Chemotherapeutic efficacy of limonin against Aflatoxin B1 induced primary hepatocarcinogenesis in Wistar albino rats

Biomedicine & Aging Pathology 2 (2012) 206–211

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Original article

Chemotherapeutic efficacy of limonin against Aflatoxin B1 induced primary hepatocarcinogenesis in Wistar albino rats K. Langeswaran a,∗ , A.J. Jagadeesan b , R. Revathy b , M.P. Balasubramanian b a b

Department of Industrial Biotechnology, Bharath University, 173, Agharam Road, Selaiyur, Tambaram, 600073 Chennai-73, Tamilnadu, India Department of Pharmacology & Environmental Toxicology, University of Madras, Taramani Campus, Chennai, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 23 July 2012 Accepted 15 August 2012 Available online 6 November 2012 Keywords: Hepatocellular carcinoma Limonin Aflatoxin B1 Alpha-fetoprotein LPO Biotransformation enzymes

a b s t r a c t Hepatocellular carcinoma (HCC) is one of the most common life-threatening cancers in the world. This cancer generally arises within the boundaries of well-defined fundamental factors. In this investigation, the defensive role of limonin on detoxification system in Aflatoxin B1 induced hepatocellular carcinoma, which is a necessary mechanism in cancer treatment, was studied in experimental rats. After 45 days, the treatment of limonin (50 mg kg bw; p.o) for 28 successive days to Aflatoxin B1 (2 mg kg bodyweight; ip) induced liver cancer bearing rats is found to be highly effective in reducing Alpha-fetoprotein level, LPO levels and enhancing both phase I and phase II enzymes to near normal levels. From this investigation, we concluded that the limonin has strong anticancer activity against Aflatoxin B1 -induced hepatocarcinogenesis, is mediated through the induction of xenobiotic enzymes and histopathological analysis. Crown Copyright © 2012 Published by Elsevier Masson SAS. All rights reserved.

1. Introduction Liver cancer is the 5th most common cancer in men and 7th in women, since of its high fatality rate, it is the 3rd most common cause of death from cancer globally [1]. Hepatocellular carcinoma (HCC) is the predominant histologic subtype compromising approximately 85–90% of all primary liver cancers [2]. In 2008, there were an estimated 695,000 deaths from HCC worldwide among whom at least two thirds of these were in the Asia Pacific region [3]. Globally, chronic infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) and prolonged dietary exposure to aflatoxin are responsible for about 80% of all HCC in human [4]. Other risks factors include primary hemochromatosis and cirrhosis of different etiologies, such as alcoholic cirrhosis and cirrhosis associated with genetic liver diseases, but the principal risk factor varies among countries [5]. Aflatoxin B1 (AFB1 ) is one of the most important mycotoxins due to its hepatotoxic and carcinogenic effects on certain animal models and humans [6–8]. Aspergillus flavus and Aspergillus parasiticus are the most important fungi responsible for its production [9]. AFB1 is the most widespread carcinogen of the aflatoxins, and the International Agency for Research on Cancer reported that there is sufficient evidence to classify AFB1 as a Group I carcinogen

(carcinogenic to humans) [10]. Drug metabolizing enzymes are important in the conversion of exogenous drugs, toxins, endogenous steroid hormones, vitamins and fatty acids [11,12]. Limonin is a bitter white crystalline substance found in orange and lemon seeds, which is the bitter principle of citrus fruits. It is also known as limonoate D-ring-lactone and limonoic acid didelta-lactone. Limonin belongs to a group of bioactive triterpenoid aglycone derivatives named limonoids which contain a furan ring attached to the D-ring at C-17 as well as oxygen containing functional groups at C -3, C -4, C -7, C -16 and C -17 and an epoxide group at C -14, C -15. Limonin has been shown to possess anticarcinogenic properties in both cell culture and in vivo rodent models [13]. In this context, the present investigation attempts to evaluate the anticancer property of limonin against Aflatoxin B1 induced experimental hepatocellular carcinoma using experimental rats. 2. Materials and methods 2.1. Chemicals Aflatoxin B1 and limonin were purchased from Sigma St Louis, MO, U.S.A. All other chemicals including solvents used were of high purity and of analytical grade. 2.2. Animals

∗ Corresponding author. Tel.: +44 22290125, +22293886; mobile: +91 9884495511; fax: +44 22290742. E-mail address: [email protected] (K. Langeswaran).

