International Immunopharmacology 30 (2016) 102–110

Contents lists available at ScienceDirect

International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Naringenin ameliorates inflammation and cell proliferation in benzo(a)pyrene induced pulmonary carcinogenesis by modulating CYP1A1, NFκB and PCNA expression Lakshmi Narendra Bodduluru a,⁎, Eshvendar Reddy Kasala a, Rajaram Mohanrao Madhana a, Chandana C. Barua b, Md Iftikar Hussain c, Prakash Haloi b, Probodh Borah c a b c

Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research (NIPER), Guwahati, Assam 781032, India Department of Pharmacology and Toxicology, College of Veterinary Science, Assam Agricultural University, Guwahati, Assam 781032, India State Biotech Hub, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, Assam 781022, India

a r t i c l e

i n f o

Article history: Received 20 August 2015 Received in revised form 22 November 2015 Accepted 30 November 2015 Available online xxxx Keywords: Lung cancer Benzo[a]pyrene Naringenin Chemoprevention NF-κB

a b s t r a c t Lung cancer is the major cause of cancer-related mortality and is a growing economic burden worldwide. Chemoprevention has emerged as a very effective preventive measure against carcinogenesis and several bioactive compounds in diet have shown their cancer curative potential on lung cancer. Naringenin (NRG), a predominant flavanone found in citrus fruits has been reported to possess anti-oxidative, anti-inflammatory and antiproliferative activity in a wide variety of cancer. The aim of the present study is to divulge the chemopreventive nature of NRG against benzo(a)pyrene (B[a]P) induced lung carcinogenesis in Swiss albino mice. Administration of B[a]P (50 mg/kg, p.o.) to mice resulted in increased lipid peroxidation (LPO), proinflammatory cytokines (TNFα, IL-6 and IL-1β) with subsequent decrease in activities of tissue enzymic antioxidants (SOD, CAT, GPx, GR, GST) and non-enzymic antioxidants (GSH and Vit-C). Treatment with NRG (50 mg/kg body weight) significantly counteracted all these alterations thereby showing potent anti-cancer effect in lung cancer. Moreover, assessment of protein expression by immunoblotting and mRNA expression by RT-PCR revealed that NRG treatment effectively negates B[a]P-induced upregulated expression of CYP1A1, PCNA and NF-κB. Further, the antiproliferative effect of NRG was confirmed by histopathological analysis and PCNA immunostaining in B[a]P induced mice which showed increased PCNA expression that was restored upon NRG administration. Overall, these findings substantiate the chemopreventive potential of NRG against chemically induced lung cancer in mice. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Lung cancer remains the most common cancer worldwide both in terms of incidence and mortality because of the high case fatality [1]. Globally, lung cancer is the largest contributor to new cancer diagnoses (1.82 million cases, 12.9% of total) and to death from cancer (1.6 million deaths, 19.4%) [1]. Even though the lung cancer incidence began declining in more developed regions as a result of reduced smoking, its incidence predominates in less developed regions where 58% (8 million) of the new cancer cases and 65% (5.3 million) of the total cancer deaths were reported as per the estimates of GLOBOCAN 2012 [1]. The 5-year total survival rate for lung cancer in the United States from 2004 to 2010 was 16.8%. Patients with localized disease at diagnosis have a 5-year survival rate of 54%; however, more than 57% of patients with distant metastasis at diagnosis have a dismal 5-year survival rate of 4% [2]. Although there has been some improvement in survival during ⁎ Corresponding author at: National Institute of Pharmaceutical Education and Research (NIPER)—Guwahati, Guwahati 32, Assam, India. E-mail address: [email protected] (L.N. Bodduluru).

http://dx.doi.org/10.1016/j.intimp.2015.11.036 1567-5769/© 2015 Elsevier B.V. All rights reserved.

