Fisetin, a novel flavonol attenuates benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice

Food and Chemical Toxicology 49 (2011) 1141–1147

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Fisetin, a novel flavonol attenuates benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice Nagaiya Ravichandran, Gopalakrishnan Suresh, Balasubramanian Ramesh, Ganesan Vijaiyan Siva ⇑ Department of Biotechnology, University of Madras, Guindy Campus, Chennai - 600 025, Tamilnadu, India

a r t i c l e

i n f o

Article history: Received 6 October 2010 Accepted 4 February 2011 Available online 17 February 2011 Keywords: Lung cancer Fisetin LPO PCNA

a b s t r a c t Lung cancer is the foremost cause of cancer mortality and is a growing economic burden worldwide. Fisetin (3,7,30 ,40 -tetrahydroxyflavone), a naturally occurring flavonoid is found in vegetables and fruits possesses anti-oxidative, anti-inflammatory and anti-proliferative effects in a wide variety of cancer. In the present study it is hypothesized that fisetin may provide chemopreventive as well as chemotherapeutic effects against experimental lung carcinogenesis. The present study was designed to investigate whether fisetin confers anti-cancer action against benzo(a)pyrene [B(a)P] induced lung carcinogenesis. Treatment with fisetin significantly reduced the degree of histological lesions, restored the levels of lipid peroxidation (LPO), enzymic and non-enzymic anti-oxidants in B(a)P-induced mice. Anti-proliferative efficacy of fisetin was assessed by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) in B(a)P induced mice showed increased PCNA expression which is restored upon fisetin administration. Together, our results depicts that fisetin can be used as chemopreventive agent against lung cancer. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Lung cancer is by far the leading cause of cancer-related deaths in the developing countries. It was estimated that 1.4 million new cases would be diagnosed and approximately 1.2 million deaths every year. There are two major types of lung cancer: small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC). Large cell lung carcinoma has the worst prognosis of all NSCLCs (Garcia-Yuste et al., 2008). Carcinogens from cigarette smoke form the link between nicotine addiction and lung cancer, contributing to a tenfold increase in risk in long-term smokers compared with non-smokers (Perera et al., 2008). Tobacco contents of smoke, the polycyclic aromatic hydrocarbons (PAHs) such as B(a)P, that play a major role in induction of lung carcinogenesis (Hecht et al., 2002). B(a)P is metabolized to (±)-B(a)P-r-7,t-8-dihydrodiol-t-9,10-epoxide (BPDE), the ultimate carcinogen. BPDE isomers then bind to the hexocyclic nitrogen of deoxyguanosine in DNA via trans-addition of the C-10 position in the epoxide molecule. This adduct might also cause activation of proto-oncogenes (Sticha et al., 2000). Superoxide and hydroxyl radicals along with hydrogen peroxide (H2O2) are collectively called as reactive oxygen species (ROS). The sources of generation of ROS in cells are various metabolic reactions with the incomplete reduction of oxygen in mitochondrial electron transport chain during respiration (Halliwell and Gutter-

⇑ Corresponding author. Tel.: +91 044 22202743. E-mail address: [email protected] (G. Vijaiyan Siva). 0278-6915/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2011.02.005

idge, 2001). Superoxide radicals in turn generate hydroxyl radicals by reacting with H2O2 in the presence of free iron by classical fenton reaction (Turrens et al., 1985). Thus generated ROS can drive the cell to a pro-oxidant state, referred as oxidative stress, affects biological molecules including membrane lipids (Halliwell and Gutteridge, 2001). The lung is exposed to higher levels of oxygen than most other tissues. The intensity of ROS in the lung is increased by cigarette smoke, inflammation, pollutants, chemicals and carcinogens (Cugell and Kamp, 2004). Accumulating evidence suggests that these free radicals and electrophile mediated oxidative stress plays an important role in all stages of chemical carcinogenesis and tumorigenesis (Sun, 1990). Chemopreventive agents have the potential to reduce the lingering lung cancer risk by rendering protection against the promotion and progression of carcinogenesis and hence chemoprevention may be considered for control of the lung cancer epidemic. Several reports suggest that phenolic compounds also act as chemopreventive agents by counteracting carcinogen-induced oxidative stress (Tanaka et al., 1998; Ashokkumar and Sudhandiran, 2009). Flavonoids are low molecular weight compounds rich in seeds, citrus fruits, red wine, tea and olive oil. Flavonoids have diverse biological effects including anti-oxidant, anti-platelet, anti-thrombotic, cytoprotective, anti-allergic, anti-viral, anti-carcinogenic activities and anti-inflammatory activities (Higa et al., 2003). Fisetin (3,7,3,4-tetrahydroxyflavone), a naturally occurring flavonoid commonly found in the smoke tree (Cotinus coggygria), is also found in fruits and vegetables such as strawberry, apple, persimmon, grape, onion and cucumber (Arai et al., 2000). It exerts a wide variety of activi-

