RESEARCH ARTICLE

PROTECTION OF LIVER FROM N-NITROSODIETHYLAMINE INDUCED HEPATOCELLULAR CARCINOGENESIS BY A NOVEL FLAVONOL–LUTEOLIN

Balamurugan, K* and Swamidoss Daniel, G. Department of Microbiology, PRIST University, Thanjavur-613403

ARTICLE INFO ABSTRACT In this study, we analyzed the anticancer properties of luteolin in N-nitosodiethylamine induced group of rats. We found that the administration of luteolin (0.2mg) for 16 weeks to N-nitosodiethylamine induced rats provides protection against the oxidative stress caused by the carcinogen and thereby prevents hepatocellular carcinogenesis. On administration of the carcinogen, the level of lipid peroxidation elevated markedly, but it was found to be significantly reduced by luteolin administration. Besides, the antioxidant activities in serum were reduced in carcinogen administered animals, which were enhanced to normal level after luteolin treatment to N-nitrosodiethylamine induced group of rats and also this luteolin prevented the elevation of marker enzymes induced by N-nitrosodiethylamine. The bodyweight of the animals decreased and their relative liver weight increased significantly on N-nitrosodiethylamine administration when compared to control group. However, Treatment with luteolin significantly prevented the decrease of the body weight and increase in relative liver weight caused by DEN. In conclusion, these findings indicate that the compound prevent lipid peroxidation, hepatic cell damage and protect the antioxidant system in N-nitrosodiethylamine-induced hepatocellular carcinogenesis.

INTRODUCTION

N-Nitrosodiethylamine (DEN) is a potent hepatocarcinogenic nitrosamine present in tobacco smoke, water,cheddar cheese, cured and fried meals, occupational settings, cosmetics, agricultural chemicals and pharmaceutical agents (Brown, 1999, Sullivan et al.,1991, Reh and Fajen, 1996 ). It has been suggested that, on metabolic

*Corresponding author: balapblr15@gmail.com

activation, it produces the pro-mutagenic products, O 6-ethyl deoxy guanosine and O 4 and O 6- ethyl deoxy thymidine in liver which are responsible for its carcinogenic effects (Verna et al.,1996). It is also reported that the generation of reactive oxygen species (ROS) by DEN causes carcinogenic effects. ROS are potentially dangerous by-products of cellular metabolism that have direct effect on cell development, growth and survival. Oxidative stress caused by ROS has been reported in membrane

ISSN: 0975-833X

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International Journal of Current Research Vol. 11, pp.016-021, December, 2010

INTERNATIONAL JOURNAL OF CURRENT RESEARCH

Key words:

Anticancer, N-nitosodiethylamine, Hepatocellular carcinogenesis. Luteolin, Lipid peroxidation, Antioxidant

Article History: Received 7th September, 2010 Received in revised form 12th October, 2010 Accepted 25th November, 2010 Published online 5th December, 2010

© Copy Right, IJCR, 2010 Academic Journals. All rights reserved.

lipid peroxidation, DNA damage and mutagenesis associated with various stages of tumor formation process (Parola and Robino 2001). Hence the model of DEN-induced HCC is considered as one of the most accepted and widely used experimental models to study hepatocarcinogenesis (Parola et al., 2001). Human liver appears to metabolize nitrosamines in a manner similar to that of rodent liver and also exhibits considerable similarities with regard to morphology, genomic alterations and gene expression, despite their different disease etiologies (Feo et al., 2000). Further, several flavonoids present naturally in food and also in plants, have been shown to modify critical reactions that cause inhibition of chemically induced hepatocarcinogenesis (Thorgeirsson et al., 2002 and Bannasch and Nehrbass, 2001). Luteolin, 3′, 4′,5,7-tetrahydroxyflavone, belongs to a group of naturally occurring compounds called flavonoids that are found widely in the plant kingdom. Flavonoids are polyphenols that play an important role in defending plant cells against microorganisms, insects, and UV irradiation (Harborne JB, Williams, 2000). Evidence from cell culture, animal, and human population studies have suggested that flavonoids are also beneficial to human and animal health. Because of their abundance in foods, e.g., vegetables, fruits, and medicinal herbs, flavonoids are common nutrients that are antioxidants, estrogenic regulators, and antimicrobial agents (Birt et al., 2001). It has been noticed that flavonoids may be a cancer preventive (Knekt et al., 1997 and Neuhouse and Dietary, 2004). Flavonoids may block several points in the progression of carcinogenesis, including cell transformation, invasion, metastasis, and angiogenesis, through inhibiting kinases, reducing transcription factors, regulating cell cycle, and inducing apoptotic cell death. In the present study to analyze the anticancer properties of luteolin in DEN induced group of rats. MATERIAL AND METHODS

Source of chemicals

Luteolin and DEN were purchased from Sigma Aldrich, USA and all other chemicals used were of analytical grade.