the past few decades, the survival advances that have been realized in other common malignancies have yet to be achieved in lung cancer. Tobacco use is the principal risk factor for lung cancer, and a large proportion of all pulmonary carcinomas are attributable to the effects of cigarette smoking. The seminal report from the US Public health service estimated that the average male smoker had an approximately 9 to 10-fold risk for lung cancer, whereas heavy smokers had at least a 20-fold risk [3]. Cigarette smoke contains many potential carcinogens, including polycyclic aromatic hydro-carbons (PAHs), aromatic amines, N-nitrosamines, and other organic and inorganic compounds, such as benzene, vinyl chloride, arsenic, and chromium [3]. The prototype PAH, B[a]P is a significant pro-carcinogenic substance, which undergoes sequential metabolic activation principally by cytochrome P450 (CYP) 1A1 to generate a highly reactive carcinogenic metabolite B[a]P-7,8diol-9,10-epoxides (BPDE) [4]. BPDE is capable of forming DNA adducts as well as chromosomal aberrations by binding to the guanine residues in DNA. Failure of the normal DNA repair mechanisms to remove these DNA adducts can lead to permanent mutations, DNA strand breaks, or other genetic alterations which contribute to the process of carcinogenesis [5].

L.N. Bodduluru et al. / International Immunopharmacology 30 (2016) 102–110

Chemoprevention has been advocated as an approach to reduce lung cancers with the idea of treating in the early steps of carcinogenesis. Chemoprevention is defined as the use of either natural or synthetic agents or their combination to interfere with the development of cancer cells by preventing the DNA damage that initiates carcinogenesis or by halting the progression of premalignant cells [6]. Flavonoids, the naturally occurring dietary antioxidants have engrossed a great deal of attention in recent years for their key role in the prevention of certain chronic diseases. Flavonoids have diverse biological activities including preventing the initiation through modulation of xenobiotic metabolism and halting the progression of carcinogenesis as a result of their antiinflammatory, antioxidant, and anticancer properties [7]. Compelling data from laboratory studies, epidemiological investigations, and human clinical trials indicate that flavonoids have important effects on cancer chemoprevention and chemotherapy [4]. Naringenin (4′, 5, 7trihydroxy flavanone; NRG (Fig. 1)), a predominant flavanone found in grape and citrus fruits has a wide spectrum of pharmacological activities, including antioxidant, free radical scavenging, anti-inflammatory, immunomodulatory, anti-mutagenic and anti-carcinogenic effects [8, 9]. Previous reports demonstrate that NRG inhibits NDEA induced hepatocarcinogenesis through the downregulation of NF-κB, VEGF and MMPs, and also induce apoptosis by modulating expression of Bax, Bcl2 and Caspase-3 [10]. NRG has also shown protection against Nmethyl-N′-nitro-N-nitrosoguanidine-induced gastric carcinoma by upregulating the antioxidant defense enzymes [11,12]. Moreover, NRG acts as chemopreventive agent against colon carcinogenesis in vivo [13]. However, the effect of NRG on lung carcinogenesis is not proven till date. Hence, in the present study we investigated the chemopreventive and therapeutic efficacy of NRG against B[a]P induced lung carcinogenesis in Swiss albino mice.

2. Materials and methods 2.1. Drugs and chemicals Naringenin, B[a]P, 2-thiobarbituric acid (TBA), trichloroacetic acid, reduced glutathione (GSH), 2,4-dinitrophenylhydrazine (DNPH), 5, 5′dithiobis-2-nitrobenzoic acid, 1-chloro-2,4-dinitrobenzene (CDNB) and bovine serum albumin were obtained from Sigma Chemical Co. (St. Louis, Mo, USA). Polyvinylidene difluoride (PVDF) membrane and 1-Step Ultra TMB blotting solution were purchased from Pierce Biotechnology, USA. Primary NF-κB (rabbit polyclonal) and PCNA (rabbit polyclonal) antibodies were purchased from Santa Cruz, USA. Primary antiβ-actin (rabbit monoclonal) and CYP1A1 (rabbit polyclonal) antibodies were procured from M/s Sigma Chemical Company, USA. The HRP-goat antirabbit IgG secondary antibody was obtained from Santa Cruz, USA. All other chemicals and reagents used were of analytical grade procured from Himedia Pvt. Ltd. (Mumbai, India).

