Modulatory effects of catechin hydrate against genotoxicity, oxidative stress, inflammation and apoptosis induced by benzo(a)pyrene in mice

Food and Chemical Toxicology 92 (2016) 64e74

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Modulatory effects of catechin hydrate against genotoxicity, oxidative stress, inflammation and apoptosis induced by benzo(a)pyrene in mice Ayaz Shahid, Rashid Ali, Nemat Ali, Syed Kazim Hasan, Preeti Bernwal, Shekh Mohammad Afzal, Abul Vafa, Sarwat Sultana* Section of Molecular Carcinogenesis and Chemoprevention Department of Medical Elementology and Toxicology, Faculty of Science Jamia Hamdard (Hamdard University), Hamdard Nagar New Delhi 110062, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 4 February 2016 Received in revised form 24 March 2016 Accepted 24 March 2016 Available online 26 March 2016

Benzo(a)pyrene [B(a)P], a polycyclic aromatic hydrocarbon (PAH) is a strong mutagen and potent carcinogen. The aim of the present study was to investigate the efficacy of catechin hydrate against B(a)P induced genotoxicity, oxidative stress, inflammation, apoptosis and to explore its underlying molecular mechanisms in the lungs of Swiss albino mice. Administration of B(a)P (125 mg/kg b. wt., p. o.) increased the activities of toxicity markers such as LPO, LDH and B(a)P metabolizing enzymes [NADPH ecytochrome P450 reductase (CYPOR) and microsomal epoxide hydrolase (mEH)] with subsequent decrease in the activities of tissue anti-oxidant armory (SOD, CAT, GPx, GR, GST, QR and GSH). It also caused DNA damage and activation of apoptotic and inflammatory pathway by upregulation of TNF-a, IL6, NF-kB, COX-2, p53, bax, caspase-3 and down regulating Bcl-2. However, pre-treatment with catechin at a dose of 20 and 40 mg/kg significantly decreased LDH, LPO, B(a)P metabolizing enzymes and increased anti-oxidant armory as well as regulated apoptosis and inflammation in lungs. Histological results also supported the protective effects of catechin. The findings of the present studies suggested that catechin as an effective natural product attenuates B(a)P induced lung toxicity. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Benzo(a)pyrene Catechin hydrate Genotoxicity Apoptosis Histopathology

1. Introduction Polycyclic aromatic hydrocarbons (PAHs) ubiquitously present as pollutants in the environment are carcinogenic in nature €m et al., 1996). Benzo(a)pyrene [B(a)P] is classified as one (Jernstro of the PAHs by IARC. B(a)P is mostly produced from cigarette smoking or car exhausts and act as a potent carcinogen which leads to lung carcinogenesis (Humans, 2010; Yeo et al., 2014). Due to lipophilic nature it is easily absorbed through biological membranes and bioactivated via a variety of metabolic pathways (Jacques et al., 2010). B(a)P binds with the aryl hydrocarbon

Abbreviations: B(a)P, Benzo(a)pyrene; PMS, Post-mitochondrial supernatant; CYPOR, NADPHecytochrome P450 reductase; mEH, Microsomal epoxide hydrolase; GPx, Glutathione peroxidase; GSH, Reduced Glutathione; GR, Glutathione reductase; GSSG, Oxidized glutathione; GST, Glutathione-S-transferase; LDH, Lactate dehydrogenase; LPO, Lipid peroxidation; MDA, Malondialdehyde; SOD, Superoxide dismutase; ROS, Reactive oxygen species; CAT, Catalase; QR, Quinone reductase. * Corresponding author. E-mail address: [email protected] (S. Sultana). http://dx.doi.org/10.1016/j.fct.2016.03.021 0278-6915/© 2016 Elsevier Ltd. All rights reserved.

receptor (AHR) and get activated, which induces the transcription of many genes that are involved in its metabolism, including cytochrome P450 1A1 (CYP1A1). It is metabolized into epoxide which induces DNA adduct formation and reactive oxygen species (ROS) production in cells (Miller and Ramos, 2001). Due to the formation of excessive free radicals, this in turn reacts with lipids causing lipid peroxidation. An enhancement in ROS also disrupts the redox homeostasis which results in an oxidative stress (Toyokuni et al., 1995). Therefore, maintaining ROS homeostasis is critical for normal development and survival. Regular exposure of agents that damage DNA may lead to chronic inflammation which has been associated with the development of several pathological conditions such as emphysema, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and lung cancer (Baumgartner et al., 1997; Jahangir and Sultana, 2008; Young et al., 2009). In-vitro and in-vivo studies have shown that B(a)P induces genetic mutations, chromosome damage and single strand breaks in DNA (AlvarezGonzalez et al., 2011; MacLeod et al., 1991). In the light of earlier studies and literature, B(a)P shows detrimental effects on lungs in the short-term exposure and thus established as a model to study