Animals

Male wistar albino rats of same age group and body weight 130-150g were selected for all the experiments. The animals were housed in polypropylene cages at an ambient temperature of 25–30ºC and 45–55% relative humidity with a 12 h each of dark and light cycle. Rats were fed pellet diet and water ad libitum. The study was approved by the Institutional Ethical Committee.

Experimental protocol

The experimental animals were divided into three groups, each group comprising of six animals for a study period of 16 weeks as follows: group 1, normal control rats fed with standard diet and pure drinking water. Group 2 rats were induced with DEN (100 mg/kg bodyweight once a week for three weeks. Ip ). In group 3 rats received 0.2 mg of luteolin was administered to DEN induced group of rats for 16 weeks. At the end of the experimental period, the rats were sacrificed by cervical dislocation. The blood was collected for further biochemical analysis. All the animal experiments were duly approved by the Institutional Animal Ethics Committee (743/03/abc/ CPCSEA dt 3.3.03) Guidelines.

Biochemical assay

Aspartate transaminase, Alanine transaminase, Acid phosphatase, alkaline phosphatase albumin, globulin and α feto protein (AFP) were estimated by using commercially available kits according to the manufacturer’s instruction.(AGGAPPE Diagnostic, Kerala, Ensure Biotech Pvt, Hyderabad, India and (ELISA KIT) UBI, MAGIWELL (USA).

Protein determination

Rat liver organs were homogenized in 10 times their weight of phosphate buffer, the homogenate centrifuged for 15 min at 4ºC and the supernatants used for measurement of protein estimation. Protein content was determined by the method of Bradford (Bradford, 1976).

Antioxidant and lipidperoxidation The activities of enzymatic antioxidants such as SOD (Kakkar et al., 1984), Catalase (Sinha, 1982), were assayed in serum of control and experimental

017 International Journal of Current Research, Vol. 11, pp.016-021, December, 2010

group of rats. Further, the Levels of lipid peroxides (Berton et al., 1998) were determined in the serum of control and experimental groups of rats. RESULTS The anticancer efficiency of luteolin against DEN induced hepatocellular cancer was analyzed in male Wistar albino rats. Fig.1 shows that the body weight reduced significantly (p < 0.05) in DEN-induced animals compared to control group of rats whereas it was normalized by rats treated with luteolin. Fig. 2 indicates that administration of DEN to animals caused a significant (p < 0.05) increase in liver weight due to appearance of liver nodules. However, rats administered with luteolin the liver weight were reversed to normal weight.

Fig. 1. Body weight of control and experimental groups of rats. Values are expressed as mean±S.D. (n = 6). a P<0.05 compare with control groups of rats, b P<0.05 compare

with DEN induced groups rats

0

50

100

150

200

250

control DEN induced

DEN + Luteolin

Body

wei

ght i

n gr

am

Initial body weight

Final body weight

b

Fig. 2. Liver weight and relative liver weight of control and experimental groups of rats. Values are expressed as mean±S.D. (n = 6). a P<0.05

compare with control groups of rats, b P<0.05 compare with DEN induced groups rats

Table 1. Effect of luteolin on serum total protein, albumin and globulin

Groups Protein(gm %/dl) albumin(gm/dl) Globulin(gm/dl)

Control 6.1±0.04 0.98±0.26 2.95±0.52

DEN induced rats 3.5±0.01a 1.85±0.31 a 1.90±0.23 a

DEN+Luteolin 5.8±0.26b 1.03±0.42 b 2.51± 0.21 b

Table 2. Effect of luteolin on serum pathophysiological enzymes

Groups AST(U/L) ALT(U/L) ALP(U/L) LDH(U/L)

Control 60±2.3 52±2.6 160±2.8 190±4.1

DEN induced 140±2.6a 132±1.2a 210±1.4a 240±4.3a

DEN+Luteolin 90±3.1b 93±1.7 b 180±2.3 b 210±3.8 b

a Comparisons are made with group 1 (control). b. Comparisons are made with group 2 (DEN-induced).

