Mutation Research 767 (2014) 13–20

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Protective effect of apigenin against N-nitrosodiethylamine (NDEA)-induced hepatotoxicity in albino rats Fahad Ali, Rahul, Falaq Naz, Smita Jyoti, Yasir Hasan Siddique ∗ Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh, Uttar Pradesh 202002, India

a r t i c l e

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Article history: Received 5 August 2013 Received in revised form 26 March 2014 Accepted 5 April 2014 Available online 19 April 2014 Keywords: Apigenin N-nitrosodiethylamine Serum enzymes Antigenotoxic Hepatoprotective

a b s t r a c t A number of pharmacological properties have been attributed to apigenin. In the present study the effect of apigenin was investigated with respect to hepatotoxicity induced by N-nitrosodiethylamine (NDEA), a compound that is present in many food stuffs and has been reported to be a hepatocarcinogen. Male rats were exposed to NDEA (0.1 mg/ml) dissolved in drinking-water separately, and with 10, 20, or 40 mg/ml of apigenin for 21 days. The activity of glutamic-oxaloacetic transaminase (SGOT), glutamic-pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) was measured in blood serum. Lipid peroxidation, protein carbonyl content and micronucleus frequency were determined in hepatocytes. To assess the effect on DNA damage, the comet assay was performed on hepatocytes, blood lymphocytes and bone-marrow cells of the exposed rats. The results of the study reveal that the treatment of NDEA together with apigenin showed a significant dose-dependent decrease in the serum concentration of the enzymes SGOT, SGPT, ALP and LDH (p < 0.05). Histological sections of the liver also showed a protective effect of apigenin. A significant dose-dependent reduction in lipid peroxidation and protein carbonyl content was observed in rats exposed to NDEA (0.1 mg/ml) together with apigenin (p < 0.05). The results obtained for the comet assay in rat hepatocytes, blood lymphocytes and bone-marrow cells showed a significant dose-dependent decrease in the mean tail length (p < 0.05). The present study supports the role of apigenin as an anti-genotoxic and hepatoprotective agent. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Apigenin is ubiquitously distributed in leaves, vegetables, stems and fruits of several vascular plants [1]. It has been shown to possess not only anti-inflammatory, antioxidant, and anticancer properties but it may also be protective in other diseases that are affected by oxidative processes such as cardiovascular and neurological disorders [2]. There are only few reports on the occurrence of adverse metabolic reactions by the consumption of apigenin and for this reason apigenin has gained more interest in recent years [3]. In our earlier studies apigenin showed antigenotoxic effects against anticancer drugs in cultured human lymphocytes [4], and in mouse

Abbreviations: NDEA, N-nitrosodiethylamine; SGOT, serum glutamicoxaloacetic transaminase; SGPT, serum glutamic-pyruvic transaminase; ALP, alkaline phosphatase; ALT, alanine transaminase; ALP, alkaline phosphatase; GGT, gamma-glutamyl transferase; LDH, lactate dehydrogenase; DNA, deoxyribonucleic acid; NOCs, N-nitroso compounds; ROS, reactive oxygen species; DMSO, dimethy sulfoxide; BNF, buffered neutral formalin; TCA, trichloroacetic acid. ∗ Corresponding author. E-mail address: yasir [email protected] (Y.H. Siddique). http://dx.doi.org/10.1016/j.mrgentox.2014.04.006 1383-5718/© 2014 Elsevier B.V. All rights reserved.

bone-marrow cells [5]. It has also shown protective effects against the genotoxicity of hydrogen peroxide and ethinylestradiol in cultured human blood lymphocytes [6,7]. In our recent study on a Drosophila model for Parkinson’s disease, apigenin showed a protective effect against PD symptoms [8]. N-nitroso compounds (NOCs) are present in the human environment and some are well-known carcinogens [9]. Nnitrosodiethylamine (NDEA) has been suggested to cause oxidative stress and cellular injury due to the involvement of free radicals [10–12]. NOCs have also been reported to induce hepatic fibrosis. The induction of hepatic fibrosis and the alterations in various biochemical parameters by nitroso compounds have been reviewed by Ahmad and Ahmad [13]. Nitroso compounds are also found predominantly in a variety of food stuffs such as milk products, meat products and preserved juices [14]. NDEA is a well-known hepatocarcinogen present in tobacco smoke, in water containing high concentration of nitrates, fried meals, cosmetics, agricultural chemicals and pharmaceutical agents. In the liver NDEA is activated by cytochrome P450 to form electrophilic and reactive oxygen species [15]. NDEA is metabolized to its active ethyl radical metabolite (CH3 CH2+ ) [16]. The nitroso compounds are considered as a group of