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ties, including neurotrophic, anti-oxidant, anti-inflammatory and anti-angiogenic effects (Maher, 2006). It has been reported to inhibit the proliferation of a wide variety of tumor cells, including prostate cancer (Haddad et al., 2006), liver cancer (Chen et al., 2002), colon cancer (Lu et al., 2005), and leukemia cells (Lee et al., 2002). However, to our knowledge, studies on the effect of fisetin on lung cancer remain unexplored. Hence, the present study was designed to elucidate the protective role of fisetin on B(a)P-induced lung cancer by assessing lipid peroxides (LPO), enzymic and non-enzymic anti-oxidants such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST), glutathione (GSH), vitamin C (Vit. C) and vitamin E (Vit. E). Histopathological study and immunohistochemical localization of PCNA in lung tissue was also done to validate the anti-cancer efficacy of fisetin against B(a)P induced lung carcinogenesis in Swiss albino mice. 2. Materials and methods 2.1. Chemicals benzo(a)pyrene and fisetin were purchased from M/s Sigma chemical company, USA. All other chemicals were procured from Sisco Research Laboratories Chemicals (Mumbai, India).

(Takahara et al., 1960), GPx (Rotruck et al., 1973), GR (Staal et al., 1969) and GST (Habig et al., 1974) were assayed. Non-enzymic anti-oxidants such as GSH (Moron et al., 1979), Vit. E (Desai, 1984) and Vit. C (Omaye et al., 1979) were also measured. 2.5. Histopathological evaluation Histological evaluation was performed on the lung tissues and a portion of specimen was fixed in 10% formalin and embedded in paraffin wax. Sections were cut at 4 lm in thickness, stained with hematoxylin and eosin and viewed under light microscope for histological changes. Histopathological evaluation for the lungs was performed according to (Booran et al., 1990). 2.6. Immunohistochemical analysis of PCNA Immunohistochemical analysis of PCNA was performed based on Wiethege et al. (1995) with some modifications. Paraffin embedded tissue sections of 4 lm thickness were dewaxed first in xylene and then rehydrated in graded ethanol. The slides were then blocked with 5% bovine serum albumin (BSA) in tris buffered saline (TBS) for 2 h. The sections were then incubated with primary antibody (Rabbit polyclonal IgG to mouse PCNA (Santacruz Biotech, USA), diluted 1:1000 with 3% BSA in TBS and incubated overnight at 4 °C. After washing the slides thrice with TBS, the sections were then incubated with goat anti-rabbit secondary antibody (1:2000) with 3% BSA in TBS and incubated for 2 h at room temperature. Sections were then washed with TBS and incubated for 5–10 min in a solution of 0.02% diaminobenzidine (DAB) containing 0.01% hydrogen peroxide. Counter staining was performed using hematoxylin, and the slides were visualized under a light microscope (Vision-2000, Labomed). To quantify the positive cells, scoring was done as arbitrary units as follows; 4 as intensely stained, 3 as moderately stained, 2 as mild staining, 1 as poorly stained in control and experimental groups. 2.7. Statistical analysis

2.2. Animals Male Swiss albino mice (6–8 weeks old) were used throughout the study. Animals were purchased from Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Madhavaram, Chennai, India and maintained in a controlled condition of temperature and humidity on alternatively 12 h light/dark cycles. All animals were fed standard pellet diet (M/s. Hindustan Lever Ltd., Mumbai) and water ad libitum. This study was conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and by Animal Ethics Committee Guidelines of our Institution (IAEC No.04/011/09).