Balamurugan and Swamidoss Daniel, Protection of liver from N-nitrosodiethylamine induced hepatocellular carcinogenesis 018

0

10

20

30

40

50

control DEN induced DEN + Luteolin

ng/m

l

a

b

content of protein in control, cancer induced, and treated groups are presented in Table 1. The serum total protein content and globulin decreased in DEN-induced animals whereas albumin level was increased as compared to normal control. However, after treatment with luteolin the altered level were normalized. With regard to serum phathophysiological enzymes, effects of luteolin on AST, ALT, ALP and LDH (Table 2). DEN-induced animals significantly increased (p < 0.05) the level of SGOT, SGPT, ALP and LDH as compared to normal control. By contrast, at a dose of 0.2 mg/kg of luteolin administration significantly reversed the altered level of the phathophysiological enzymes in serum as compared to the DEN-induced rats. Table 3 shows the production of lipid peroxides in serum carcinogen-administered groups of animals. A significant increase in LPO level (p < 0.05) did not occur at treatment with Luteolin. Besides, the activity of catalase and SOD was significantly

lower in carcinogen-administered group of animals when compared with control groups of rats, Nevertheless, rats administered with Luteolin significant enhancement were observed when compared with in carcinogen-administered group of rats. The Fig. 3 depicts the level of the tumor markers protein α feto protein (AFP). Their levels were found to be elevated significantly (p < 0.05) in DEN-induced animals where as their levels were significantly lower on treatment with luteolin. DISCUSSION Liver damage caused by DEN generally reflects instability of liver cell metabolism which leads to distinctive changes in the serum enzyme activities (Plaa and Hewitt, 1989). Serum SGOT, SGPT, ALP and ACP are representative of liver function; their increased levels are indicators of liver damage. The elevation of ALT activity is

Table 3. The production of lipid peroxides in serum carcinogen-administered group of rats

Groups MDA(nm/mg of protein)

Catalase (U/mg of protein)

SOD (U/mg of protein)

Control 2.13±0.41 17.54±.0.42 415.06±0.43

DEN induced 3.85±0.24a 10.13±0.42a 356.64±0.42a

DEN+Luteolin 2.10±0.13 b 14.42±0.14b 396.01±0.12b

Values are expressed as mean±S.D. (n = 6). a P<0.05 compare with control groups of rats, b P<0.05 compare with DEN induced groups of rats

Fig. 3. Values are expressed as mean±S.D. (n = 6). Statistical significance p < 0.05. Activity is expressed as ng/ml for α feto protein

a Comparisons are made with group 1 (control). b. Comparisons are made with group 2 (DEN-induced).

019 International Journal of Current Research, Vol. 11, pp.016-021, December, 2010

repeatedly credited to hepatocellular damage and is usually accompanied by a rise in AST (Plaa and Hewitt, 1989). Increase in ALP reflects the pathological alteration in biliary flow.In the present study, treatment with luteolin attenuated the increased activities of these enzymes were normalized. It suggested that the luteolin assist in parenchymal cell regeneration in liver, thus protecting membrane integrity, thereby decreasing enzyme leakage. Reactive oxygen species degrade polyunsaturated lipids to produce the malondialdehyde. This compounds is a reactive aldehyde and is one of the many reactive electrophiles species that cause toxic stress in cells and form covalent protein adducts which are termed to as advanced lipoxidation end products in analogy to advanced glycation end products The production of this aldehyde is used as a biomarker to measure the level of oxidative stress in an organism. Lipid peroxidation is one of the major mechanisms of cellular injury caused by free radicals(Esterbauer and Chesseman, 1990). Administration of DEN has been reported to generate lipid peroxidation products like MDA and 4-hydroxy nonenal that may interact with various molecules leading to oxidative stress and carcinogenesis (Hietanen, et al., 1987).The level of LPO increases with the administration of DEN during hepatocarcinogenesis. SOD is an important defense enzyme which catalyses the dismutation of superoxide radicals to hydrogen peroxide thereby reducing the likelihood of superoxide anion interacting with NO to form reactive peroxynitrite (Maritim et al., 2003). Hydrogen peroxide is successively metabolized into water and non reactive oxygen species by the activities of catalase and GPx (Matés and Sánchez-Jiménez, 1999). Catalase a tetrameric enzyme which is that decomposes the hydrogen peroxide into the harmless products such as water and molecular oxygen. Hydrogen peroxide toxicity is enhanced to the formation of reactive hydroxyl radical on capture of an electron from Fe (II) or Cu (I). The hydroxyl radical reacts instantly with all cellular components resulting in the modifications of protein and nucleic acids. Catalase is one of the most competent enzymes so that it cannot be saturated by hydrogen peroxide at any