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carcinogens that may be formed endogenously in the human body [17]. NDEA has been suggested to cause oxidative stress and cellular injury by generating reactive oxygen species [18]. The reactive products and free radicals also result in an increase in the serum indices of liver function such as alanine transaminase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT) and total bilirubin, and in severe histopathological lesions in the liver, which may lead to neoplastic transformation [19]. The presence of reactive oxygen species (ROS) also results in carbonylation of protein, peroxidation of lipids and DNA damage leading to cytotoxicity, carcinogenicity and mutagenicity [1]. ROS are potentially dangerous by-products of cellular metabolism that can lead to the development of cancer [20]. The supplementation of dietary antioxidants in addition to the cellular defense system has been proposed for the protection against oxidative stress [15]. A number of epidemiological and animal studies have shown that high intake of natural products may be associated with a decreased risk of many cancers [19]. Flavonoids are ubiquitously present in fruits, vegetables and are suggested to have various pharmacological properties without causing significant toxicity [21]. In the present study the effect of apigenin was investigated in respect of the NDEA-induced hepatotoxicity in Swiss albino rats.

supernatant was removed, and the pellet re-suspended in homogenization buffer and kept at −20 ◦ C for further analyses. 2.6. Estimation of protein carbonyl content The protein carbonyl content was estimated according to the method described by Hawkins et al. [23]. The liver homogenate was diluted to a protein concentration of approximately 1 mg/ml. About 250 ␮l of each diluted homogenate was taken in separate Eppendorf centrifuge tubes. Then 250 ␮l of 10 mM 2,4-dinitrophenyl hydrazine (dissolved in 2.5 M HCl) was added, and the mixture was vortexed and kept in the dark for 20 min. About 125 ␮l of 50% (w/v) trichloroacetic acid (TCA) was added, mixed thoroughly and incubated at −20 ◦ C for 15 min. The tubes were then centrifuged at 4 ◦ C for 10 min at 9000 × g. The supernatant was discarded and the pellet was washed twice in ice-cold ethanol:ethylacetate (1:1). Finally the pellets were re-dissolved in 1 ml 6 M guanidine hydrochloride, and the absorbance was read at 370 nm. 2.7. Estimation of lipid peroxidation The method described by Siddique et al. [24] was used for the estimation of lipid peroxidation in liver cells. Reagent 1 (R1) was prepared by dissolving 0.064 g of 1methyl-2-phenylindole in 30 ml of acetonitrile. The preparation of 37% HCl served as the reagent R2. About 200 ␮l of diluted liver homogenate (protein concentration approximately 1 mg/ml) was mixed with 300 ␮l of R1. Then 300 ␮l of R2 was added, and the mixture was vortexed, incubated at 45 ◦ C for 40 min, cooled on ice, and centrifuged at 11,000 × g at 4 ◦ C. The absorbance was read at 586 nm. 2.8. Micronucleus assay