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adverse effects on pulmonary system (Baumgartner et al., 1997; Qamar et al., 2012). Currently, scientists all over the world adopted a newer strategy which involves the use of herb and dietary agents such as flavonoids, terpenoids, and polyphenol so as to combat various kinds of pathological activities (Newman and Cragg, 2007). Natural products are known to exert their protective effects by removing free radicals and modulating antioxidant defense system and carcinogen detoxification. Many epidemiological studies have suggested that natural products rich in flavonoids has reduced risks of diabetes, neurodegenerative disorder, cardiovascular disorder including cancer (Clere et al., 2011; Fu et al., 2011; García-Lafuente et al., 2009; Hoensch et al., 2010; Mandel et al., 2008; Wang and Morris, 2007). Catechin, a member of flavan family is a mixture of chemicals such as gallocatechin gallate, epicatechin gallate, and epigallocatechin gallate (Fukuda et al., 2009). It is categories a natural phenol having antioxidant activity and found as a secondary metabolite in plants which shows free radical scavenging property by removing reactive oxygen and nitrogen species that leads to protection of lipid membranes, proteins, and nucleic acids (Lobo et al., 2010). Beneficial effects of catechin on human health have been shown and that proved by both epidemiological and in vitro studies (Goh et al., 2015; Higdon and Frei, 2003). It can decline generation of malondialdehyde level (MDA) of the platelets, signifying the protective role of peroxidative stress and prevention of platelet aggregation (Neiva et al., 1999). Catechin possesses several biological properties such as antioxidant and anti-inflammatory which have shown chemopreventive activities in animal models (Alshatwi, 2010; Caporali et al., 2004; Scaltriti et al., 2006). Several studies of pharmacological properties of catechin have also been studied, including protection against inflammation and in coronary heart disease (Middleton et al., 2000; Ogata et al., 1995; Vinson et al., 1995). Tea catechin has also shown in vitro genotoxic effects which is suggested by previous studies (Savi et al., 2006). Some studies have reported hepatic toxicity with or without any morphological changes (Takami et al., 2008; Molinari et al., 2006). Based on above studies and literature reported, we have taken catechin to check its efficacy against B(a)P induced lung damage. Till date, no reports exist on the protective efficacy of catechin against B(a)P-induced lung genotoxicity, oxidative stress, inflammation and apoptosis. 2. Materials and methods

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obtained from the Central Animal House Facility of Jamia Hamdard (Hamdard University), New Delhi. Animals were housed in polypropylene cages in groups of six mice per cage under room temperature 25 ± 2
C with a 12 h light/dark cycles and were given free access to water ad libitum. Prior to the treatment, mice were left for one week to acclimatize. All the animals used in this study were reviewed and approved (Approval ID/Project Number: 1045) by the Institutional Animal Ethical Committee, accredited by the Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Government of India.

2.3. Treatment regimen The treatment regimen of the prophylactic effect of catechin against lung toxicity was selected on the basis of previous studies (Parvez et al., 2006; Uzun and Kalender, 2013) while the dose of B(a)P was selected in accordance with (Sehgal et al., 2012). Thirty male Swiss albino mice were randomly allocated into five groups having six animals in each group (Fig. 1).  Group I: Served as control and administered normal saline (10 ml/kg b. wt.) by oral gavage for 7 days and corn oil at the dose of 10 ml/kg b. wt. orally at 7th day only.  Group II: Served as toxicant group and received a single dose of B(a)P (125 mg/kg b. wt. in corn oil) by oral gavage on the 7th day only.  Group III: Pre-treated with catechin hydrate at the dose of 20 mg/kg b. wt. by oral gavage from day 1 to day 7 and B(a)P (125 mg/kg b. wt. in corn oil) was given on 7th day orally after 2 h of the pre-treatment with catechin.  Group IV: Pre-treated with catechin hydrate at the dose of 40 mg/kg b. wt. by oral gavage from day 1 to day 7 and B(a)P (125 mg/kg b. wt. in corn oil) was given on 7th day orally after 2 h of the pre-treatment with catechin.  Group V: Received only higher dose of catechin hydrate (40 mg/ kg b. wt.) by oral gavage up to 7 days. All animals were sacrificed by cervical dislocation 24 h after the treatment regime and processed for subcellular fractionation. Lungs were collected for examination of various biochemical and others parameters. Before sacrifice, mice underwent mild ether anesthesia. Later on, blood was drawn from the retro-orbital sinus and serum was obtained.

2.1. Chemicals 2.4. DNA isolation Benzo(a)pyrene, catechin hydrate, reduced glutathione (GSH), oxidized glutathione (GSSG), NADPH, NADP, sodium pyruvate, EDTA, cytochrome c, styrene oxide, DMSO, 4,5-(p-nitrobenzyl) pyridine, thiobarbituric acid, pyrogallol, peroxidase from horseradish, dextrose, poly-L-lysine, xanthine, bovine serum albumin, mayer's haematoxylin, 5,5-dithio-bis-(2-nitrobenzoic acid), 1chloro-2, 4-dinitrobenzene and glutathione reductase (GR) were obtained from SigmaeAldrich Chemical Co. (St. Louis, MO, USA). H2O2, K2CO3, sulphosalicylic acid, perchloric acid, tri-sodium citrate, trichloroacetic acid, tween-20, Folin-Ciocalteau reagent, sodium azide, phenol red, sodium potassium tartarate, di-sodium hydrogen phosphate, copper sulphate, sodium di-hydrogen phosphate and sodium hydroxide were purchased from E. Merck Limited. 2.2. Experimental animals Male Swiss albino mice, 8e10 weeks, weighing (25e30 g) was

DNA extraction from lung tissues was done by a standard chloroform isoamyl method (Ahmad and Sultana, 2012). The quality and quantity of DNA extracted was measured using a Thermoscientific Nano drop spectrophotometer 2000c (Wilmington, DE, USA) and 1% agarose gel electrophoresis. The amount of DNA was quantitated spectrophotometrically at 260 and 280 nm.

