmol of CDNB conjugated/min/mg protein.b nmol of NADPH oxidized/min/mg protein.c μmol of H2O2 consumed/min/g wet wt tissue.PM, plant material.• p < 0.001 compared with control. ••• p < 0.01 compared with control. † p < 0.001 compared withDEN treated rats. †† p < 0.005 compared with DEN treated rats. ††† p < 0.01 compared with DENtreated rats. * p < 0.001 compared with DEN + DDT treated rats. ** p < 0.005 compared withDEN + DDT treated rats. *** p < 0.01 compared with DEN + DDT treated rats.



DISCUSSION AND CONCLUSION

DEN and DDT are effective mutagens causing seriousdamage in biological systems. DEN is a low molecularweight carcinogen, which produces ethyl adducts,primarily N7- guanyl, O6- guanyl and O4- thymidyladducts with DNA. The O6- guanyl adducts are stronglyimplicated in the carcinogenic lesions because of theirincorrect base pairing properties (Sancar and Sancar,1988; Pegg, 1990). DDT is extremely toxic, eliciting anincrease in lipid peroxidation and causing oxidativeDNA damage (Sukata et al., 2002). In this study DENhas been used as an initiator and DDT as a promoterof hepatocarcinogenesis in a rat model. Earlier studiesin various laboratories have shown that a single i.p.administration of DEN for 2 weeks can cause liver can-cer in 22 weeks. DDT was given after DEN to promotethe process of hepatocarcinogenesis.

A rat group in which only DDT was administeredwas not studied, during the course of the present study.This would have served as a better comparison betweenthe cellular response to DDT and DEN treatment.However, it is a known fact that DDT is a nongenotoxicpromoter of hepatocarcinogenesis. According to stud-ies by various scientists it takes 2 years for DDT toinduce preneoplastic lesions in the liver of rats. Ourobjective was to observe the effect of the aqueous extractof Asparagus racemosus on DEN initiated hepatocar-cinogenesis. So this study included an animal group inwhich only DEN has been administered, while in anothergroup the effect of pretreating the animals with an aque-ous extract of the plant was observed. Post-treatmentwith the aqueous extract following DEN (initiator) andDDT (promoter) treatment was also studied.

The rationale for administering DEN and DDT incombination to group II was to develop a good animalmodel for hepatocarcinogenesis. DEN is an initiatorand DDT is a promoter, of hepatocarcinogenesis. Itwas found that both are required to develop a goodanimal model for hepatocarcinogenesis.

The best route for causing hepatocarcinogenesis, asgiven in the literature is intraperitoneally for DEN andorally for DDT. The extract of Asparagus racemosuswas administered orally because it the simplest andmost convenient route for administering the extract. Achemical/drug administered orally passes through theintestinal wall and liver before it is transported viathe bloodstream to the target site. Since this was a studyof the prevention of hepatocarcinogenesis, an oral routeof administration was chosen for the plant extract.

The mechanism of DEN-induced hepatocarcinogen-esis was shown to be due to a hydroxylation of DENby cytochrome P-450 isozymes, to become bioactive.Bioactivated DEN reacts with DNA , causing ethylationof the bases. The ethyl DNA adducts can interrupt basepairing, resulting in mutation and the activation ofproto-oncogenes and the inhibition of tumor suppres-sor genes such as p53, resulting in hepatocarcinogenesis.While previous studies have reported that continuousintrahepatic necroinflammatory changes were observedduring the process of DEN initiated carcinogenesis, thepathogenetic importance of the inflammatory responsehas not been well defined (Matsuda et al., 2005)

DDT may induce eosinophilic altered hepatocellularfoci and promote the progression of preneoplastic into

hepatocellular tumors. The production of oxidativestress which might be secondary to metabolic activa-tion could be a key factor in hepatocarcinogenesis byDDT and play an important role in tumor promotionand progression (Harada et al., 2006).