2. Materials and methods 2.1. Chemicals N-nitrosodiethylamine and apigenin were purchased from Sigma Chemicals Co. (USA). Agarose (normal and low-melting), Triton X, ethidium bromide, dimethyl sulfoxide (DMSO), Tris, EDTA and all other chemicals were purchased from SISCO Research Laboratories, India. May-Gruenwald’s stain and Giemsa stain were procured from Merck Ltd. (India). 2.2. Animals and treatment Male Wistar rats weighing 100–120 g were used in the study. The animals were divided over nine groups (5 rats/group). The first group received Nnitrosodiethylamine (NDEA) dissolved in drinking-water (0.1 mg/ml), other groups received the same solution of NDEA (0.1 mg/ml) plus apigenin at 10 mg/ml (group 2), apigenin at 20 mg/ml (group 3), or apigenin at 40 mg/ml (group 4). The fifth group served as a control (normal drinking-water), and the sixth, seventh and eighth group received drinking-water with apigenin at final concentrations of 10, 20 and 40 mg/ml, respectively. The ninth group (negative control) received drinking-water containing DMSO (3 ␮l/ml). Apigenin was first dissolved in 0.03% DMSO and the final concentrations of 10, 20 and 40 mg/ml in drinking-water were established. The rats were allowed to feed ad libitum for 21 days and were sacrificed under mild ether anaesthesia. All animal care procedures and animal ethics were taken into consideration while performing the experiments. The study was approved by an ethical committee. 2.3. Histological evaluation of the liver A portion of liver was removed and washed thoroughly with 0.9% saline. The tissue was kept in 10% buffered neutral formalin (BNF) for 24 h. Then the fixed liver specimens from each group were embedded in paraffin and processed for light microscopy by staining individual sections with haematoxylin–eosin. 2.4. Biochemical analysis The blood samples were collected directly by cardiac puncture in a vacutainer with a clot activator (AKÜret, Medkit). The serum was collected for biochemical analyses of glutamic-oxaloacetic transaminase (SGOT), glutamic-pyruvic transaminase (SGPT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). The enzyme concentrations were estimated according to the method described in the commercial kits (Crest Biosystems, India).

The micronucleus assay was performed according to the method of Igarashi and Shimada [25]. The supernatant was removed and fresh homogenizing buffer was used to re-suspend the pellet. A drop of suspension was placed at one end of a precleaned, grease-free microscope slide and spread-out with a cover slip held at an angle of 45◦ to obtain a smooth layer of cells. Before staining, the slides were allowed to air-dry in a dust-free environment for at least 12 h. The slides were then stained for 2 min in May-Gruenwald stain (0.25% in methanol) followed by staining with 10% Giemsa for 10 min. The slides were rinsed twice in distilled water, dried, rinsed with methanol, cleared in xylene, and mounted in DPX. A total of 500 cells were counted per animal for the presence of micronuclei by use of a light microscope at 40× magnification [26]. 2.9. Comet assay The comet assay was performed according to the method described by Singh et al. [27], with modifications as suggested by Dhawan et al. [28]. Frosted microscope slides were dipped in 1% normal-melting agarose and the underside was wiped to remove the agarose (dissolved in PBS). The slides were allowed to dry for 24 h. For the liver cells, 40 ␮l of the cell suspension were mixed with 60 ␮l of 0.5% lowmelting agarose (dissolved in PBS) and layered on the prepared slides. For the blood lymphocytes, 20 ␮l of whole blood in 1 ml of RPMI 1640 was mixed with 100 ␮l of Ficoll histopaque and centrifuged at 1500 rpm 300 g for 15 min, and the pellet was re-suspended in 70 ␮l of LMPA. For bone-marrow cells, the femurs were perfused with 1 ml of cold homogenization buffer and 10 ␮l of the cell suspension was mixed with 70 ␮l of LMPA. A cover slip was placed on top to spread the suspension evenly. The agarose was allowed to solidify at 4 ◦ C for 10 min. The cover slip was removed and a third layer of 1% low-melting agarose was added and allowed to solidify on ice for 5 min. The slides were then immersed in cold lysis solution (2.5 M NaCl, 100 mM EDTA, 10 mM Tris and 1% triton X-100, pH 10) at 4 ◦ C, overnight. Next day the slides were kept for 30 min in alkaline electrophoresis buffer (300 mM NaOH, 1 mM EDTA; pH > 13) for unwinding of the DNA. Electrophoresis was performed at 4 ◦ C at 24 V for 45 min. The slides were washed with neutralization buffer (0.4 M Tris) and stained with ethidium bromide (20 ␮g/ml). Three slides were prepared per rat, and a total of 50 randomly captured comets per slide were analysed under a fluorescence microscope for scoring comet-tail length by use of Comet 1.5 software (TriTek Corporation). 2.10. Statistical analysis All data were expressed as the mean ± standard error and Student’s t-test was used for the analysis. Statistical significance was considered at the 5% level.