2.5. Gel electrophoresis and DNA fragmentation The sample was mixed with 10 ml of loading solution (10 mM EDTA (pH 8.0), 1% (w/v) bromophenol blue and 40% (w/v) sucrose) preheated to 70
C. The DNA samples were loaded onto a 1.8% (w/v) agarose gel and sealed with 0.8% (w/v) low melting point agarose. The DNA fragments were separated by electrophoresis at 25 V for 12 h at 4
C in Tris Borate EDTA (TBE) buffer. The DNA was visualized using ethidium bromide and photographed using a digital camera.

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Fig. 1. Schematic representation of experimental design.

2.6. Alkaline unwinding assay

2.8. Biochemical analysis

The fluorescence of double stranded DNA was determined by placing a 100 mmol DNA sample, 2 ml SDS (0.5%) and 100 ml NaCl (25 mM) in a prechilled test tube, followed by the addition of 3 ml potassium phosphate (0.2M, pH 9) and 3 ml bisbenzamide (1 mg/ ml). All were mixed and permitted to react in the dark (15 min) and wait for fluorescence to stabilize. The fluorescence of single stranded DNA was determined as above but using a DNA sample that had been boiled for 30 min to completely unwind the DNA. NaOH (50 ml, 0.05 N) was rapidly mixed with 100 ml of a DNA sample in a pre-chilled test tube. The mixture was incubated on ice in the dark for 30 min followed by rapid addition and mixing of 50 ml HCl (0.05N). This was followed immediately by the addition of 2 ml SDS (0.5%) and the mixture was forcefully passed through a 21G needle six times. Fluorescence of the alkaline unwound DNA sample was measured as described above. The ratio between double stranded DNA to total DNA (F value) was determined as follows:

LPO was estimated by the method of (Wright et al., 1981). LDH activity has been estimated in serum by the method of (Kornberg, 1955). Glutathione reductase activity was estimated by the method of (Carlberg and Mannervik, 1975). The activity of NADPHecytochrome P450 reductase activity was determined by the method of (Guengerich et al., 2009). The activity of SOD was measured by the method of (Marklund and Marklund, 1974). The QR activity was determined by the method of (Benson et al., 1980). Glutathione-S-transferase activity was assayed by the method of (Habig et al., 1974). Microsomal epoxide hydrolase (mEH) activity was assayed by the method of (Cedrone et al., 2005). Catalase activity was measured by the method of (Claiborne, 1985). Glutathione peroxidase activity was assayed by the method of (Mohandas et al., 1984). Reduced glutathione was determined by the method of (Jollow et al., 1974). The protein concentration in all samples was determined using bovine serum albumin (BSA) as standard following the method of (Lowry et al., 1951).

F value ¼ ðauDNA  ssDNAÞ=ðdsDNA  ssDNAÞ where auDNA, ssDNA and dsDNA are the degree of fluorescence from the partially unwound, single stranded and double stranded DNA determinations, respectively (Shugart, 1988).

2.7. Preparation of post-mitochondrial supernatant and cytosolic fractions Lung tissue homogenates were prepared in chilled phosphate buffer (0.1 M, pH 7.4) using polytron homogenizer (PT-MR 3000, Kinematica AG, Littau, Switzerland) and filtered through muslin cloth. The homogenized tissue was centrifuged in different steps to obtain different fractions by methods of (Daniels et al., 1990) as follows (i) at 300  g for 5 min at 4
C to collect and discard cellular debris, (ii) at 3000  g for 10 min at 4
C to collect nuclear pellet, (iii) supernatant was centrifuged at 10,000  g for 30 min at 4
C to obtain the post-mitochondrial supernatant (PMS) and (iv) a part of PMS was ultracentrifuged at 100,000  g for 1 h at 4
C (Beckman Coulter L80, CA, USA), pellets and supernatants (cytosolic fraction) were collected and stored at 20
C along with other cell fractions for further evaluations.

2.9. Immunohistochemical staining for the detection of NF-kB, IL-6, TNF-a, COX-2, Bcl-2, p53, Bax and Caspase-3 The protective effects of catechin on B(a)P induced inflammation and apoptosis in lung tissue was assessed by immunohistochemical staining. The lung tissues were fixed in formalin and embedded in paraffin. 5 mm thickness sections of lungs were cut on to poly-lysine coated glass slides. Sections were de-paraffinized three times (5 min) in xylene followed by dehydration in graded ethanol and finally rehydrated in running tap water. For antigen retrieval, sections were boiled in 10 mM citrate buffer (pH 6.0) for 5e7 min. Sections were incubated with hydrogen peroxide for 15 min to minimize non-specific staining and then rinsed three times (5 min each) with 1  PBST (0.05% Tween-20). Blocking solution was applied for 10 min then sections were incubated with diluted rabbit polyclonal antibodies namely anti-COX-2 (dilution 1:200, Santa Cruz), anti- NF-kB (p65) (1:300, Bio legend), anti-IL-6 (1:200, ebioscience), anti-TNF-a (1:200, ebioscience), anticaspases-3 (1:300, Bio legend), anti-p53 (1:200, ebioscience), Anti-Bcl-2 (1:300, Bio legend) and anti-Bax (1:200, santa cruz) overnight at 4
C in humid chamber. Further processing was done according to the instructions of Ultra Vision plus Detection System