2.3. Experimental protocol The experimental animals were divided into five groups, each group comprising of six animals. Group 1 served as normal control. Group 2 animals were administered with B(a)P (50 mg/kg body weight dissolved in corn oil, orally) twice a week for 4 successive weeks to induce lung cancer by 16th week. Group 3 animals were pre-treated with fisetin (25 mg/kg body weight, dissolved in 0.1% DMSO, twice a week, orally) (according to the optimum dosage fixation study) one week before the first dose of B(a)P induction and continued for 16 weeks. Group 4 animals were post-treated with fisetin (as in Group 3) from 8th week of B(a)P induction till the end of the experiment (16th week). Group 5 animals were treated with fisetin alone (as in Group 3) for 16 weeks to study the cytotoxicity (if any) induced by fisetin. The pre and post treatment groups were used to study the chemopreventive and chemotherapeutic efficacies of fisetin in the experimental animals.

2.4. Biochemical analysis At the end of the experimental period, the animals were sacrificed by cervical decapitation. Lung tissues were immediately excised, weighed and then homogenized in 0.1 M Tris–HCl buffer (pH 7.4). Both homogenate and serum were taken for the analysis as described below. Total protein was estimated by the method of Lowry et al. (1951). Lipid peroxides was estimated by the method of Ohkawa et al. (1979). Enzymic anti-oxidants such as SOD (Misra and Fridovich, 1972), CAT

All the groups of data were evaluated for statistical significance with SPSS v.10 software. Hypothesis testing methods included one way analysis of variance followed by least significant difference test. Probability values of less than 0.05 were considered to indicate statistical significance. All these results were expressed as mean ± S.D for six animals in each group.

3. Results Table 1 shows the effect of fisetin on the body weight and lung weight in control and experimental groups of animals. Induction of Lung cancer by B(a)P in mice (Group 2) resulted in loss of body weight when compared to control mice. On the contrary, the lung weight was significantly increased (p < 0.05) than that of control group of (Group 1) animals. Administration of fisetin to B(a)P-induced mice (Groups 3 & 4) showed significant (p < 0.05) increase in the body weight and lowered lung weight when compared with B(a)P induced mice (Group 2). No obvious changes were observed between the control and fisetin alone treated group which is indicative of nontoxic nature of fisetin. The effect of fisetin on the activities of enzymic anti-oxidant in lungs of control and experimental groups of animals is depicted in Table 2. The enzymic anti-oxidants such as SOD, CAT, GPx, GR and GST were found to be significantly reduced in B(a)P induced (Group 2) animals (p < 0.05). Treatment with fisetin to animals (Group 3 and 4) significantly reduced the enzymic anti-oxidants to near normal levels when compared to Group 2 animals. No adverse effect was observed in Group 5 animals. The extent of LPO in the serum and lungs of control and experimental groups of animals was analyzed for oxidative stress

Table 1 Effect of fisetin on the body weight and lung weight control and experimental group of animals. Parameters

Group 1

Group 2

Group 3

Group 4

Group 5

Body weight (g) Lung weight (mg)

26.27 ± 2.10 278 ± 22.24

19.64 ± 2.06a 316 ± 37.92a

24.18 ± 2.29b,c 289 ± 30.34b,c

22.67 ± 2.24b 297 ± 32.67b

27.35 ± 2.21 281 ± 22.76

Each value is expressed as mean ± S.D for four determinations in each experimental group. Statistical significance: p < 0.05. a Group 2 compared with Group 1. b Group 3 and Group 4 compared with Group 2. c Group 3 compared with Group 4.