2.5. Preparation of liver homogenate

3. Results

The homogenate was prepared according to the procedure described by Singh et al. [22] with minor modifications. The livers were washed thoroughly with chilled 0.9% saline. The final wash was given with cold homogenizing buffer (0.024 M EDTA, 0.075 M NaCl, 10% DMSO; pH 7.5). After weighing, the liver was mixed, suspended in cold homogenizing buffer at a concentration of 1 g/ml, and was homogenized on ice at 300 × g. The homogenate was then centrifuged at 5500 × g for 10 min at 4 ◦ C. The

The histomorphological study of the liver sections revealed a normal structure of the hepatocytes in the control group (Fig. 1a). The rats exposed to NDEA (0.1 mg/ml) showed sinusoidal dilution, swollen and empty hepatocytes. The nuclei were also enlarged and the cytoplasm showed several tiny vacuoles, indicative of vacuolar

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Fig. 1. Microscope images of rat livers stained with haematoxylin–eosin (400× magnification) (PLN: partially lysed nuclei; PN: pyknotic nuclei; BD: ballooning degeneration of hepatocytes. (a) Control; (b) rats exposed to NDEA (0.1 mg/ml) for 21 days. (c) Rats exposed to 0.1 mg/ml of NDEA together with 10 mg/ml of apigenin, and (d) rats exposed to 0.1 mg/ml of NDEA together with 20 mg/ml of apigenin. (e) Rats exposed to 0.1 mg/ml of NDEA together with 40 mg/ml of apigenin.

degeneration in the liver cells (Fig. 1b). The rats exposed to various concentrations of apigenin together with NDEA (0.1 mg/ml) showed a normal structure of hepatocytes (Fig. 1c–e). The results obtained for various enzyme activities in the blood serum are shown in Table 1. The mean SGOT activity in the group exposed to NDEA (0.1 mg/ml) was 86 ± 1.97 U/ml, which was nearly three times the control value (p < 0.05). The rats exposed to NDEA (0.1 mg/ml) together with 10, 20 or 40 mg/ml of apigenin showed a steady decrease of mean SGOT activity, which was significant already at the lowest concentration (p < 0.05). The value at the highest concentration of apigenin was 1.2-fold the control (p < 0.05). The mean SGPT activity in the group exposed to NDEA (0.1 mg/ml) was 52 ± 1.30 U/ml, which was more than two times the control value (p < 0.05). The rats exposed to NDEA (0.1 mg/ml) together with 10, 20 or 40 mg/ml of apigenin showed a steady decrease of mean SGPT activity, which was significant already at the lowest concentration (p < 0.05). The value at the highest concentration of apigenin was

1.8 times that of the control (p < 0.05). The mean ALP activity in the group exposed to NDEA (0.1 mg/ml) was 350 ± 1.14 U/L, which was 1.84-fold the control value (p < 0.05). The rats exposed to NDEA (0.1 mg/ml) together with 10, 20 or 40 mg/ml of apigenin showed a steady decrease of mean ALP activity, which was significant already at the lowest concentration (p < 0.05). The value at the highest concentration of apigenin was 1.52-fold the control value (p < 0.05). The mean LDH activity in the group exposed to NDEA (0.1 mg/ml) was 520 ± 1.37 U/L, which was nearly 1.48-fold the control value (p < 0.05). The rats exposed to NDEA (0.1 mg/ml) together with 10, 20 or 40 mg/ml of apigenin showed a steady decrease of mean LDH activity, which was significant already at the lowest concentration (p < 0.05). The value at the highest concentration of apigenin was 1.36-fold the control (p < 0.05) (Table 1). The results obtained for the lipid-peroxidation assay are shown in Fig. 2a. Samples from rats exposed to NDEA (0.1 mg/ml) showed a mean absorbance value of 0.147 ± 0.0018 (p < 0.05) (Fig. 2a). The

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Table 1 The activity of serum-enzyme markers in different groups of rats exposed to NDEA alone and together with various amounts of apigenin. Treatments

SGOT (U/ml) (mean ± SE)