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Anti-Polyvalent, HRP/DAB (Ready-To-Use) staining kit (Thermo scientific system). The peroxidase complex was visualized with 3, 3’-diaminobenzidine (DAB). Lastly the slides were counterstained with hematoxylin and dried. Finally the sections were mounted with DPX and covered with cover slips. The slides were ready to be observed under microscope (BX 51 Olympus). Analysis was done at 40 magnification. 2.10. Histopathological analysis The lung tissues were excised out and fixed in 10% neutral formalin and embedded in paraffin. Sections of 5 mm thickness were cut from the middle lobe of lungs of animals of each group more or less from similar positions. The paraffin embedded tissues were then de-paraffinized using xylene and ethanol. Slides were then washed using phosphate buffer saline (PBS) and permeabilized with 0.1 M citrate, 0.1% Triton X-100 permeabilization solution. The deparaffinized sections were stained with hematoxylin and eosin. Lung sections were evaluated at 40 magnification using fluorescent microscope (Olympus).To avoid any type of bias, slides were coded and examined by histopathologist in blinded manner.

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compared to B(a)P treated Group II (Table 1). No significant difference was found in the only D2 dose (group V) when compared with control group. 4.3. Effect of pre-treatment of catechin on the CYPOR It has been observed that B(a)P treatment significantly increased in CYPOR formation (***p < 0.001) as compared to control. Treatment with catechin by both the doses (20 mg/kg b.wt. and 40 mg/ kg b.wt.) significantly decreased (#p < 0.01, ###p < 0.01) CYPOR formation in the groups (III and IV) when compared to B(a)P treated Group II (Table 1). Catechin alone exhibits no significant difference as compared to control. 4.4. Effect of catechin pre-treatment on the microsomal epoxide hydrolase activity B(a)P administration leads to significant (***p < 0.001) increased as compared to control. However, both the doses of catechin significantly decreased (***p < 0.001) in Group III and Group IV as compared with Group II. No significant change observed in the Group V as compared to Group I (Table 1).

3. Statistical analysis The data from individual groups were presented as the mean ± SD. Differences between groups were analyzed using analysis of variance (ANOVA) followed by TukeyeKramer multiple comparisons test and minimum criterion for statistical significance was set at p < 0.05 for all comparisons. 4. Results 4.1. Effect of pre-treatment of catechin on LDH activity B(a)P treated group showed significantly increase in serum LDH (***p < 0.001) when compared to control group. Catechin pretreatment significantly decreased towards the normal in Group III and Group IV (###p < 0.001, ###p < 0.001) as compared with Group II. No significant change observed in the Group V as compared to Group I (Table 1). 4.2. Effect of pre-treatment of catechin on lung MDA content MDA production in lungs was measured to show the oxidative damage in the B(a)P-induced lung injury of Swiss albino mice. A significantly increased (***p < 0.001) in the level of MDA was found in B(a)P treated mice (group II) when compared with control mice (Group I). We observed that both the doses of catechin significantly restorated membrane integrity in group (III and IV) when

4.5. Effect of catechin on enzymatic and non-enzymatic antioxidants Table 2 represents the status of enzymatic and non-enzymatic antioxidants in lung tissues of control and experimental groups. The activities of cellular enzymatic antioxidants (SOD, CAT, GR, GPx, GST, QR) and the levels of non-enzymatic antioxidant (GSH) were found to be significantly decreased in Group II mice when compared to Group I mice. Pre-treatment of catechin offered significant elevation in levels of antioxidants status. However, the catechin alone treated mice (Group V) did not show marked differences when compared with the control animals (Group I). 4.6. Effect of catechin on B(a)P induced DNA damage In the DNA alkaline unwinding assay (Fig. 2), In B(a)P treated group, F-value was significantly decreased when compared with the control group. Treatments with catechin hydrate on both the doses significant increase in the F-value. DNA damage was also evaluated in terms of smearing pattern. B(a)P treated group showed more smearing pattern as compared with control group (Fig. 3), thus validating B(a)P induced DNA damage, which may be due to ROS generation. Groups III and IV showed the protective effects of catechin against B(a)P induced DNA damage. There was no significant DNA damage in group I and group V.

Table 1 Effect of catechin hydrate and B(a)P on the activities of lipid peroxidation (LPO), lactate dehydrogenase (LDH), NADPHecytochrome P450 reductase (CYPOR) and microsomal epoxide hydrolase (mEH). Treatment groups

LPO

Group Group Group Group Group

2.9 8.17 5.6 4.02 3.66

I Vehicle only II [B(a)P only] III [Dose 1 þ B(a)P] IV [Dose 2 þ B(a)P] V [Dose 2]

LDH ± ± ± ± ±

0.49 0.12*** 0.4726## 0.322### 0.48

229.11 443.73 290.16 260.9 225.59

CYPOR ± ± ± ± ±

2.67 6.91*** 4.43### 2.23### 1.923

3.87 4.07 3.67 3.52 3.15

± ± ± ± ±

mEH 0.03 0.0176** 0.014### 0.049### 0.045

87.6 119.67 110.8 100.67 87.2

± ± ± ± ±

0.91 1.2*** 2.3## 1.42### 0.66

Values of LPO, LDH, CYPOR and mEH are expressed as mean ± S.D, (n ¼ 6). LPO was measured as nmol MDA formed/min/g tissue, LDH as nmol NADH oxidized/min/mg protein, CYPOR as measured as pmol cytochrome c reduced/min/mg protein and mEH was measured as nmol styrene oxide hydrolyzed/mg protein. Significant differences were indicated by ***p < 0.01, **p < 0.05 when compared with vehicle group (Group I) and ##p < 0.01, ###p < 0.001 when compared with B(a)P group (Group II).