Healthy adult Wistar albino rats of male sex weighing between 160 ± 20 g were used for the study. They were obtained from

2210-5220/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biomag.2012.08.002

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Fig. 1. The levels of Alpha-Feto Protein (AFP) in serum of control and experimental animals. Each bar expressed as mean ± SD for six animals in each group: a: Group I Vs Group II, III and IV; b: Group II Vs Group III and IV; c: Group III Vs Group IV. The significance at the level of P < 0.05.

the Central Animal House Facility, Dr. ALMPGIBMS, University of Madras, Chennai (IAEC No: 07/012/08). The animals were kept in polypropylene cages and received standardized rat pellet and water ad libitum. All the procedures were done in compliance with the guidelines issued by the Institutional Animal Ethics Committee.

2.6. Estimation of ˛-fetoprotein (AFP) Alpha-fetoprotein (AFP) was measured quantitatively by the method of solid phase Enzyme Linked Immunosorbent Assay (ELISA). 2.7. Estimation of macromolecular damages

2.3. Experimental protocol The rats were divided into four groups of six animals each. Group I served as control animals were administered orally with 1 ml of Dimethyl sulfoxide (DMSO). Group II animals were administrated of Aflatoxin B1 (2 mg kg bw; i.p) in a single dose by dissolving in DMSO (1 ml) to induce primary hepatocellular carcinoma. After 45 days, Group III primary hepatocellular carcinoma bearing rats were treated with limonin (50 mg kg bw p.o) dissolved in 1 ml of DMSO for a period of 28 successive days. Group IV animals received limonin (50 mg kg bw p.o) dissolved in 1 ml of DMSO for a period of 28 successive days.

2.4. Collection of blood and organs At the end of the experiment, all the animals were sacrificed by cervical decapitation. Animals were fasted overnight before sacrifice. Blood was collected in tubes containing EDTA and centrifuged at 3,000 rpm for 15 min. The buffy coat was removed and the packed cells were washed thrice with physiological saline. The washed cells were lysed by suspending in hypotonic buffer and centrifuged at 15,000 g for 30 min. The resulting pellet is the erythrocyte membrane and the supernatant represent the hemolysate. Liver were perfused in situ with cold 0.15 M NaCl at 37 ◦ C.

2.5. Preparation of tissue homogenate One hundred milligram of liver samples were homogenized in 10 ml of ice-cold 0.1 M Tris-HCl buffer (pH 7.4) to give 10% homogenate. It was then subjected to differential centrifugation and mitochondria, microsomes and cytosolic fractions were isolated. Total homogenate and subcellular fractions were used for the assay of the following parameters in serum, plasma and liver samples.

Assay of lipid peroxidation (LPO) [14], Peroxide induced lipid peroxidation, Ascorbate induced lipid peroxidation [15]. 2.8. Estimation of drug metabolizing enzymes Preparation of Liver Microsomes [16,17], Estimation of Cytochrome b5 [18], Assay of NADPH-Cytochrome P450 Reductase [19], Assay of Glutathione-S-Transferase [20], Assay of UDPGlucuronyl Trasnferase [21,22]. 2.9. Tissue processing for histological studies Immediately after sacrifice, the liver was rapidly dissected out and washed in saline and fixed in 10% formalin. Liver was taken from right portion of the median lobe, since this portion of the rat liver has been found to be a site of more apparent histological lesions [23]. The samples were dehydrated in alcohol series 30%, 50%, 70%, 90% and 100% and cleared in xylene. The clean tissues were embedded with molten paraffin at 58 ◦ C. Consecutive sections were taken at 7 ␮m thickness and stained with heamatoxylin and eosin (H and E). 2.10. Statistical analysis Data are presented as the mean ± standard deviation (SD). One way analysis of variance (ANOVA) followed by Tukey’s multiple comparison method was used to compare the means of different groups of by using SPSS 12.5 student’s versions. Comparisons were made between group II and IV with group I and III for animal studies. P < 0.05 was considerable statistically significant in all cases. 3. Results Fig. 1 represents the alpha-fetoprotein levels in serum of control and experimental animals. The AFP level got elevated in