The possible healing effect of Asparagus racemosusas discussed subsequently could be due to the presenceof saponins and anthraquinones in the aqueousextract, which have cytostatic effects. In addition tothis, the absence of p53+ foci in rats administered withthe aqueous plant extract prior to DEN and DDT treat-ment indicates that the active principles present in theextract have some effect on the p53 gene. This datawarrants further investigation.

The p53 protein is involved in a great variety ofregulating pathways, mainly through transcriptionalactivation, but also through protein–protein interactions.In response to DNA damage or cellular stress, p53protein activates a gene that induces cell cycle arrest toallow DNA repair to take place or induce apoptosis inthe case of overly damaged DNA (Staib et al., 2003;Attardi and Jacks, 1999). Clusters of cells that expressp53 in the mutant conformation can be detected byimmunohistochemical staining (Rebel et al., 2001).

Analysis of the results obtained after immunohisto-chemical staining confirmed evidence of pre-malignancyin these groups of Wistar rats as shown by p53 reactiv-ity. An important guideline for the evaluation of p53reactivity is the type of pattern obtained qualitatively,i.e. dispersed or compact. We interpret from our resultsthat pretreatment with the aqueous extract of Asparagusracemosus roots leads to significant prevention ofhepatotoxicity as well as malignancy induced by DENand a combination of DEN and DDT, when comparedwith the controls. The results of the histopathologicalstudies revealed that Wistar rats treated with DEN anda combination of DEN and DDT developed pre-neoplasticlesions in the liver. Histopathology of livers from DENand DDT administered Wistar rats pretreated with theaqueous extract showed no inflammation or dysplasia.The state of the liver cells in group III (rats treatedwith DEN, followed by DDT) was not estimated afterDEN treatment as the histopathology cannot be per-formed without killing the rats.

Phytochemical screening of the aqueous extract ofAsparagus racemosus showed positive results for thepresence of saponins and anthraquinones. The saponinspresent in plants and plant products have diverse bio-logical effects. Saponins have been found to show wide-ranging cytostatic effects against cancer cells (Franciset al., 2002). Anthraquinones have also been found tohave antitumor activity. The anthraquinones stop theprogression of the cell cycle at the G1 phase (Alves et al.,2004). The presence of saponins and anthroquinones inthe aqueous extract of Asparagus racemosus may pos-sibly account for the fact that the aqueous extract ofthe plant has anticarcinogenic activity.

The results of the biochemical determinations alsoshow that pretreatment of Wistar rats with the aqueousextract of Asparagus racemosus leads to an ameliora-tion of the oxidative stress and hepatotoxicity broughtabout by the treatment with DEN.

The liver microsections of Wistar rats treated withDEN (200 mg/kg bwt) followed by treatment with aque-ous extract of Asparagus racemosus (200 mg/kg bwt)showed marked fatty changes in the liver tissue. No



inflammation or malignancy was noted in these sec-tions. However, the histopathological changes indicatediffuse steatosis. No p53+ foci were seen in the liversections of Wistar rats treated with DEN followed bytreatment with the aqueous extract of Asparagusracemosus. These results suggest that rats treated withDEN followed by treatment with the aqueous extractof Asparagus racemosus did not show evidence ofmalignancy.

The effect of the aqueous extract of Asparagusracemosus in xenografts of cancer cells in nude micewould be a useful study and is being planned in ourlaboratory.

The present results thus indicate that the aqueousextract of Asparagus racemosus is useful for preventinghepatocarcinogenesis induced by DEN and also for thetreatment of DEN induced hepatocarcinogenesis.

Acknowledgements

The authors wish to acknowledge the financial support received fromthe Defence Research and Development Organization (DRDO), India.The author SKR wishes to acknowledge the Council of Scientific andIndustrial Research for the award of the Junior and Senior ResearchFellowship. The facilities provided by Dr B. R. Ambedkar Center forBiomedical Research, University of Delhi are gratefully acknowledged.

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