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N. Ravichandran et al. / Food and Chemical Toxicology 49 (2011) 1141–1147 Table 2 Effect of fisetin on the activities of enzymic anti-oxidant in lung of control and experimental group of animals. Parameters SOD CAT GPx GST GR

Group 1 6.42 ± 0.25 219 ± 17.52 7.81 ± 0.32 25.32 ± 2.02 8.67 ± 0.35

Group 2

Group 3 a

3.25 ± 0.19 112 ± 13.44a 4.10 ± 0.24a 18.64 ± 1.86a 4.28 ± 0.33a

Group 4 b,c

5.62 ± 0.28 182 ± 18.2b,c 5.82 ± 0.29b,c 23.48 ± 2.11b,c 6.25 ± 0.31b,c

Group 5 b

4.38 ± 0.24 160 ± 17.6b 4.18 ± 0.22b 21.41 ± 1.96b 5.28 ± 0.26b

6.37 ± 0.26 220 ± 17.82 7.76 ± 0.31 25.72 ± 2.10 8.56 ± 0.34

Each value expressed as mean ± S.D for four determinations in each experimental group. SOD-units/min/mg protein, CAT-lmoles of H2O2 consumed/min/mg protein, GPxlmoles of GSH oxidized/min/mg protein, GST-lmoles of 1-chloro-2,4 dinitrobenzene conjugated/min/mg protein; GR-lmoles NADPH oxidized/min/mg protein. Statistical significance: p < 0.05. a Group 2 compared with Group 1. b Group 3 and Group 4 compared with Group 2. c Group 3 compared with Group 4.

(Fig. 1). In B(a)P induced (Group 2) animals, there was a significant (p < 0.05) increase in the levels of lipid peroxides when compared with normal control (Group 1) animals. Where as in Group 3 (fisetin pre-treated) and Group 4 (fisetin post-treated) animals there was a significant (p < 0.05) decrease in the levels of lipid peroxides when compared with tumor bearing (Group 2) animals. However,

animals treated with fisetin alone (Group 5) did not show any significant change when compared with control animals (Group 1). The effect of fisetin on the levels of non-enzymic anti-oxidant in lungs of control and experimental groups of animals is shown in Fig. 2. Non-enzymic anti-oxidants such as GSH, Vit. E and Vit. C were found to be significantly reduced in group 2 animals

Fig. 1. Effect of fisetin on LPO in lungs and serum of control and experimental groups of animals. Each value is expressed as mean ± S.D of four determinations in each experimental group. LPO, nmol of MDA released/mg protein. Statistical significance: p < 0.05. (a) Group 2 compared with Group 1. (b) Group 3 and Group 4 compared with Group 2. (c) Group 3 compared with Group 4.

Fig. 2. Effect of fisetin on the level of non-enzymic anti-oxidants in lung tissue of control and experimental groups of animals. Each value expressed as mean ± S.D of four determinations in each experimental group. GSH, Vitamin E and Vitamin C are expressed as lg/mg protein. Statistical significance: p < 0.05. (a) Group 2 compared with Group 1. (b) Group 3 and Group 4 compared with Group 2. (c) Group 3 compared with Group 4.

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Fig. 3. Histopathological observation of lung tissue viewed under light microscope. H & E staining (40  H & E) (A) Control animals showed normal architecture of the lung tissue. (B) B(a)P administrated animals showed alveolar damage and the histological appearance of lung carcinoma is also extremely variegated. In addition, many different histological patterns may be seen from hyperchromatic and irregular nuclei in the cells of alveolar wall. (C) Fisetin pre-treated cancer bearing animals showed near normal architecture. (D) Fisetin post-treated showed slightly reduced alveolar damage. (E) Drug control animals showed normal architecture as that of control animals.

(p < 0.05). Supplementation with fisetin to animals (Group 3 and 4) significantly restored the anti-oxidant enzymes activities to near normal levels compared to Group 2 animals. No adverse effect was observed between control (Group 1) and fisetin treated (Group 5) animals. The histological examination of lung section of control and experimental groups of animals related that, lungs from control (Group 1) animals showed a normal architecture of cells with small uniform nuclei (Fig. 3A). Lung cancer bearing animals (Fig. 3B) showed loss of architecture with distorted alveoli as seen from increased number of hyper chromatic nuclei in the cells of alveolar wall with extensive proliferation of alveolar epithelium (Group 2). Cancer bearing animals pre-treated (Fig. 3C) with fisetin (Group 3) exhibited reduced alveolar damage with near normal architecture (Fig. 3A). Group 4 animals post-treated with fisetin showed slightly reduced alveolar damage (Fig. 3D).