103

2.2. Animal model Adult male Swiss albino mice weighing between 20 and 25 g (6– 8 weeks old) were obtained from the central animal house facility of the institute. The animals were housed in plastic cages and maintained under standard conditions of temperature (25 ± 3 °C) and humidity (50 ± 10%) with a 12 h light–dark cycle. The animals had free access to a standard pellet diet and water ad libitum. All the procedures with animals were strictly conducted in accordance with approved guidelines by the Institutional Animal Ethical Committee regulated by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India. 2.3. Experimental design Experimental animals were randomly divided into five groups, with six mice in each group. Group I (Normal control): Mice received corn oil throughout the course of the experiment. Group II (Drug control): Mice received NRG (50 mg/kg b.wt. dissolved in corn oil) orally, thrice in a week for 16 weeks. Group III (B[a]P control): Mice treated with B[a]P (50 mg/kg b.wt. dissolved in corn oil) orally twice a week for four successive weeks. Group IV (Pre-treatment): Mice received B[a]P (as in Group III) along with NRG (as in Group II) orally. NRG treatment was started one week prior to the first dose of B[a]P administration and continued for 16 weeks. Group V (Post-treatment): Mice received B[a]P (as in Group III) but NRG treatment was started from 8th week till the end of the experiment. The dose and dosing regimens of benzo(a)pyrene and hesperetin were fixed based on previous published literature [14–17]. Mice from each group were euthanized after 16 weeks by cervical decapitation under ether anesthesia. After euthanizing, lungs were immediately excised and washed with ice-cold saline. A 10% homogenate of the washed tissue was prepared in 0.01 M phosphate buffer (pH 7.4). The homogenate was centrifuged at a speed of 12,000 ×g for 15 min in a refrigerated high-speed centrifuge at 4 °C and the supernatant collected was stored at −80 °C until analysis. 2.4. Biochemical estimations The following biochemical estimations were carried out in lung homogenate. Lipid peroxides were estimated by the method of Ohkawa et al. in which the malondialdehyde (MDA) released served as the index of LPO [18]. GSH was assayed by the method as described by Ellman [19]. Superoxide dismutase (SOD) activity was determined using SOD assay kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer specifications. Catalase (CAT) activity was assayed by the method of Sinha [20], Glutathione peroxidase (GPx) was determined by the method of Rotruck et al. [21]. Glutathione reductase (GR) was assayed by the method of Carlberg and Mannervik [22]. Glutathione-S-transferase (GST) was assayed by the method of Habig et al. [23]. Vitamin C (Vit C) was measured by the method of Omaye et al. [24]. Total protein was estimated by the method of Bradford [25]. 2.5. Estimation of pro-inflammatory cytokines

Fig. 1. Chemical structure of naringenin.

For estimation of proinflammatory cytokines (TNF-α, IL-6 and IL1β) in lung tissues, a 10% tissue homogenate was prepared with phosphate buffer saline (0.01 M, pH 7.4) containing 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). Then, the homogenates were centrifuged at 10,000 ×g for 20 min and the supernatant obtained was used for the estimation of TNF-α, IL-6 and IL-1β using mouse TNFα, IL-6 and IL-1β ELISA kits (Pierce Biotechnology, Rockford, IL, USA). The concentration of cytokines in lung tissue was expressed as ρg/mg protein.

104

L.N. Bodduluru et al. / International Immunopharmacology 30 (2016) 102–110

2.8. Lung histology and immunohistochemistry of PCNA

Table 1 Oligonucleotide primer sequences for target genes used in RT-PCR. Gene of interest CYP1A1 PCNA NFκB-p65 GAPDH

Primer sequences FP: 5′-GACCCTTACAAGTATTTGGTCGT-3′ RP: 5′-GGTATCCAGAGCCAGTAACC-3′ FP: 5′-TTTGAGGCACGCCTGATCC-3′ RP: 5′-GGAGACGTGAGACGAGTCCAT-3′ FP: 5′-CTTCCTCAGCCATGGTACCTCT-3′ RP: 5′-CAAGTCTTCATCAGCATCAAACTG-3′ FP: 5′-AGGTCGGTGTGAACGGATTTG-3′ RP: 5′-GGGGTCGTTGATGGCAACA-3′