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Table 1 The levels of lipid peroxidation in serum of control and experimental animals. Parameters (n moles of TBARS formed/mg protein/min)

Group I Control

Group II AFB1

Group III AFB1 + Limonin

Group IV Limonin

Basal H2 O2 induced Ascorbic acid induced FeSO4 induced

2.79 ± 0.19 5.70 ± 0.17 4.34 ± 0.14

4.38 ± 0.18a 11.40 ± 0.20a 9.39 ± 0.19a

3.49 ± 0.17a,b 7.47 ± 0.18a,b 7.60 ± 0.16a,b

2.68 ± 0.18b,c 5.47 ± 0.18b,c 4.15 ± 0.23b,c

6.01 ± 0.46

10.70 ± 0.16a

8.17 ± 0.22a,b

6.06 ± 0.17b,c

Values are expressed as mean ± SD for six animals in each group. The significance at the level of P < 0.05. a Group I Vs Group II, III and IV. b Group II Vs Group III and IV. c Group III Vs Group IV.

Table 2 The levels of lipid peroxidation in liver of control and experimental animals. Parameters (n moles of TBARS formed/mg protein/min)

Group I Control

Group II AFB1

Group III AFB1 + Limonin

Group IV Limonin

Basal H2 O2 induced Ascorbic acid induced FeSO4 induced

2.38 ± 0.17 3.80 ± 0.21 2.93 ± 0.17

4.38 ± 0.18a 6.06 ± 0.26a 5.96 ± 0.16a

3.08 ± 0.22a,b 4.59 ± 0.18a,b 3.59 ± 0.18a,b

2.30 ± 0.16b,c 3.58 ± 0.16b,c 2.69 ± 0.16b,c

3.11 ± 0.20

5.60 ± 0.18a

4.69 ± 0.17a,b

2.90 ± 0.19b,c

Values are expressed as mean ± SD for six animals in each group. The significance at the level of P < 0.05. a Group I Vs Group II, III and IV. b Group II Vs Group III and IV. c Group III Vs Group IV

hepatocellular carcinoma bearing animals as compared to control animals. In Group III cancer bearing animals, the levels were lowered to near normal value due to the treatment of limonin. Tables 1 and 2 show the effect of limonin on LPO in the serum and liver of control and experimental animals. The levels of LPO were found to be significantly increased in group II cancer bearing animals (P < 0.05) when compared with control animals under basal conditions and also in the presence of inducers. Conversely, the administration of limonin significantly reduces the peroxidation reaction in group III drug treated animals (P < 0.05). However, no significant changes were observed in group IV drug control animals when compared to the control animals.

Table 3 represents the effect of limonin on drug metabolizing enzymes in liver of control and experimental animals. In Group II HCC bearing animals, the levels of phase I biotransformation enzymes such as Cytochrome P450 , Cytochrome b5 , NADPH Cytochrome “C” reductase, were found to be decreased significantly (P < 0.05) when compared to the control animals. On the other hand, phase II biotransformation enzymes such as glutathioneS-transferase and UDP-Glucronyl transferase were significantly increased in group II cancer bearing animals when compared to the control. On contrary, a significant increase in phase I enzymes and a concomitant decrease in phase II enzymes were observed in limonin treated group III animals when compared to Group II cancer bearing animals. However, there were no significant changes in

Table 3 Effect of Limonin on biotransformation enzymes in liver of control and experimental animals. Parameters (mg/g wet + tissue) Phase I enzyme (Cytochrome P450 n mole/mg microsomal protein/min) Cytochrome b5 (n mole/mg microsomal protein/min) NADPH Cyt “C” Reductase (n mole Cytochrome “C” reduced/mg microsomal protein/min) Phase II enzyme glutathione-S-transferase (␮ mole of CDNB conjugated/mg microsomal protein/min) UDP Glueronyl transferase (units/min/mg microsomal protein)