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Table 2 Effects of catechin hydrate and B(a)P on the activities of glutathione peroxidase (GPx), glutathione-S-transferase (GST), glutathione reductase (GR), reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and Quinone reductase (QR). Parameters

Group I (vehicle only)

GPx GST GR GSH CAT SOD QR

1.12 220.63 391.23 0.931 77.53 89.73 161.03

± ± ± ± ± ± ±

0.1068 9.619 37.65 0.029 6.30 1.61 3.03

Group II [B(a)P only] 0.518 130.83 152.5 0.533 35.50 41.06 97.13

± ± ± ± ± ± ±

0.052*** 3.874*** .15.67*** 0.013*** 3.37*** 0.70*** 3.37***

Group III [catechin 1 þ B(a)P] 0.784 170.5 260.33 0.694 56.67 55.43 115.96

± ± ± ± ± ± ±

0.0271# 3.031## 18.77# 0.0168## 1.73# 2.25### 2.51##

Group IV [catechin 2 þ B(a)P] 0.87 189.2 384.9 0.819 65.45 75.80 135.93

± ± ± ± ± ± ±

0.0288## 2.918### 14.11### 0.026### 2.77## 1.88### 2.32###

Group V [catechin 2] 1.08 211.84 386.83 0.916 75.96 83.40 156.9

± ± ± ± ± ± ±

0.023 3.189 14.24 0.031 6.37 1.32 2.33

Values of anti-oxidant armory (GPx, GST, GR, GSH CAT, SOD and QR) are expressed as mean ± S.D, (n ¼ 6). GPx was measured as mmol NADPH oxidized/min/mg protein, GST as mmol CDNB conjugate formed/min/mg protein, GR as nmol NADPH oxidized/min/mg protein, GSH as nmol GSH/g in tissues tissue, CAT was measured as nmol H2O2 consumed/min/mg protein, SOD as units/mg protein and QR as mmol DCPIP reduced/min/mg protein. Significant differences were indicated by ***p < 0.001 when compared with vehicle group (group I) and #p < 0.05, ##p < 0.01, ###p < 0.001 when compared with B(a)P group (group II).

alveolar epithelium (Fig. 4B). Infiltration of inflammatory cells can also be seen. These results correlate with the cytotoxicity markers level. Catechin at both the doses (20 and 40 mg/kg b.wt.) showed protection against B(a)P in terms of lung histology (Fig. 4C and D). Only higher dose of catechin (Fig. 4E) of 40 mg/kg b. wt. showed normal histology was observed as in (Fig. 4A). Original magnifications 40. 4.8. Effect of catechin on the expression of NF-kB, IL-6, TNF-a, COX2, Bcl-2, p53, Bax and Caspase-3

Fig. 2. The effect of catechin on F-value. The results represent mean ± SE of six animals in each group. B(a)P treatment decreased the level of F-value in Group II significantly ***P < 0.001 as compared with control Group I. Catechin pretreatment attenuates the level of F-value #P < 0.05 and ##P < 0.01 in Group III and Group IV. There was no significant difference in the level of F-value in Group I and Group V.

4.7. Effect of catechin on the histology of the lung Oral administration of B(a)P caused disruptions of epithelium (Fig. 4B) when compared with control group (Fig. 4A). It also caused severe destruction of alveolar architecture and necrosis of the

Fig. 3. The effect of catechin on DNA damage. Agarose gel electrophoresis of DNA obtained from mouse lung. Lane 1, control; lane 2, B(a)P only; lane 3, catechin (20 mg/ kg) and B(a)P; lane 4, catechin (40 mg/kg) and B(a)P; lane 5, catechin acid only (40 mg/ kg). B(a)P treatment caused DNA fragmentation as indicated by smearing of DNA compared with the control. There was a decrease in DNA smearing as a result of catechin pretreatment at both doses.