Annealing temperature

Product size

58 °C

145 bp

60 °C

135 bp

60 °C

167 bp

60 °C

95 bp

2.6. Gene expression analysis Phenol–guanidinium thiocyanate-based TRIzol reagent was used for extraction of total RNA following the manufacturer instructions. The RNA's concentration and purity were determined by its absorbance at 260/280 nm. To amplify the cDNA, PCR reaction mixture consists of 10 μl of 2 × PCR master mix, 0.5 μl of forward and reverse primer (10 pmol/μl), 1 μl of template and nuclease free water to a total volume of 20 μl [26]. Oligonucleotide primers used for amplification were shown in Table 1. All PCR samples were denatured at 95 °C for 3 min before cycling and were extended for 10 min at 72 °C after cycling. The PCR assay using primers was performed for 30 cycles at 95 °C for 30 s, annealing temperature varies for different primers for 45 s (Table 1) and 72 °C for 45 s. GAPDH served as internal control to check for equal loading. PCR products were analyzed using the Image Lab 5.1 (Bio Rad Co).

Histopathological analyses were performed to determine the incidence of lung carcinogenesis and the activity of NRG. A portion of lung tissue was fixed in 10% buffered formalin for 48 h, dehydrated in graded series of alcohol, cleaned in xylene and embedded in paraffin to prepare the block. Sections were cut at 4–5 μm in thickness, stained with hematoxylin and eosin and examined under light microscope for histological changes. Immunohistochemistry was performed following the methods used by Ramakrishnan et al. [28]. Briefly, the tissue sections were deparaffinized first in xylene and hydrated through a graded series of alcohol, the slides were incubated in citrate buffer (pH 6.0) for three cycles of 5 min each in a microwave oven for antigen retrieval. The sections were cooled, rinsed with Tris-buffered saline (TBS) and then treated with freshly made 0.3% H2O2 in methanol for 20 min to quench endogenous peroxidase activity. After blocking with 3% BSA for 1 h, the sections were then incubated with PCNA antibody at a dilution 1:50 for 1 h in room temperature. The slides were washed with TBS gently and subsequently incubated with anti-rabbit HRP labeled secondary antibody at a 1:500 dilution for 1 h. Sections were washed with TBS and incubated for 5–10 min in TBS containing 0.02% DAB and 0.01% H2O2 for visualizing peroxidase activity. Counter staining was performed using Meyer's hematoxylin. Quantitative analysis was made in a blinded manner to illustrate the total number of positively stained cells under a light microscope. The labeling index was expressed as the number of cells with positive staining per 100 counted cells in ten randomly selected fields at high magnification (40×). 2.9. Statistical analysis

2.7. SDS-PAGE and Western blot analysis Approximately, 50 mg of lung tissue sample was subjected to lysis in RIPA buffer and the protein concentration of lysates was determined using the BCA method. Lung homogenate samples with 40 μg of total protein were mixed with an equal volume of 2× Laemmli buffer, boiled for 5 min at 95 °C, cooled, loaded on each lane of 10% polyacrylamide gel and separated by SDS-PAGE following the method described in Laemmli [27]. The resolved proteins were electrophoretically transferred to PVDF membranes (Pierce Biotechnology, Rockford, IL, USA) for immunoblot analysis. After blocking with 3% bovine serum albumin in 1× TBST for 1 h at room temperature, the membranes were incubated overnight at 4 °C with rabbit polyclonal anti-PCNA, anti-NF-κB antibody (1:500; Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit anti-CYP1A1, anti-β-actin (1:250; Sigma-Aldrich). The membranes were then washed with diluted TBST gently and subsequently incubated with appropriate secondary antibodies (goat anti-rabbit IgG) conjugated to horse-radish peroxidase at a 1:3000 dilution for 1 h. β-actin served as internal control to check the equal loading of the protein. The bands were visualized by treating the membranes with 1-Step Ultra TMB blotting solution (Pierce Biotechnology, Rockford, IL, USA). The membranes were scanned and band intensities were quantified using image analysis software, Image J (NIH, Bethesda, MD, USA). Densitometric data is presented as fold change compared with control.