NDEA NDEA + A1 NDEA + A2 NDEA + A3 Control Negative control A1 A2 A3

86 56 42 36 30 30 29 30 31

± ± ± ± ± ± ± ± ±

1.97a 1.09a , b 1.51a , b 1.04a , b 1.09 1.41 1.34 1.26 1.00

SGPT (U/ml) (mean ± SE) 52 48 40 38 21 21 20 18 20

± ± ± ± ± ± ± ± ±

1.30a 1.14a , b 1.04a , b 1.14a , b 0.70 0.61 1.76 1.37 0.70

ALP (U/L) (mean ± SE) 350 302 301 290 190 179 182 180 178

± ± ± ± ± ± ± ± ±

1.14a 1.26a , b 0.77a , b 0.63a , b 0.31 1.13 1.04 0.83 1.26

LDH (U/L)(mean ± SE) 520 500 490 482 352 349 351 350 348

± ± ± ± ± ± ± ± ±

1.37a 0.70a , b 1.34a , b 0.89a , b 0.94 0.78 0.77 1.26 0.89

NDEA: N-nitrosodiethylamine (0.1 mg/ml); A1 = apigenin (10 mg/ml); A2 = apigenin (20 mg/ml); A3 = apigenin (40 mg/ml). SGOT: serum glutamic-oxaloacetic transaminase; SGPT: serum glutamic-pyruvic transaminase; ALP: serum alkaline phosphatase; LDH: lactate dehydrogenase: SE: standard error. Negative control: dimethyl sulfoxide (3 ␮l/ml). a Significant compared with control (p < 0.05). b Significant compared with NDEA treatment (p < 0.05).

rats exposed to NDEA (0.1 mg/ml) along with 10, 20 and 40 mg/ml of apigenin yielded mean values of 0.092 ± 0.0011, 0.088 ± 0.008 and 0.069 ± 0.0015, respectively (Fig. 2a). The control samples showed a mean absorbance value of 0.051 ± 0.0008. The results obtained for protein carbonyl content are shown in Fig. 2b. The treatment of NDEA (0.1 mg/ml) resulted in a mean absorbance value of 0.270 ± 0.001 (p < 0.05) (Fig. 2b). The exposure of rats to NDEA (0.1 mg/ml) along with 10, 20

and 40 mg/ml of apigenin showed mean absorbance values of 0.182 ± 0.001, 0.172 ± 0.0004 and 0.152 ± 0.0011, respectively (p < 0.05) (Fig. 2b). The control samples had a mean absorbance value of 0.118 ± 0.0008. The micronucleus formation in rat hepatocytes is shown in Fig. 3a. The results obtained for the micronucleus frequency in hepatocytes are shown in Fig. 3b. Exposure of rats to NDEA (0.1 mg/ml) produced a mean value of 0.036 ± 0.54/cell (p < 0.05)

Fig. 2. Lipid peroxidation (a) and protein carbonyl content (b) measured in rat liver after 21 days of treatment with NDEA alone, or together with different amounts of apigenin. NDEA: N-nitrosodiethylamine; A1 = apigenin (10 mg/ml); A2 = apigenin (20 mg/ml); A3 = apigenin (40 mg/ml); negative control: dimethyl sulfoxide (3 ␮l/ml); SE: standard error. a Significant compared with control (p < 0.05); b significant compared with NDEA treatment (p < 0.05).

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Fig. 3. (a) Hepatocytes showing micronuclei: (i) control; (ii) treated with NDEA (0.1 mg/ml). (b) Micronucleus formation in hepatocytes after 21 days of treatment of rats with N-nitroso-diethylamine alone or together with different amounts of apigenin. NDEA: N-nitrosodiethylamine; A1 = apigenin (10 mg/ml); A2 = apigenin (20 mg/ml); A3 = apigenin (40 mg/ml); negative control: dimethyl sulfoxide (3 ␮l/ml); SE: standard error. a Significant compared with control (p < 0.05); b significant compared with NDEA treatment (p < 0.05).

(Fig. 3b). The exposure of rats to NDEA (0.1 mg/ml) together with 10, 20 and 40 mg/ml of apigenin resulted in mean values of 0.019 ± 0.31, 0.016 ± 0.54 and 0.014 ± 0.70, respectively (Fig. 3b). Fig. 4 shows results of the comet assay performed on rat-liver cells, blood lymphocytes and bone-marrow cells. Samples from rats exposed to NDEA (0.1 mg/ml) produced comets with mean tail length of 99.36 ± 0.454 au (p < 0.05) (Fig. 5a). The rats exposed to NDEA (0.1 mg/ml) together with 10, 20 and 40 mg/ml of apigenin showed mean tail lengths of 54 ± 0.200, 43 ± 0.270 and 32 ± 0.288, respectively (Fig. 5a).