Group I Control

Group II AFB1

Group III AFB1 + Limonin

Group IV Limonin

0.87 ± 0.09

0.41 ± 0.05a

0.76 ± 0.07a,b

0.88 ± 0.08b,c

0.78 ± 0.44

0.30 ± 0.18a

0.51 ± 0.26a,b

0.74 ± 0.18b,c

48.03 ± 2.17

25.97 ± 1.82a

33.99 ± 1.84a,b

50.07 ± 2.01b,c

25.10 ± 1.86

38.95 ± 1.99a

30.74 ± 1.66a,b

23.93 ± 1.67b,c

0.68 ± 0.04

1.25 ± 0.03a

1.00 ± 0.02a,b

0.67 ± 0.05b,c

Values are expressed as mean ± SD for six animals in each group. The significance at the level of P < 0.05. a Group I Vs Group II, III and IV. b Group II Vs Group III and IV. c Group III Vs Group IV.

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Plate 1. Histopathological observation of liver of control and experimental animals stained with Hematoxylin and Eosin: (a) Group I control (40×, HE); (b) Group II AFB1 induced cancer bearing animals were showed loss of architecture with enlarged central vein and sinusoids with the trend to spread via intrahepatic veins with significant deformities (40×, HE); (c) Group III Limonin treated animals were showed almost normal hepatocytes along with congested sinusoids and loosely arranged normal hepatocytes (40×, HE); (d) Group IV Limonin alone treated animals were showed normal architecture of hepatocytes with normal cytoplasm and small uniform nuclei (40×, HE).

Group IV, drug control animals when compared to group I control animals. The histological analysis of liver tissues of control and experimental animals are shown in Plate 1 4. Discussion Alpha-fetoprotein (AFP) is the major protein expressed by the foetus during gestation but not expressed after birth. In the majority of HCC, AFP is re-expressed, whereas in normal hepatocytes or in regenerating cirrhotic liver, AFP is rarely expressed or expressed to a significantly lesser degree [24]. Serum AFP concentration tends to increase with disease progression and high levels of AFP are found in patients with advanced HCC. In rats, with partial hepatectomy, chemical injury, exposure to chemical carcinogens or with hepatic cancer, AFP production is roughly proportional to the amount of transplantable mRNA part. The major control of AFP production is therefore probably a level of gene transcription [25]. In the present study, increased concentration of AFP in hepatoma bearing animals suggests the risk factor of hepatocellular carcinoma. It was brought back to near normal value by administration of limonin indicating the antitumorogenic nature of limonin. Lipid peroxidation of polyunsaturated fatty acids which is an important consequence of oxidative stress and has been investigated extensively [26]. Reactive oxygen species and organic free radical intermediates formed as a result of biotransformation are suggested to initiate various macromolecular changes like LPO, DNA damage, necrosis and apoptosis. These macromolecular changes are reflection of xenobiotics interfering with normal cellular metabolism, the enzyme regulation, damage nucleic acids, membranes and proteins [27]. Malondialdehyde (MDA), which is a

major end product and an index of LPO, cross-links DNA, protein and nucleotides on the same and opposite strand [28,29]. Furthermore, reported that MDA is mutagenic in mammalian system, which readily reacts with deoxyribonucleosides to produce adducts and cause DNA damage [30]. MDA is a genotoxic agent that may contribute to the development of human cancer [31]. Aflatoxin B1 (AFB1 ), a potent hepatotoxic and hepatocarcinogenic mycotoxin, induces lipid peroxidation (LPO) in rat liver and this may constitute an underlying mechanism of carcinogenesis caused by AFB1 [32]. These increased levels of LPO under basal and also in the presence of inducers (H2 O2 , ascorbate and FeSO4 ) in group II cancer-bearing animals may be due to free radicals produced by AFB1 administration. On the other hand, the administration of limonin decreased the LPO levels in drug treated animals which may be due to the scavenging activity of the citrus limonin. Naturally, there is a dynamic balance between the amount of free radicals generated in the body and antioxidant defence system that scavenge them and protect the body against their deleterious effects [33]. Xenobiotics may exert their pathological effects through generation of ROS, which is related to the aetiology of cancer [34]. Xenobiotic metabolizing enzymes can be divided into phase I (functionalisation) and phase II (conjugation) reaction enzymes. The cytochrome P450 system is a collection of isoenzymes, which catalyze different types of oxidation reactions. Phase II reactions, also known as conjugation reactions, involve the addition of a polar group to the foreign molecule. There are a number of phase II enzymes but glutathione conjugation catalysed by glutathioneS-transferase (GST) is a particularly important route of phase II metabolism as it is often involved in the removal of reactive intermediates [35]. Phase II enzymes, conjugated with various molecules, such as glucuronic acid, sulfate, acetyl, or methyl group,