Fisetin treated animals (Group 5) (Fig. 3E) showed no appreciable change of histopathological features as that of control animals. Fig. 4 shows the immunohistochemical analysis of PCNA in the control and experimental groups of animals. Induction with B(a)P (Group 2) (Fig. 4B) increased the expression of PCNA which was observed as dense brown colour dots. Administration of fisetin (pre-treated, Group 3 and post-treated, Group 4) (Fig. 4C and D) extensively abridged the expression of PCNA. Control (Fig. 4A) and fisetin treated (Fig. 4E) mice, however, showed few positive expressions of PCNA. The quantification of PCNA is presented in Fig. 4F. In B(a)P-induced mice, there was a significant (p < 0.05) increase in PCNA formation as compared to control mice. In case of fisetin administration to B(a)P-induced mice, it significantly (p < 0.05) decreased the number of PCNA as compared to B(a)P alone induced mice (Fig. 5).

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Fig. 4. Effect of fisetin on immunohistochemical localization of PCNA in control and experimental groups of animals (A) Normal expression of PCNA (Control), (B) B(a)Pinduced animal showed extensive expression of PCNA, (C) B(a)P + Fisetin pre-treated group showed a very week expression of PCNA, (D) B(a)P + Fisetin post-treated group showed somewhat abridged expression of PCNA, (E) Fisetin alone group resembles control section, (F) Represent the bar graph of percentage of PCNA-positive cells in control and experimental groups, ? Indicating the expression of PCNA (Values are expressed as mean ± SD for six mice each group. (a) Group 2 compared with Group 1. (b) Group 2 compared with Group 3 and Group 4. (c) Group 3 compared with Group 4).

4. Discussion and conclusion Cancer chemoprevention has become an important area of cancer research. Natural flavonoids have been extensively studied as chemopreventive agent as they do not possess any side effects. Fisetin, a novel compound available in many fruits and vegetables, such as strawberry, apple, persimmon, grape, onion and cucumber (Arai et al., 2000), recently allured much attention as a developing drug in cancer prevention. ROS and organic free radical intermediates formed from many carcinogens are suggested to be involved in the initiation and progression of carcinogenic transformation (Ramakrishnan et al., 2006). The aromatic hydrocarbon B(a)P is a very effective carcinogen with ability to generate large amounts of free radicals, which in turn reacts with lipids causing LPO (Kim et al., 2000). The products of LPO include malondialdehyde,

has been reported to be involved in formation of tumors (Ramakrishnan et al., 2007). A significant increase in the levels of LPO was observed in the lung tissues of cancer bearing mice. This may be due to either a consequence of increased level of superoxide radicals, which are produced in significant amounts in response to B(a)P exposure or inhibition of free radical scavenging enzymes (Kim et al., 2000). Decreased levels of lipid peroxides were seen in fisetin treated Group 3 and 4 animals. This clearly shows that fisetin inhibits LPO thereby limiting the formation of LPO products which are involved in carcinogenesis. Anti-oxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes (Bagchi et al., 2000). The antioxidant defense system includes SODs which convert superoxide radical (O 2 ) into hydrogen peroxide (H2O2). Accumulation of excess H2O2 causes toxic effects cellular system. In this regard GPx

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Fig. 5. Schematic representation of the possible mechanism of action of Fisetin during B(a)P induced lung carcinogenesis.