All the results were expressed as mean ± S.D. The statistical significance of differences among various experimental groups was calculated by one-way ANOVA followed by Tukey's post hoc test. Analysis was performed using the statistical software Graph Pad version 5.0 (San Diego, CA, USA). Results were considered statistically significant if the p b 0.05. 3. Results 3.1. Body weight and lung weight of mice treated with B[a]P and NRG Table 2 shows the body weight, lung weight and tumor incidence of control and experimental animals. The mean body weights were significantly (p b 0.01) decreased while the lung weights were significantly increased (p b 0.01) in B[a]P-treated animals (Group-III) compared with the untreated control animals (Group-I). Pre- and post-treatment of NRG to B[a]P-treated mice significantly (p b 0.05) improved the body weight and decreased the lung weight compared to B[a]P-treated animals (Group III). Conceptually, the increase in body weight after the administration of NRG in groups IV and V mice suggests a protective effect of the flavonoid on body mass. No significant difference was found in the body weights and lung weights of control and NRG alone treated group which is indicative of nontoxic nature of NRG.

Table 2 Effect of NRG on body weight, lung weight and tumor incidence in control and experimental animals. Results are expressed as mean ± S.D. (n = 6), where **p b 0.01 vs normal control; #p b 0.05 vs B[a]P control. Parameters

Group I (vehicle control)

Group II (drug control)

Group III (B[a]P control)

Group IV (B[a]P + NRG pretreated)

Group V (B[a]P + NRG posttreated)

Number of mice examined Body weight (g) Lung weight (mg) Tumor incidence

6 31.6 ± 1.82 269 ± 15.22 0

6 32.4 ± 1.75 276 ± 12.80 0

6 22.4 ± 1.40** 368 ± 18.48** 6

6 30.1 ± 1.52# 287 ± 14.20# 1

6 28.7 ± 1.91 302 ± 17.40# 3

L.N. Bodduluru et al. / International Immunopharmacology 30 (2016) 102–110

105

Table 3 Effect of NRG on LPO, enzymic and non-enzymic antioxidants in control and experimental animals. All the values are expressed as mean ± S.D. (n = 6), where ***p b 0.001 vs normal control; ###p b 0.001, ##p b 0.01, #p b 0.05 vs B[a]P control; @@@p b 0.001, @@p b 0.01, @p b 0.05 vs posttreatment group. Units: LPO—nM of MDA released/mg protein; SOD—units/mg protein; Catalase—μM of H2O2 consumed/min/mg protein; GPx—μM of GSH oxidized/min/mg protein; GR—μM of NADPH oxidized/min/mg protein; GST—μM of CDNB conjugated/min/mg protein, GSH—μM/mg protein; Vit C—μg/mg protein. Parameters

Group I (vehicle control)

Group II (drug control)

Group III (B[a]P control)

Group IV (B[a]P + NRG pretreated)

Group V (B[a]P + NRG posttreated)

LPO SOD CAT GPx GR GST GSH Vit C

0.69 ± 0.08 6.14 ± 0.38 221 ± 13.56 8.32 ± 0.26 5.49 ± 0.47 24.96 ± 2.24 1.54 ± 0.14 0.46 ± 0.06

0.73 ± 0.09 5.98 ± 0.39 217 ± 19.2 8.16 ± 0.22 5.65 ± 0.57 25.12 ± 2.30 1.49 ± 0.18 0.48 ± 0.02

1.74 ± 0.21*** 3.74 ± 0.28*** 123 ± 12.12*** 4.51 ± 0.38*** 2.43 ± 0.34*** 14.86 ± 1.85*** 0.93 ± 0.11*** 0.30 ± 0.04***

0.82 ± 0.16## 5.22 ± 0.33###, @ 193 ± 15.2###, @@ 6.71 ± 0.31###, @@ 4.92 ± 0.30###, @@@ 21.12 ± 1.41### 1.32 ± 0.18## 0.42 ± 0.05###

1.04 ± 0.19# 4.53 ± 0.35## 156 ± 14.1## 5.95 ± 0.33### 3.56 ± 0.38### 18.23 ± 1.12# 1.20 ± 0.12# 0.37 ± 0.02#

3.2. Effect of NRG on lipid peroxidation, enzymic and non-enzymic antioxidants Table 3 represents the status of LPO, enzymic and nonenzymic antioxidants in lung tissues of control and experimental groups. A significant increase (p b 0.001) in the level of LPO was observed in the tumor bearing animals (Group III) when compared with control animals (Group I). The activities of cellular enzymatic antioxidants (SOD, CAT, GR, GPx, GST) and the levels of non-enzymatic antioxidants (GSH and Vit C) were found to be significantly decreased in Group III cancer bearing mice when compared to Group I mice. Pre- and post-treatment of NRG offered significant reduction and elevation in levels of LPO and antioxidants status respectively. However, the NRG alone treated mice (Group II) did not show marked differences when compared with the control animals (Group I).