concentration (Lledías et al., 1998). Decreases in the activities of SOD, CAT are observed in tumor cells. The luteolin that can scavenge excessive free radicals in the body are suggested to hinder the process of carcinogenesis. Such studies support our findings that activities of the enzymic antioxidants are reverted to near normal in Luteolin treated animals and hence prevent the initiation of carcinogenesis by DEN. The protection offered by luteolin to the enzymatic antioxidant system may be explained by the increase in the level of these antioxidants probably due to the direct reaction of luteolin with ROS. Luteolin may also protect the membrane and antioxidants from ROS. α Feto protein (AFP) an oncofetal serum protein, progressively lost during development, such that it is virtually absent from the healthy adult (Sell et al., 1983). It has long been recognized that exposure of rats to certain carcinogens like DEN causes an elevation of circulating AFP levels. This corroborates with the results showing the significant rise in levels of AFP obtained in DEN-induced animals (Becker and Sell, 1979) that were found to be reduced in luteolin treated animals. Our results highlight the ability of luteolin to change the levels of LPO and significantly increase the endogenous antioxidant defense mechanisms in DEN induced hepatocellular carcinogenesis. Our results also show that the significant increase in the levels of serum markers and tumor markers was prevented by luteolin treatment. From the results obtained, we suggest that luteolin may be developed as an effective chemotherapeutic agent. Further studies are underway to elucidate the molecular mechanisms involved to prove luteolin efficacy as an anti-hepatocarcinoma agent. Acknowledgement

My sincere thanks to PRIST University for providing all facilities REFERENCES Bannasch, P., Nehrbass, D. 2001. A. Kopp-

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021 International Journal of Current Research, Vol. 11, pp.016-021, December, 2010

*****

ORIGINAL ARTICLE

Evaluation of Luteolin in the Preventionof N-nitrosodiethylamine-induced HepatocellularCarcinoma Using Animal Model System

K. Balamurugan • J. Karthikeyan

Received: 6 July 2011 / Accepted: 12 September 2011

� Association of Clinical Biochemists of India 2011

Abstract Hepatocellular carcinoma (HCC) is one of the

commonest tumors worldwide. The treatment of HCC is vital

for disease diagnosis and prognosis, as the liver is the most

important organ controlling metabolic functions. Now-a-

days, western folklore medicines are largely dependent on

the phyto compounds which are highly effective in therapy

and with low side effects. Luteolin is a flavonoid (3,4,5,7-

Tetrahydro flavones) possess anti-inflammatory, anticancer

and anti allergic property. The present study evaluates the

efficacy of luteolin against N-nitrosodiethylamine (DEN)

induced HCC in albino rats. In the highlight of the above,

luteolin was evaluated for its efficacy against DEN induced

HCC in male Wistar albino rats. The Biochemical parame-

ters such as tissue damaging enzymes viz., AST, ALP, LDH

and c-GT, enzymatic antioxidants viz., SOD, CAT, GSH and

GPx and histopathological changes have been estimated.

The tissue damaging enzymes were found to be high in DEN

alone treated group whereas the enzymatic antioxidants

decreased destructively. Severe lesions and cirrhosis were

observed in the toxin (DEN alone) treated group. The lute-

olin treated DEN group altered the tissue damaging enzymes

and the enzymatic antioxidants. The damaged lesion in the

histoarchitecture of DEN treated rat liver was almost com-

pletely restored. Finally this study strongly demonstrates that

luteolin has potent curative property against HCC in albino

rats.

Keywords Hepatocellular carcinoma � Luteolin � DEN �Enzymatic/Non-enzymatic Antioxidants � Ultrastructural

study

Introduction

Hepatocellular carcinoma (HCC) is a major malignancy

worldwide and is increasingly associated with cancer

related death [1]. The treatment of cancer is still a big

challenge in medicine. HCC is a heterogeneous disease in

the expression of etiology and underlying associations as

well as biological and clinical behavior.