Fig. 5. Comet tail-length in rat hepatocytes (a), rat blood lymphocytes (b) and rat bone-marrow cells (c) after 21 days of treatment of rats with N-nitrosodiethylamine alone, and together with different doses of apigenin. NDEA: N-nitrosodiethylamine; A1 = apigenin (10 mg/ml); A2 = apigenin (20 mg/ml); A3 = apigenin (40 mg/ml); negative control: dimethyl sulfoxide (3 ␮l/ml); SE: standard error. a Significant compared with control (p < 0.05); b significant compared with NDEA treatment (p < 0.05).

Fig. 4. Comet assay performed with blood lymphocytes: (a) control; (b) treated with NDEA (0.1 mg/ml); with bone-marrow cells: (c) control; (d) treated with NDEA (0.1 mg/ml); with hepatocytes: (e) control; (f) treated with NDEA (0.1 mg/ml).

The results of the comet assay with rat blood lymphocytes are shown in Fig. 5b. Samples from rats exposed to NDEA (0.1 mg/ml) showed a mean tail length of 10 ± 0.173 (au ± SE) (p < 0.05) (Fig. 5b). Those from rats exposed to NDEA (0.1 mg/ml) together with 10, 20 and 40 mg/ml of apigenin had mean tail lengths of 8 ± 0.331, 6 ± 0.238 and 5 ± 0.300, respectively (p < 0.05) (Fig. 5b). The control samples showed a mean tail length of 2 ± 0.163. The results obtained for the comet assay performed on bonemarrow cells of rats are shown in Fig. 5c. Samples from rats exposed to NDEA (0.1 mg/ml) had a mean tail length of 25 ± 0.321 (p < 0.05) (Fig. 5c). Those from rats exposed to NDEA (0.1 mg/ml) together with 10, 20 and 40 mg/ml of apigenin showed mean tail lengths of 18 ± 0.509, 14 ± 0.282 and 13 ± 0.294, respectively (p < 0.05) (Fig. 5c). The control group showed a mean tail length of 3 ± 0.341. On the basis of above results a possible mechanism for the reduction of damage induced by NDEA by apigenin is proposed (Fig. 6). The metabolic activation of NDEA may lead to the formation of an ethyl-radical metabolite that causes toxicity as well

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Fig. 6. Possible mechanism of the metabolic activation of N-nitrosodiethyl amine (NDEA) and the protective role of apigenin.

as genotoxicity. Apigenin may enhance the antioxidant enzymes or directly scavenge the ethyl radical, thus providing protection against the toxicity of NDEA (Fig. 6). 4. Discussion The results of the present study reveal that apigenin has a protective effect against NDEA-induced toxicity in rats. Exposure of rats to 0.1 mg/ml of NDEA for 21 days showed liver damage, as was clear from the increase in the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP) and lactate dehydrogenase (LDH). The exposure of rats to NDEA along with various doses of apigenin showed a dose-dependent decrease in the levels of these enzymes, thereby conforming a protective role of apigenin against NDEA-induced liver damage. A significant increase of these enzymes in serum serum is an indication of damage in the liver plasma membrane, due to the oxidation of polyunsaturated fatty acids in the plasma membrane by ROS generated by the metabolism of NDEA [16]. The potential of apigenin to reduce the serum levels of the above-mentioned enzymes may be attributed to the free-radical scavenging potential of the flavonoids, thus preventing damage of the plasma membrane of the liver cells [29]. The increase in lipid peroxidation and protein carbonyl

content in liver after treatment with NDEA has been reported earlier [16]. They both are markers of oxidative stress. The treatment of apigenin results in a dose-dependent decrease in lipid peroxidation in the liver. This may be due to the ROS-scavenging potential of apigenin, or to the induction of antioxidant enzymes in the liver cells [30]. Protein carbonyl content indicates the protein oxidation by free radicals or ROS generated during NDEA metabolism [16]. The reduction in protein carbonyl content after treatment with apigenin suggests its ROS-scavenging property or its potential to promote the detoxification of ROS by inducing antioxidant enzymes [1]. The generation of ROS has been reported to occur through a large number of physiological and non-physiological processes, which include their production as by-products of normal cellular metabolism, primarily in the mitochondria [31]. Lipid peroxidation provides a reliable marker of free-radical generation and is indicative of membrane damage [32]. In the present study, apigenin was mixed with NDEA and the results obtained show that the damaging effects of NDEA are curtailed as the dose of apigenin increases. Treatment with apigenin at 25 mg/kg body weight for two weeks resulted in protection against NDEA-induced and phenobarbital-promoted carcinogenesis in Wistar albino rats [33]. Apigenin reduced the radiation-induced oxidative damage and inflammatory responses when administered to mice after a