supplementation of NRG significantly reduced the levels of inflammatory cytokines in both pre- and post-treated mice when compared with B[a]P induced mice. However, no significant changes were observed in the NRG alone-treated animals when compared with that of control animals. 3.4. RT-PCR analysis of CYP1A1, NF-κB and PCNA Fig. 3 represents the RT-PCR analysis of CYP1A1, NF-κB and PCNA. A significant (p b 0.001) increase in the gene expression of CYP1A1, NF-κB and PCNA was found in B[a]P administered animals (lane 4) compared with the control (lane 2). NRG treatment caused a significant reduction in the levels of these expressions in pretreated (lane 5) and post-treated (lane 6) mice when compared with B[a]P-treated animals (Group III). Administration of NRG alone did not significantly affect the expression of the markers analyzed compared with the control.

3.3. Effect of NRG on proinflammatory cytokines (TNF-α, IL-6 and IL-1β) 3.5. Immunoblotting analysis of CYP1A1, NF-κB and PCNA The levels of pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in lung tissues of control and experimental groups are presented in Fig. 2. These proinflammatory cytokine levels were significantly elevated in B[a]P-induced lung cancer bearing animals (Group-III) when compared with normal control animals (Group I). Conversely,

The immunoblotting analysis of CYP1A1, NF-κB and PCNA is depicted in Fig. 4. A significant increase (p b 0.001) in the expression of CYP1A1, NF-κB and PCNA was observed in the lung tissue of B[a]P-administered animals (lane 3) when compared with control (lane 1). NRG

Fig. 2. Effect of NRG on pro-inflammatory cytokines in lung tissue of control and experimental mice. All the values are expressed as mean ± S.D. for six mice in each group. ⁎⁎⁎p b 0.001 compared with the normal control group; ###p b 0.001, #p b 0.05 compared with the B[a]P group; @@@p b 0.001 compared with the posttreatment group.

106

L.N. Bodduluru et al. / International Immunopharmacology 30 (2016) 102–110

Fig. 3. Effect of NRG and B[a]P on CYP1A1, NFκB and PCNA mRNA expression in the lung of control and experimental groups of animals. (A) Lane 1-DNA marker, Lane 2-Vehicle control, Lane 3-NRG alone treated group, Lane 4-B[a]P-induced group, Lane 5-B[a]P + NRG (Pre-treated), Lane 6-B[a]P + NRG (Post-treated). GAPDH was used as a positive control to assess the equal loading of sample. (B, C and D) Quantitative data expressing the corresponding mRNA levels were assessed using densitometry and is expressed as ‘fold changes’ as compared with control. Values represent the mean ± S.D. (n = 3) in each group. Statistical significance: ⁎⁎⁎p b 0.001 compared with the normal control group; ###p b 0.001, #p b 0.01 compared with the B[a]P group; @p b 0.05 compared with the posttreatment group.

treatment caused a significant decrease in the levels of CYP1A1, NF-κB, PCNA protein expression in pre-treated mice (lane 4) and post-treated mice (lane 5) when compared to B[a]P-induced mice (lane 3). There was no significant change observed between control and the animals treated with NRG alone.

3.6. Assessment of histological changes in the lung Fig. 5 shows the histological analysis of lung section of control and experimental groups of animals. Tissue sections of control (Group-I) animals showed intact architecture with no apparent signs of any

Fig. 4. Effect of NRG and B[a]P on CYP1A1, PCNA and NFκB protein expression in the lung of control and experimental groups of mice. (A) Lanes 1–5 correspond to the lung lysate of Groups I to V respectively. β-actin was used as internal control to assess the equal loading of sample. (B, C and D) Quantitative data expressing the corresponding protein levels was assessed using NIH Image J software and is expressed as fold change compared with control. Values represent the mean ± S.D. for three mice in each group.⁎⁎⁎p b 0.001 compared with the normal control group; ###p b 0.001, ##p b 0.01 compared with the B[a]P group; @@@p b 0.001, @p b 0.05 compared with the posttreatment group.