Several chemicals are known to possess chemo preventive

property against a broad spectrum of cancers. Chemopre-

vention serves as an attractive alternative to control malig-

nancy [2]. Several herbal drugs have been evaluated for their

potential as liver protectant against NDEA-induced hepato-

toxicity in rats [3]. Luteolin is a bioactive flavonoid, chem-

ically (3,4,5,7-Tetrahydroxy flavones) an important member

of the flavoniod family. It is present in various fruits, and

vegetables. It exhibits a wide spectrum of pharmacological

property including anti-inflammatory and anti allergic

property [4, 5]. Much attention has been recently paid to its

antioxidant property and to its proliferative effect.

DEN (N-Nitrosodiethylamine) is a strong hepatocarcin-

ogenic dialkylnitrosoamine present in tobacco smoke,

water, cheddar cheese, curd and fried meals and in a

number of alcoholic beverages [6].NDEA is known to

cause perturbations in the nuclear enzymes involved in

DNA repair/replication and producing reproducible HCC

after repeated administration in experimental animals [7].

Plenty of reports give evidence that that NDEA causes a

wide range of tumors in all animal species and such com-

pounds are hazardous to human health [8]. The formation

K. Balamurugan

Department of Biochemistry, PRIST University, Thanjavur,

South India

J. Karthikeyan (&)

Centre for Research and Development, PRIST University,

Thanjavur, South India

e-mail: karthikoushik@gmail.com

123

Ind J Clin Biochem

DOI 10.1007/s12291-011-0166-7

of reactive oxygen species (ROS) is apparent during the

metabolic biotransformation of NDEA resulting in oxida-

tive stress. Oxidative stress leads to carcinogenesis by

several mechanisms including DNA, lipid and protein

damage, change in intracellular signaling pathways and

even changes in gene expression. Lipid peroxidation (LPO)

may also result in several changes, including structural and

functional membrane modifications, protein oxidation and

generation of oxidation products such as acrolein, croton-

aldehyde, malondialdehyde (MDA) and 4-hydroxy-2-non-

enal (HNE), which are considered strong carcinogens.

Efforts to develop less toxic drugs that affect antioxidant

system, malignant cells and mechanism-based approach are

necessary in prevention and therapy of cancer.

The majority of HCC occurs in patients with liver cir-

rhosis and consequent hepatic dysfunction, which compli-

cates safe administration of systemic therapy and poses a

challenge to conducting clinical trials in this patient pop-

ulation. Therefore this paper emphasis the in vivo antitu-

mor efficacy of the phytal compound luteolin (flavonol)

against DEN induced HCC in male albino Wistar rats.

Materials and Methods

Animals

Male Wistar albino rats (130–150 g) were procured from

Kerala Agriculture University, Thrissur, Kerala. The ani-

mals were housed in polypropylene cages at an ambient

temperature of 25–30�C and 45–55% relative humidity

with a 12 h each of dark and light cycle. Rats were fed

pellet diet and water ad libitum. The study was approved

by the Institutional Ethical Committee. (743/03/abc/CPC-

SEA dated 3.3.03).

Source of Chemicals

Luteolin and DEN were purchased from Sigma Aldrich,

USA and all other chemicals used were of analytical grade.

Experimental Design

The experimental animals were divided into four groups,

each group comprising of six animals for a study period of

16 weeks. Group I, control rats (untreated) were fed with

standard diet and water ab libitum. Group II rats were

administered with luteolin alone (0.2 mg/kg b.w.daily) was

administered by Intraperitonially. Group III rats were

induced intraperitonially with DEN (100 mg/kg b.w.) once

in a week for a period of 6 weeks. To the group IV

rats, luteolin (0.2 mg/kg b.w.daily) was administered by

intraperitonially to DEN induced group of rats after the

proliferation tumor from 6th week onwards up to

16 weeks. At the end of the experimental period, the rats

were euthanized by cervical dislocation. The blood was

collected, processed and stored for further analysis. The

liver was excised immediately, rinsed in ice cold saline and

was homogenized in 0.1 M Tris buffer (pH.7.4) for further

biochemical analysis.