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with products of phase I reaction are leading to excretion form the body [36]. The CYP enzyme family plays an important role in phase-I metabolism of many drugs. These enzymes are expressed in many tissues but the highest levels are found in liver [37]. Reduced hepatic concentration of cytochrome P450 was assumed to be caused by LPO induced by AFB1 , since it has been noted that LPO caused the degradation of cytochrome P450 [38,39]. In this present investigation, the level of cytochrome P450 was decreased in group II HCC bearing experimental animals. Cytochrome b5 is involved in the activation of molecular oxygen by Cytochrome P450 requires electrons from the donor NADPH-Cytochrome P450 reductase (NADH-cytochrome b5 reductase) [40]. In the present study, decreased level of Cytochrome b5 was observed in Aflatoxin B1 induced liver cancer bearing animals. This inhibition may be due to the alteration of metabolism by Aflatoxin B1 . It was reported that stimulation of NADPH-dependent microsomal lipid peroxidation was proposed to be mediated by NADPH-Cytochrome P450 reductase and Cytochrome P450 [41]. Thus, it was assumed that the NADPH cytochrome reductase is involved in the metabolism of carcinogen and facilitated the microsomal peroxidation. The increase in TBARS levels in aflatoxin B1 treated animals is in agreement with findings reported that decreased level of NADPH cytochrome reductase in rat liver [42,43]. In the present study, the levels of NADPH “C” reductase was decreased in group II cancer bearing animal. Glutathione-S-transferase (GST) is known to play a key role in the detoxification of both xenobiotics and endogeneous compound and in reduction of ROS and DNA adduct formation [44]. An increase in GST activity caused by a substance is therefore an elevation in the mechanism that protects against the noxious effects of xenobiotics, including carcinogens. Limonoids may protect against a variety of cancers by inducing GST activity to neutralise carcinogenic free radicals [45,46]. The balance between the rate of AFB1 -8,9-epoxide production and the rate of inactivation by GST-conjugation determines the susceptibility of the individual and species to AFB1 carcinogenesis [47]. It is reported that dietary supplementation with the citrus limonoids, limonin and nomilin, activates glutathione-S-transferase in the liver and small intestine of the rat [48], suppresses carcinogenesis [49,50]. UDPglucronyl transferase (UDPGT) is a family of integral proteins of the endoplamic reticulum membrane and the nuclear envelope, which are present in many tissues of vertebrates [51]. Early report suggested that synthesis of glucuronides by microsomal UDPGT is a major pathway for the inactivation and subsequent excreation of both endogeneous and xenobiotic organic compounds [52]. Phase II enzymes GST and UDPGT was increased in group II cancer bearing animals. A large number of studies on laboratory animals have also demonstrated that a wide range of non-nutritive dietary bioactive compounds derived from fruit and vegetables inhibit chemical carcinogenesis caused by electrophiles and reactive oxygen species arising from endogenous and exogenous sources [53,54]. In our present investigation, the reduced activity of phase I enzymes were observed in Group-II cancer bearing animals, this may be due to utilization of these enzymes to excrete the AFB1 metabolites. UDP-GT and GST are known to be significant proneoplastic and neoplastic markers to evaluate the extent of free radical damage caused by exposure to various carcinogens. Limonin administration to AFB1 treated animals restored the activities of phase I and phase II enzymes to near those of the control animals, by elevating the phase I enzyme activities and lowering the phase II enzyme activities. The probable mechanism behind this could be that due to the efficiency of limonin to arrest the formation of free radicals and thereby oxidative threat to the animals generated by AFB1 .