and CAT converts H2O2 into water. Therefore two toxic species O 2 and H2O2 are converted into water (Li et al., 2000). Several reports have reported that, decreased activities of SOD and CAT in various carcinogenic conditions that may be due to the increased compensation of these anti-oxidant enzymes to neutralize the oxidative stress (Ramakrishnan et al., 2006). The anti-oxidant enzymes SOD, CAT, GPx and GR that are activated against cell injury also limit the effects of oxidant molecules on tissues (Lee et al., 2010). The induction of GPx and CAT, which are of central importance in the detoxification of peroxides and hydroperoxides, was measured in the lung tissue where these processes have fundamental importance (Gaetani et al., 1989). In the present study, the lung cancer bearing animals showed decrease in the activities of enzymic anti-oxidants SOD, CAT, GPx, GST and GR. Fisetin supplementation significantly increased all above enzymic anti-oxidants. This clearly shows the ability of fisetin to protect lung from oxidative damage via increasing the levels of enzymic anti-oxidants. The non-enzymic anti-oxidants such as GSH, Vit. C and E comprise a protective system in the cells against ROS. GSH is found to be present in high concentration in the cells, and it protects cells from free radical generation (Farombi et al., 2000). GSH acts directly as a free radical scavenger by donating a hydrogen atom and thereby neutralizing the hydroxyl radical. It also reduces peroxides and maintains protein thiols in the reduced state (Nwanjo and Oze, 2007). GPx uses GSH as a substrate to catalyze the reduction in organic hydroperoxide and H2O2 (Bebe and Panemangalore, 2003). Non-enzymic anti-oxidants such as vitamins have a number of biological activities including immune stimulation, scavenging free radicals and alteration in metabolic activation of carcinogens (Yeung and Or, 2007). Vitamin E scavenges free radicals to prevent lipid peroxidation of polyunsaturated fatty acids, which can act as promoters of carcinogenesis (Adly, 2010). Vitamin C scavenges ROS generated during the metabolism of carcinogen and thus possibly protects the genetic material at the initiation and promotion stages of carcinogenesis (Karaoz et al., 2002). The decreased level of these non-enzymic anti-oxidants in the B(a)P-induced animals shows the increased requirement of the cells to neutralize the developed oxidative stress. However, the animals treated with fisetin showed restoration of these enzymic and non-enzymic anti-oxidants levels which might be due to the significant inhibition of tumor burden as well as tumor incidence by fisetin. The histopathological observations clearly indicate that fisetin administration at pre-initiation stage greatly influences lung carcinogenesis by altering the efficacy at which B(a)P can initiate histological changes. Well differentiated signs of increased number of

hyperchromatic nuclei in the cells of alveolar wall with extensive proliferation of alveolar epithelium were observed in lung tissue sections due to B(a)P induction that was restored to normal in fisetin pre-treated mice. The ability of fisetin to restore the B(a)P induced histological changes, clearly indicates the anti-carcinogenic potential of this flavonoid. A previous report also suggested that rats exposed to cigarette smoke rapidly causes increased levels of cell proliferation in the epithelium and walls of bronchioles and in the walls of associated pulmonary arteries (Li et al., 2009). Fisetin ameliorated the histopathological alterations in B(a)P-induced mice probably by extenuating the levels of LPO. Thus, the histological findings clearly support the biochemical data and suggest that fisetin may play a promising anti-cancer role with respect to lung carcinogenesis. Cell proliferation plays an important role in multi-stage carcinogenesis with multiple genetic changes (Kunnumakkara et al., 2007). Increased cell proliferation has been proposed to be a biomarker of increased susceptibility to lung cancer (Kamaraj et al., 2009). Thus, over expression of PCNA observed in the current study reflects increased cell proliferation, which was confirmed by immunohistochemical analysis in lung tumors. B(a)P induced animals treated with fisetin showed a decline in the number of PCNApositive cells that in turn reflects a decrease in S phase cells and thus reduced proliferative activity. Most potential chemopreventive agents against chemical-induced lung carcinogenesis suppress cell proliferation activity through PCNA index (Tanaka et al., 2000). It was reported that natural components and major flavonoids such as morin and capsaicin suppress various cancers by altering cell proliferation (Yoshitani et al., 2001). In addition, there are several reports stating that fisetin controls cell proliferation in various experimental cancer studies (Haddad et al., 2006; Khan et al., 2008). This is in accordance with the present study which shows that the fisetin has the ability to suppress the cell proliferation, as confirmed by PCNA index. In conclusion, the present study demonstrates that the fisetin possesses potent free radical scavenging and anti-oxidant activities. From the results it is evident that fisetin is capable of protecting the lungs against oxidative damage. In addition, it also maintains the level of anti-oxidant molecules and enzymes in vivo. Further, the study clearly shows the ability of fisetin in inhibiting cell proliferation and tumor development. The preliminary studies in the present investigation depicts that fisetin effect is more pronounced when used as a chemo preventive agent against B(a)P induced lung carcinogenesis.

5. Conflict of interest The authors declare that there are no conflicts of interest.

Acknowledgments One of the author N. Ravichandran gratefully acknowledges the University Grants Commission, New Delhi, India, for the financial assistance given in the form of Junior Research Fellow.

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