Preparation of Hepatic Tissue Homogenate

Hepatic tissues from control and experimental group of rats

were excised, rinsed with ice-cold saline and homogenized

in Tris–HCl buffer (100 mM, pH 7.4) using Teflon

homogenizer and centrifuged at 12,0009g for 30 min at

4�C. The supernatant was pooled and used for the further

estimations. The protein content in the tissue homogenate

was measured by the method of [9].

Liver Injury Markers

The activities of aspartate transaminase (AST) and Alanine

transaminase (ALT) were estimated by the method of [10],

while alkaline phosphatase (ALP) and glutamyl transferase

(GT) were estimated by methods of [11, 12].

Antioxidant Enzyme Assay

The activity of superoxide dismutase (SOD) in the hepatic

tissue was assayed by the method of [13]. Catalase (CAT)

activity was measured according to the method of [14].

Reduced glutathione (GSH) level was measured by the

method of [15]. Glutathione peroxidase (GPx) activity was

measured by the method of [16] and the activity of gluta-

thione-S-transferase (GST) was measured according to the

method of [17].

Histopathological Assessment

Liver sections were prepared from different groups of rats,

fixed in 10% formalin, dehydrated in gradual ethanol

(50–100%), cleared in xylene, and embedded in paraffin.

The pathological changes were observed microscopically

after staining with hematoxylin and eosin (H–E).

Transmission Electron Microscopic Study

For Ultrastructural study, a portion of liver (about 1 mm)

from control and experimental groups of rats were fixed in

3% glutaraldehyde in sodium phosphate buffer (200 mM,

pH 7.4) for 3 h at 4�C. Tissue samples were washed with

the same buffer, placed in 1% osmium tetroxide and

sodium phosphate buffer (200 mM, pH 7.4) for 1 h at

4�C.The samples were again washed with the same buffer

Ind J Clin Biochem

123

for 3 h at 4�C, dehydrated with graded series of ethanol

and embedded in Araldite. Thin sections were cut with

LKBUM4 ultra microtome using a diamond knife

(Diatome, Aldermaston, Berkshire, England), mounted on

a copper grid and stained with 2% uranyl acetate and

reynolds lead citrate [18]. The grids were examined under a

Philips EM201C transmission electron microscope (TEM)

(Philips, Eindhoven, Netherlands).

Statistical Analysis

The data were analysed using SPSS/16.0 software.

Hypothesis testing methods were included with analysis of

variance (ANOVA) followed by least significance differ-

ence (LSD). P values of [ 0.05 were considered statisti-

cally significant. The data were expressed as mean ± S.D

with six animals in each group.

Results

Table 1 illustrates the activity of liver enzymes LDH, ALT,

AST and ALP in experimental group of rats. DEN alone

treated group-III showed significant increase in the liver

enzymes viz., LDH, ALT, AST and ALP compared to

group-I control and group-II luteoloin alone treated group.

The enzyme activities in the DEN induced luteolin treated

group was similar to that of the group-I (control) rats.

Table 2 shows the content of antioxidants in liver of

treated group. The levels of antioxidant enzymes viz.,

CAT, Glutathione peroxidase (GPx), SOD were found to

be more in the DEN induced group-III treated rats, whereas

glutathione reductase (GR) and GST levels significantly

decreased in these groups. In the luteolin treated group,

the antioxidants enzyme levels were slightly lower than the

control group. The DEN induced luteolin treated group

restores the changes to near normalcy by its antioxidant

efficacy.

The antioxidant potential was further confirmed by the

non-enzymatic antioxidants such as Vitamin-C, Vitamin E,

GSH and MDA levels. The DEN induced treated group

showed significantly decreased levels of Vitamin-C,

Vitamin-E, GSH and MDA (Table 3). The luteolin treated

group possessed slightly elevated levels of non-enzymatic

antioxidants than the control group-I untreated rats. The

non enzymatic antioxidants in the DEN induced luteolin

treated group IV rats were found to be similar to that of

control (normal) rats.

The histology of the liver tissue was examined under a

light microscope and represented in Fig. 1 (a–d). Group I

(control rats) indicated in the Fig. 1a revealed the normal

architecture of the liver cells and group-II (Fig. 1b) rats

treated with luteolin alone showed the normal histological

appearance of liver cells further ascertaining its nontoxic

property. Group-III (Fig. 1c) DEN alone treated groups

depicted the area of severe hepatocellular necrosis with the

adjacent liver cells. It is interesting to observe that, in the

DEN induced luteolin treated group (Fig. 1d) the damaged

liver architecture was altered, necrosis healed and the

cellular degeneration was found to be lower.