Immunohistochemical analysis (Original magnification: 40) of NF-kB, IL-6, TNF-a, COX-2, Bcl-2, p53, Bax and Caspase-3 has been shown in Figs. 5, 6, 7, 8, 9, 10, 11 and 12 respectively. No detectable NF-kB, IL-6, TNF-a, COX-2, Bax, p53 and Caspase-3 staining was observed in the lung of control mice. However, B(a)P treated lung cell stained positive by antibodies against NF-kB, IL-6, TNF-a, COX2, Bax, p53 and Caspase-3. Catechin pretreatment significantly reduced expression in a dose dependent manner in Figs. 5, 6, 7, 8, 10, 11 and 12. Immunostaining with anti-bcl-2 antibody showed moderate expression in the control mice lung (Fig. 9). This immunoreactivity was found to be reduced in B(a)P treated group. In contrast, catechin administered mice showed positive staining and increased immunoreactivity. 5. Discussion Presently, the most significant treatment option for cancer patients is chemotherapy. Although it's therapeutic treatment usage is limited because of its severe clinical side effects (Naidu et al., 2004; Ramadori and Cameron, 2010). Flavonoids, the ubiquitous dietary phenolic antioxidants have diverse pharmacological activities such as anti-inflammatory, antiallergic, antibacterial, antioxidant, antimutagenic and anticancer properties (Kasala et al., 2015). In this study, we have examined the protective effects of catechin against B(a)P-induced lung genotoxicity, oxidative stress, inflammation and apoptosis in mice. Although mechanism underlying catechin efficacy is not fully clear. B(a)P induced lung toxicity is well documented, which leads to several pathological conditions, including cancers (Kumar et al., 2012; Yeo et al., 2014). Increased in an LPO activity is one of the toxic consequences by the B(a)P exposure and this is measured in terms of MDA levels and it acts as a mutagen and tumor promoter (Tandon et al., 2013). In the present study, B(a)P treated group of animals showed increased levels of MDA compared to control. However, pre-treatment of catechin significantly decreased the level of MDA which showed that catechin restored the intensity of antioxidants along with its anti-lipid peroxidative action. The correlation exists between

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Fig. 4. Effect of catechin against B(a)P-induced histopathological alterations. A. Showing normal histology of control mice lung. B. B(a)P (125 mg/kg b. wt.) treated group shows severe destruction of alveolar architecture and necrosis of the alveolar epithelium of the lungs. C and D. Catechin at both the doses (20 and 40 mg/kg b. wt.) showed protection against B(a)P induced lung histopathology. E. Only higher dose of catechin of 40 mg/kg b. wt. showed normal histology was observed as in (Fig. 4A). (Original Magnification 40).

cellular injuries and leakage of enzymes, as shown by the high levels of serum marker enzymes (Sehrawat and Sultana, 2006). The levels of serum toxicity marker (LDH) in our studies showed a significant increase in B(a)P treated group. Though, pre-treatment with catechin significantly restored the level towards normal. B(a)P have deleterious effects on lung tissue after its metabolic conversion to its epoxides that form DNA adducts and are carcinogenic in nature. Cytochrome P450, NADPHecytochrome P450 reductase, microsomal epoxide hydrolase (mEH), and glutathioneS-transferase (GST) are the enzymes involved in B(a)P metabolism. NADPHecytochrome P450 reductase is play important role in donating electrons via the oxidoreductase system and is essential for the biocatalytic activities of cytochrome P450 which play

central role in metabolic activation (bioactivation) of B(a)P to B(a)P €m and Gra €slund, 1994). Although mEH perform a epoxides (Jernstro protective role by hydrolyzing genotoxic epoxides, in case of B(a)P, it gives an metabolite (intermediate) that eventually get converted into benzo(a)pyrene 7,8-dihydrodiol 9,10-epoxide (BPDE) by the action of cytochrome P450. GST catalyses the of B(a)P metabolites conjugation with GSH and facilitates its detoxification and excretion (Hu et al., 1999). B(a)P induced the activities of NADPHecytochrome P450 reductase and mEH in lung tissue along with a significant reduction in GSH content and GST activity. Catechin pre-treatment significantly restored the activities of NADPHecytochrome P450 reductase, mEH, GST activity and GSH content in lung tissue.

Fig. 5. Photomicrographs depicting immunohistochemical staining of NF-KB. Effect of catechin pretreatment on B(a)P-induced NF-KB expression. Photomicrographs of lung sections depicting (A) vehicle treated control group (Group I), (B) B(a)P treated group (125 mg/kg b. wt.) (Group II), (C) dose 1 of catechin (20 mg/kg b. wt.)þB(a)P (Group III), (D) dose 2 of catechin (40 mg/kg b. wt.)þB(a)P (Group IV), and (E) only dose 2 of catechin (40 mg/kg b. wt.) (Group V). For immunohistochemical analyses, dark brown color indicates specific immunostaining of NF-KB and blue color indicates nuclear hematoxylin staining. The lung section of B(a)P treated group (Group II) has more NF-KB immunopositive staining (arrows) as indicated by brown color as compared to control group (Group I) while pretreatment of catechin in Groups III and IV reduced NF-KB immunostaining as compared to Group II. No significant difference was observed between groups V and I. Original magnification: 40. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Photomicrographs depicting immunohistochemical staining of IL-6. Effect of catechin pretreatment on B(a)P-induced IL-6 expression. Photomicrographs of lung sections depicting (A) vehicle treated control group (Group I), (B) B(a)P treated group (125 mg/kg b. wt.) (Group II), (C) dose 1 of catechin (20 mg/kg b. wt.)þ B(a)P (Group III), and (D) dose 2 of catechin (40 mg/kg b. wt.) þ B(a)P (Group IV), and (E) only dose 2 of catechin (40 mg/kg b. wt.) (Group V). For immunohistochemical analyses, brown color indicates specific immunostaining of IL-6 and blue color indicates nuclear hematoxylin staining. The lung section of B(a)P treated group (Group II) has more IL-6 immunopositive staining (arrows) as indicated by brown color as compared to control group (Group I) while pretreatment of catechin in Groups III and IV reduced IL-6 immunostaining as compared to Group II. No significant difference was observed between groups V and I. Original magnification: 40. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