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whole-body exposure to radiation [29]. Apigenin has very low intrinsic toxicity and has been recommended by the National Toxicology Program (NTP) to be included in toxicological studies. DNA is susceptible to mutagens and carcinogens that may cause changes in DNA structure leading to mutations or cell death [34]. The micronucleus assay is a useful end-point for bio-monitoring and ecotoxicology, and has found a wide application in the assessment of chromosomal damage in vitro and in vivo caused by different mutagens [35]. In the present study, micronucleus formation was evaluated in liver cells of treated and control rats. The data obtained for the MN clearly demonstrate the protective effect of apigenin against NDEA. The selected doses of apigenin did not induce MN at a significant level compared with the Mn level in untreated controls. The MN test was coupled with the comet assay. The comet assay was performed on hepatocytes, blood lymphocytes and bone-marrow cells. The selected dose of NDEA showed the DNA damage in all the three cell types. The treatment of apigenin showed a dose-dependent protective effect and reduced the DNA damage in the hepatocytes, blood lymphocytes and bone-marrow cells, as was evident from the decrease in the tail length. The metabolic activation of nitrosoamines has been reported to generate ROS, which are capable of initiating DNA damage in the cell [36]. The reduction in the mean tail length in hepatocytes, blood lymphocytes, and bone-marrow cells, and the micronucleus frequency in hepatocytes after the supplementation of apigenin clearly demonstrates the anti-genotoxic role of apigenin. The free-radical scavenging property of apigenin may be attributed to the hydroxyl groups in the 4th, 5th and 7th position of the molecule [37]. The antioxidant property has been attributed to the double bond between carbon atoms 2 and 3 of the carbon ring [38]. Plant-based diets, especially fruits and vegetables are rich in carotenoids, flavonoids, polyphenols, isoflavones, catechins and other compounds that reduce the risk of various types of cancer [39]. There are no reports to date showing adverse metabolic reactions for apigenin in vivo when consumed as part of a normal diet [2]. Most of the biological effects of apigenin in vitro as well as in vivo are due to its antioxidant effects and its role in scavenging free radicals [40]. It has been reported to modulate the genotoxic effects of nitropyrenes in Salmonella and in CHO cells [41], and its prior exposure to a carcinogenic insult showed a protective effect in murine skin and colon cancer models [42,43]. In one study it has been reported to enhance the intracellular concentration of glutathione against the oxidative stress [44]. The results from the present study clearly demonstrate that apigenin prevents NDEA-induced hepatotoxicity in rats. Oral administration of apigenin together with NDEA significantly reduced the toxic effects of NDEA. Hence, it is suggested that apigenin is not only a hepato-protective but also an anti-genotoxic agent. Conflict of interests The authors declare that there are no conflicts of interests. Acknowledgements We are gratefully thankful to Science and Engineering Research Board (SERB), Department of Science & Technology, Technology Bhavan, New Delhi, India for the sanction of the research project (No. SR/FT/LS-60/2011) to Dr. Yasir Hasan Siddique. We are also thankful to the Chairman, Department of Zoology, AMU, Aligarh for providing laboratory facilities. References [1] J.P.V. Singh, K. Selvendiran, S.M. Banu, R. Padmavathi, Protective role of apigenin on the status of lipid peroxidation and antioxidant defense against hepatocarcinogenesis in Wistar albino rats, Phytomedicine 11 (2004) 309–331.

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Protective effect of apigenin against N-nitrosodiethylamine (NDEA)-induced hepatotoxicity in albino rats.

A number of pharmacological properties have been attributed to apigenin. In the present study the effect of apigenin was investigated with respect to ...
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