The ultra structural changes occurred in hepatocytes of

control and experimental groups of rats were shown in

Fig. 2 (a–d). The Fig. 2a depicts the electron micrograph of

hepatocyte of control group of rats. From the figure, it

could be seen that the normal cellular organelles, mito-

chondria (M), rough endoplasmic reticulum, golgi complex

(GC), nucleus (N) with intact nuclear membrane (NM) and

nuclear chromatin were visible. Similar architecture was

observed in the electron micrograph of control group of

rats treated with luteolin Fig. 2b. The electron micrograph

of hepatocyte of DEN induced HCC group III of rats in

Fig. 2c revealed the decrease of organelles regeneration,

swelling in the cisternae of the rough endoplasmic reticu-

lum and mitochondrial cristae, fusion or disappearance of

mitochondrial crests, degranulation of rough endoplasmic

reticulum, pyknotic nuclei with damaged NM, extensions

in perinuclear area, increased smooth ER and lipid accu-

mulation, cells with dark and light cytoplasms.

Figure 2d shows an apparent appearance of NM and

chromatin, either absent or significant reduction in the

swelling in the cisternae of the rough endoplasmic reticu-

lum and mitochondrial cristae, dilation in the perinuclear

space, presence of few pyknotic nuclei and reduction of

smooth ER. This finding shows the in vivo antitumor

Table 1 Effect of luteolin on liver enzyme profiles of untreated and treated rat groups

Group LDH (U/l) ALT (U/l) AST (U/l) ALP (u/l)

Control 182 ± 5.6 32.21 ± 3.2 74.21 ± 4.5 128.22 ± 0.9

Luteolin alone 178 ± 2.6 31.66 ± 3.2 76.52 ± 3.5 130.47 ± 2.6

DEN alone 230 ± 13.5a 192.20 ± 4.8a 273. ± 10.5a 242.6 ± 0.6a

DEN ? luteolin 162 ± 3.9b 69.00 ± 5.1b 123 ± 4.89b 163.14 ± 14b

Values of results are expressed as mean ± .SD for six rats. In a column, means followed by a common letter are not significantly different at the

5% level by DMRT

Ind J Clin Biochem

123

Table 2 Effect of luteolin on liver antioxidant enzymes of untreated and treated rat groups

Groups Catalase GPx GRD SOD GST

Control 18.52 ± 0.0.52 437.84 ± 2.0 25.07 ± 0.45 468.04 ± 23 0.34 ± 0.01

Luteolin alone 17.61 ± 0.42 440.12 ± 27 24.97 ± 0.21 465.65 ± 15 0.33. ± 0.04

DEN alone 13.58 ± 0.21a 262.61 ± 4.1a 15.42 ± 0.41a 269.84 ± 16a 0.15 ± 0.08a

DEN ? luteolin 16.85 ± 0.17b 390.45 ± 3.6b 22.15 ± 0.45b 442.23 ± 11b 0.28 ± 0.01b

Catalase (U/mg of protein), GPx (U/mg of protein), GR (nmol NADPH/min/mg protein), SOD (U/mg of protein), GST (lmol 1-chloro-2,

4-dinitrobenzene conjugate formed/min/mg of protein)

Values of results are expressed as mean ± .SD for six rats. In a column, means followed by a common letter are not significantly different at the

5% level by DMRT

Table 3 Effect of luteolin on non-antioxidant enzymes in liver of untreated and treated rat groups

Groups Vitamin C (lg/mg of protein) Vitamin E (lg/mg of protein) GSH (mg/100 g of wet tissue MDA (nm/mg of protein)

Control 1.23 ± 0.05 1.56 ± 0.04 40.23 ± 1.6 2.17 ± 0.41

Luteolin alone 1.28 ± 0.02 1.26 ± 0.01 46.21 ± 1.8 2.04 ± 0.12

DEN alone 0.56 ± 0.08a 0.62 ± 0.07a 20.56 ± 2.1a 8.16 ± 0.41a

DEN ? luteolin 1.16 ± 0.06b 1.38 ± 0.09b 38.26 ± 2.4b 4.12 ± 0.36b

Values of results are expressed as mean ± .SD for six rats. In a column, means followed by a common letter are not significantly different at the

5% level by DMRT

Fig. 1 Light microscopic

analysis of control and treated

group of rats

Ind J Clin Biochem

123

activity of luteolin in the experimental group of animals.