To confirm the anticancer effect of limonin, histopathological studies were also carried out. In the present study, manifest changes were observed in liver architecture of cancer bearing animals. Hepatocytes undergo a sequence of alterations recognized morphologically by a progression from altered foci to nodules and from nodules to cancer [55]. The nodules appearing in Group II animals represent transitional stages from normal liver parenchyma to HCC. The development of liver cell hyperplastic foci and nodules in AFB1 induced carcinogenesis [21]. The normalization of adverse histologic patterns observed in Group III animals may be due to the treatment of limonin which reduce the nodular formation in hepatocellular carcinoma bearing animals. The treatment with limonin reduces the extent of Aflatoxin B1 induced DNA strand breaks in group III drug treated animals. 5. Conclusion Hence, the present study suggested that limonin has been shown to prevent Aflatoxin B1 induced hepatocarcinogenesis presumably by decreasing alpha-fetoprotein, LPO levels and by the regulation of biotransformation enzymes and by recovering histological changes altered by potent hepatocarcinogen AFB1 . Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. References [1] Ferlay J, Shin H, Bray F, Forman D, Mathers C, Parkin D. GLOBOCAN 2008. Cancer Incidence and Mortality Worldwide: IARC Cancer Base No. 10. Lyon, France: International Agency for Research on Cancer; 2010. [2] El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007;132(7):2557–76. [3] Asia-Pacific Working Party on Prevention of Hepatocellular Carcinoma. Prevention of hepatocellular carcinoma in the Asia-Pacific region: consensus statements. J Gastroenterol Hepatol 2010;25(4):657–63. [4] Bosch FX, Ribes J, Borras J. Epidemiology of primary liver cancer. Semin Liver Dis 1999;19:271–85. [5] El-Serag HB. Epidemiology of hepatocellular carcinoma. Clin Liver Dis 2001;5:87–107. [6] McLean M, Dutton MF. Cellular interactions and metabolism of aflatoxin: an update. Pharmacol Ther 1995;65:163–92. [7] Wild CP, Turner PC. The toxicology of aflatoxins as a basis for public health decisions. Mutagenesis 2002;17:471–81. [8] Williams JH, Phillips TD, Jolly PE, Stiles JK, Jolly CM, Aggarwal D. Human aflatoxicosis in developing countries: a review of toxicology, exposure, potential health consequences, and interventions. Am J Clin Nutr 2004;80:1106–22. [9] Sargeant KA, Sheridan J, O’Kelly, Carnaghan RBA. Toxicity associated with certain samples of groundnuts. Nature 1961;192:1096–7. [10] IARC. Aflatoxin. IARC Monographs 1985;Suppl. 7:83–7. [11] Gonzalez FJ, Gelboin HV. Role of human cytochromes p450 in the metabolic activation of chemical carcinogens and toxins. Drug Met Rev 1994;26:165–83. [12] Lu H, Li Y. Cytochrome P450 and cancer. Sheng Li Ke Xue Jin Zhan 1997;28: 178–80. [13] Lam LKT, Zang J, Hasegawa S. Citrus limonoid reduction of chemically induced tumorigenesis. Food Technol 1994;13:104–8. [14] Hogberg J, Larson RE, Kristoferson A, Orrenius S. NADPH-dependent reductase solubilised from microsomes by peroxidation and its activity. Biochem Biophys Res Commun 1974;56:836–42. [15] Devasagayam TPA, Tarachand U. Decreased lipid peroxidation in the rat kidney during gestation. Biochem Biophys Res Commun 1987;145:134–8. [16] Boyd MR, Burka LT. In vitro studies on the relationship between target organ alkylation and the pulmonary toxicity of a chemically reactive metabolite of 4-ipomeanol. J Pharmacol Exp Ther 1978;207:687–97. [17] Kamath SA, Narayan KA. Interaction of Ca2+ with endoplasmic reticulum of rat liver. A standardized procedure for the isolation of rat liver microsomes. Anal Biochem 1972;48:53–61. [18] Omura T, Sato R. The carbon monoxide binding pigment of liver microsomes. J Biol Chem 1964;239:2370–8. [19] Phillips AH, Langdon RG. Hepatic triphosphopyridine nucleotide cytochrome C reductase: isolation, characterization and kinetic studies. J Biol Chem 1962;237:2652–60. [20] Habig WH, Pabste MJ, Jakboy WB. Glutathione-s-transferase: the firest enzymatic step in mercapturic acid formation. J Biol Chem 1974;249:7130–9.

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