A reduction in endogenous damage can indicate increased protection of DNA by catechin against free radical attack and/or increased rates of repair of damaged DNA. In our studies catechin was successful to prevent DNA fragmentation by reducing cellular injuries induced through B(a)P. The F value was also come to normal in the catechin pre-treated groups. These results have shown that catechin may also protect against B(a)P-induced lung toxicity. We found some smear of DNA in only catechin hydrate group (group V) as compared to control group (group I), showing non-significant genotoxicity which is also reported in previous

studies (Savi et al., 2006). Cells have different antioxidant systems to defend against the toxic effects of oxygen-derived species. Cellular and sub-cellular antioxidants such as SOD, CAT, GPx, QR and GR are critical in prevention of reactive oxygen species-induced cell death and tissue injury. The activities of these enzymes were depleted in the B(a)P treated group but pre-treatment with catechin have shown amelioration in these activities. Previous findings matched with the outcome of our study which showed that the antioxidant enzyme activities decreased in lung tissue after B(a)P treatment (Kamaraj

Fig. 7. Photomicrographs depicting immunohistochemical staining of TNF-a. Effect of catechin pretreatment on B(a)P-induced TNF-a expression. Photomicrographs of lung sections depicting (A) vehicle treated control group (Group I), (B) B(a)P treated group (125 mg/kg b. wt.) (Group II), (C) dose 1 of catechin (20 mg/kg b. wt.)þ B(a)P (Group III), and (D) dose 2 of catechin (40 mg/kg b. wt.)þ B(a)P (Group IV), and (E) only dose 2 of catechin (40 mg/kg b. wt.) (Group V). For immunohistochemical analyses, dark brown color indicates specific immunostaining of TNF-a and blue color indicates nuclear hematoxylin staining. The lung section of B(a)P treated group (Group II) has more TNF-a immunopositive staining (arrows) as indicated by brown color as compared to control group (Group I) while pretreatment of catechin in Groups III and IV reduced TNF-a immunostaining as compared to Group II. No significant difference was observed between groups V and I. Original magnification: 40Х. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 8. Photomicrographs depicting immunohistochemical staining of COX-2. Effect of catechin pretreatment on B(a)P-induced COX-2 expression. Photomicrographs of lung sections depicting (A) vehicle treated control group (Group I), (B) B(a)P treated group (125 mg/kg b. wt.) (Group II), (C) dose 1 of catechin (20 mg/kg b. wt.)þ B(a)P (Group III), and (D) dose 2 of catechin (40 mg/kg b. wt.)þ B(a)P (Group IV), and (E) only dose 2 of catechin (40 mg/kg b. wt.) (Group V). For immunohistochemical analyses, dark brown color indicates specific immunostaining of COX-2 and blue color indicates nuclear hematoxylin staining. The lung section of B(a)P treated group (Group II) has more COX-2 immunopositive staining (arrows) as indicated by brown color as compared to control group (Group I) while pretreatment of catechin in Groups III and IV reduced COX-2 immunostaining as compared to Group II. No significant difference was observed between groups V and I. Original magnification: 40. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

et al., 2009). The p53 protein acts as a tumor suppressive protein and has a role as transcription factor to regulate the cell cycle, DNA repair and apoptosis (Riley et al., 2008). In the cell, p53 level maintained by the ubiquitin mediated proteasomal degradation and Mdm-2 act as a corepressor (Haupt et al., 1997). p38MAPK activated p53 by phosphorylation in response to DNA damage and therefore, the cellular level of p53 protein increases (Maya et al., 2001). p53 causes apoptosis by cytochrome c release from mitochondrial leading to caspases activation (Scheller et al., 2011). Activation of caspase-3

causes DNA fragmentation and cleavage of specific cellular proteins such as actin, lamin, poly (ADP-ribose) polymerase (PARP) and fodrin in apoptosis (Sakahira et al., 1998). However, in some studies it has been reported that p53 also requires Bax to induced apoptosis (Chipuk et al., 2003). Granzyme B (GzmB) also prompts apoptosis in the presence of Bax (Heibein et al., 2000), but over expression of anti-apoptotic protein such as Bcl-2 blocked apoptosis (Pinkoski et al., 2001). In the current study, B(a)P increased expression of p53, Bax and Caspase-3, however the Bcl-2 expression was decreased as compared to control which was regulated with

Fig. 9. Photomicrographs depicting immunohistochemical staining of Bcl-2. Effect of catechin pretreatment on B(a)P-induced Bcl-2 expression. Photomicrographs of lung sections depicting (A) vehicle treated control group (Group I), (B) B(a)P treated group (125 mg/kg b. wt.) (Group II), (C) dose 1 of catechin (20 mg/kg b. wt.)þ B(a)P (Group III), and (D) dose 2 of catechin (40 mg/kg b. wt.)þ B(a)P (Group IV), and (E) only dose 2 of catechin (40 mg/kg b. wt.) (Group V). For immunohistochemical analyses, dark brown color indicates specific immunostaining of Bcl-2 and blue color indicates nuclear hematoxylin staining. The lung section of B(a)P treated group (Group II) has more Bcl-2 immunopositive staining (arrows) as indicated by brown color as compared to control group (Group I) while pretreatment of catechin in Groups III and IV reduced Bcl-2 immunostaining as compared to Group II. No significant difference was observed between groups V and I. Original magnification: 40. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 10. Photomicrographs depicting immunohistochemical staining of p53. Effect of catechin pretreatment on B(a)P-induced p53 expression. Photomicrographs of lung sections depicting (A) vehicle treated control group (Group I), (B) B(a)P treated group (125 mg/kg b. wt.) (Group II), (C) dose 1 of catechin (20 mg/kg b. wt.)þ B(a)P (Group III), and (D) dose 2 of catechin (40 mg/kg b. wt.)þ B(a)P (Group IV), and (E) only dose 2 of catechin (40 mg/kg b. wt.) (Group V). For immunohistochemical analyses, brown color indicates specific immunostaining of p53 and blue color indicates nuclear hematoxylin staining. The lung section of B(a)P treated group (Group II) has more p53 immunopositive staining (arrows) as indicated by brown color as compared to control group (Group I) while pretreatment of catechin in Groups III and IV reduced p53 immunostaining as compared to Group II. No significant difference was observed between groups V and I. Original magnification: 40. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