Specifically it confirms the HCC protective nature of

luteolin in DEN induced HCC group of rats.

Discussion

Luteolin is a naturally occurring flavonoid, is a known

biochemical target other than the fact that it induces

topoisomerase-II mediated apoptosis. Similar activity has

been reported by inhibition of invasive activity in Mia-

PaCa-2 cancer cells [19–21]. Luteolin exhibits wide spec-

trum of anti-tumor activities, but little is known about its

anti-cancer mechanisms.

Liver damage caused by DEN generally reflects insta-

bility of liver cell metabolism which leads to distinctive

changes in the serum enzyme activities [22]. AST, ALT,

LDH and ALP are representative of liver function. Their

increased levels are indicators of liver damage. The ele-

vation of ALT activity is repeatedly credited to hepato-

cellular damage and is usually accompanied by a rise in

AST. Increase in ALP reflects the pathological alteration in

biliary flow. In the present study, treatment with luteolin

induced the increased activities of these enzymes and were

normalized. This suggested that the luteolin played a role

in parenchymal cell regeneration in liver, thus protecting

membrane integrity, thereby decreasing enzyme leakage.

SOD acts as the first line of defense against superoxide

free radicals, which dismutates two superoxide radicals to

H2O2 and O2. Besides CAT and GPx act as supporting

antioxidant enzymes by converting H2O2 to H2O, thereby

providing protection against ROS [23]. The reduction in

activity of these enzymes may be caused by the increase in

radical production during DEN metabolism. In the present

Fig. 2 TEM image of HCC in

control and experimental rats.

a Control. b Luteolin alone.

c HCC. d HCC ? luteolin.

Hepatic tissue sections were

showed at 150009

magnification. The organelles

such as ER, GC, N, NM and M

were displayed

Ind J Clin Biochem

123

investigation, a raise in MDA formation was presumably

associated with increased ROS, consistent with the obser-

vation that these free radicals reduce the activity of hepatic

SOD [24] In the present study the reduction in these anti-

oxidant enzymes was due to the action of luteolin on the

DEN induced HCC. Biochemical results of hepatic SOD

showed a decrease in activity of SOD in DEN-induced rats

compared to control and luteolin treatment on the DEN

induced HCC group rats. GPx is one more endogenous

antioxidant seleno protein present in the cytosol and

mitochondrial matrix that participate in the defense

mechanism. GPx was activated before the initiation of

chronic oxidative stress and catalyzes the reduction of lipid

and non-lipid hydro peroxides using two molecules of GSH

and thereby curtails the quantity of biomolecules having

destructive properties [25]. Similarly, GST is a soluble

protein situated in cytosol and plays a vital role in detox-

ification and excretion of xenobiotics [26].

GST catalyzes the conjugation of the thiol functional

groups of GSH to electrophilic xenobiotics and results in

escalating solubility. The xenobiotic–GSH conjugate is

then either eliminated or converted to mercapturic acid

[27]. Since GST increases solubility of hydrophobic sub-

stances, it plays an important role in storage and excretion

of xenobiotics. Induction of xenobiotic detoxifying

enzymes is an additional mechanism by which antioxidant

rich extracts may act as anticarcinogens as they compete

with steps in xenobiotic activation and metabolize toxic

compounds to non-toxic ones [28].As the activity of GST

increased in luteolin treated rats, it appears that the drug

induces greater coupling of electrophilic intermediates with

GSH.

In summary, luteolin stabilizes and restores the antiox-

idant defense system viz., GSH, CAT, SOD, GPx and GST.

These antioxidant enzymes protect cells from ROS damage

in DEN-induced HCC. Luteolin, a bioflavonoid protects the

activities of liver injury and tumor markers by decreasing

MDA. The research findings clearly indicate that luteolin

eliminates the oxidative state induced by the initiator DEN,

and it interacts directly with ROS (e.g., OH•), as well as

indirectly by activating the antioxidant defense system.

Luteolin thus reduces the DEN induced increased ROS

generation during hepatocarcinogenesis and promotes the

enzymatic and non-enzymatic antioxidant defense system

and has potentiality in chemoprevention.

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