catechin pre-treatment in a dose-dependent manner. These results further supported the involvement of apoptosis in lung damage and oxidative stress in B(a)P induced toxicity. Inflammation is an adaptive reaction that is generated by toxic stimulus and conditions like infection and injuries (Majno and Joris, 1996; Kumar et al., 2003). It is favorable if the inflammation response is in controlled manner like in providing protection against infection, but it can be damaging if dysregulated such as septic shock. It also plays an important role in lung toxicity induced

by the B(a)P leading to ROS production, hence causes oxidative stress which is closely connected with carcinogenesis (Anand et al., 2013). NF-kB and COX-2 are the important enzymes which have key positions in inflammatory signaling pathways. NF-kB activation causes transcriptional up-regulation of COX-2 and proinflammatory cytokines, such as IL-6 and TNF-a (Wu et al., 2008). In our study, we have observed high levels of TNF-a, IL-6, COX-2 and NF-kB immunopositive staining in B(a)P treated group as compared to control while catechin significantly ameliorated expression of

Fig. 11. Photomicrographs depicting immunohistochemical staining of Bax. Effect of catechin pretreatment on B(a)P-induced Bax expression. Photomicrographs of lung sections depicting (A) vehicle treated control group (Group I), (B) B(a)P treated group (125 mg/kg b. wt.) (Group II), (C) dose 1 of catechin (20 mg/kg b. wt.)þ B(a)P (Group III), and (D) dose 2 of catechin (40 mg/kg b. wt.)þB(a)P (Group IV), and (E) only dose 2 of catechin (40 mg/kg b. wt.) (Group V). For immunohistochemical analyses, dark brown color indicates specific immunostaining of Bax and color indicates nuclear hematoxylin staining. The lung section of B(a)P treated group (Group II) has more Bax immunopositive staining (arrows) as indicated by brown color as compared to control group (Group I) while pretreatment of catechin in Groups III and IV reduced Bax immunostaining as compared to Group II. No significant difference was observed between groups V and I. Original magnification: 40. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 12. Photomicrographs depicting immunohistochemical staining of caspases-3. Effect of catechin pretreatment on B(a)P-induced caspases-3 expression. Photomicrographs of lung sections depicting (A) vehicle treated control group (Group I), (B) B(a)P treated group (125 mg/kg b. wt.) (Group II), (C) dose 1 of catechin (20 mg/kg b. wt.)þ B(a)P (Group III), and (D) dose 2 of catechin (40 mg/kg b. wt.)þB(a)P (Group IV), and (E) only dose 2 of catechin (40 mg/kg b. wt.) (Group V). For immunohistochemical analyses, dark brown color indicates specific immunostaining of caspases-3 and blue color indicates nuclear hematoxylin staining. The lung section of B(a)P treated group (Group II) has more caspases-3 immunopositive staining (arrows) as indicated by brown color as compared to control group (Group I) while pretreatment of catechin in Groups III and IV reduced caspases-3 immunostaining as compared to Group II. No significant difference was observed between groups V and I. Original magnification: 40. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

these inflammatory markers which supported its anti-oxidative and anti-inflammatory properties. In the similar way, the histological findings supported the above results and shown that the catechin protect against B(a)P induced lung toxicity. Oral administration of B(a)P induced distorted lung architecture and induced necrosis of the alveolar epithelium when compared with the control group. These results associated with the cytotoxicity markers like LPO and LDH support as well as the other parameters in the present investigation that catechin at both the doses (20 and 40 mg/kg b. wt.) showed protection against B(a)P induced lung toxicity. We have also found non-significant changes in histology in lungs of mice in only catechin hydrate group (group V) as compared to control group (group I). Higher dose of catechin showed some toxicity which is also reported in previous studies (Takami et al., 2008; Molinari et al., 2006).

6. Conclusion The findings of the present study concluded that genotoxicity, oxidative stress, inflammation and apoptosis are directly connected with B(a)P-induced lung toxicity. Our results have shown protective efficacy of catechin hydrate as a lung protective agent against B(a)P possibly via attenuating tissue damage and might be of great interest clinically. However, the precise mechanism of action of catechin is not completely defined yet. Therefore, further investigations are needed to explain the exact mechanism of action of catechin.

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

Acknowledgment The authors acknowledge the help provided by University Grants Commission (UGC), New Delhi, India for carrying out this research work.

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