L.N. Bodduluru et al. / International Immunopharmacology 30 (2016) 102–110

107

Fig. 5. Figure presents the histological examination of lung sections from control and experimental animals (H & E staining; 40×). Control mice lung sections showing normal architecture of alveoli with uniform nuclei (Fig. 5A). Animals treated with NRG alone showing normal architecture of alveolus and bronchioles similar to control animals (Fig. 5B). B[a]P-administered animals showed loss of architecture and alveolar damage as seen from the increased number of hyperchromatic nuclei (indicated by arrows) in the cells of alveolar wall (Fig. 5C,D). Animals pre-treated with NRG exhibited reduced alveolar damage with near normal architecture (Fig. 5E), and animals post-treated with NRG showed slightly reduced alveolar damage (Fig. 5F).

abnormality. NRG alone treated animals (Group-II) showed no appreciable change of histopathological appearance from control animals. Lung cancer bearing animals (Group-III) revealed loss of architecture and alveolar damage as seen from hyperchromatic and irregular nuclei with extensive proliferation of alveolar epithelium. Animals pre-treated with NRG (Group IV) exhibited reduced proliferative lesions with near normal architecture and animals post-treated (Group V) with NRG showed slightly reduced alveolar damage with few proliferative lesions.

3.7. Immunohistochemical analysis of PCNA Fig. 6 shows the immunohistochemical analysis of PCNA in the lungs of control and experimental groups of animals. B[a]P-administered lung cancer bearing animals (Group III) revealed a significant (p b 0.001) increase in the PCNA expression and the total number of PCNA positive cells when compared with normal control animals. Whereas, pre- and post-treatment of NRG to tumor bearing animals significantly decreased the expression of PCNA and the number of PCNA positive cells. However, control and NRG alone treated mice exhibited very low expression of this marker.

4. Discussion Cancer chemoprevention using naturally occurring phytochemicals gained a lot of attention in recent years. Epidemiological and animal studies have conclusively proved that a high intake of fruits and vegetables rich in antioxidants decreases the risk of many cancers. Flavonoids, the ubiquitous dietary phenolic antioxidants have diverse pharmacological activities such as anti-inflammatory, antiallergic, antibacterial, antioxidant, antimutagenic and anticancer properties [7]. NRG, a flavanone predominantly found in citrus fruits has received considerable attention because of its positive health effects in various disease conditions with particular interest as anticancer compound. Pharmacologically, it has anticancer, antimutagenic, anti-inflammatory, antioxidant, antiproliferative and antiatherogenic activities [6]. Although the effect of NRG against different cancer cell lines and experimental cancer models is known, to the best of our knowledge its effect against experimental lung carcinogenesis is not known till date. Therefore, this study was undertaken to assess the chemopreventive effect of NRG in B[a]P-induced lung carcinogenesis and to determine the involvement of oxidative stress and inflammation in this regard.

108

L.N. Bodduluru et al. / International Immunopharmacology 30 (2016) 102–110

Fig. 6. Immunohistochemical analysis of PCNA in the lung of control and experimental group of animals (40×). Plates A and B represent the lung sections of normal control and NRG-alone treated animals. Plate C shows augmented protein expression of PCNA in B[a]P-induced lung cancer bearing animals. Plates D and E shows reduced protein expression of PCNA in NRG preand post-treated animals respectively. Plate F shows quantitative analysis of PCNA expression, the number of stained (positive) cells/100 cells was counted across 10 fields/section. The data are expressed as mean ± SD for six mice in each group. ***p b 0.001 vs vehicle control; ###p b 0.001 vs B[a]P group; @@@p b 0.001 vs posttreatment group.

The regression in the body weight observed in cancer bearing mice could be because of cancer cachexia, anorexia, or malabsorption, which reportedly contributes to progressive wasting of host body compartments such as skeletal muscle and adipose tissue [29]. Further, an increased lung weight in B[a]P-induced animals could be due to increased incidence of lung nodules and inflammation as reported previously [30]. Supplementation with NRG in the pre- and post-treatment groups restored the body, lung weight changes and reduced the nodule incidence indicating the protective efficacy of the flavonoid. The aromatic hydrocarbon B[a]P is a potent carcinogen with an ability to induce enormous amounts of free radicals, which in turn react with lipids causing LPO [31]. MDA formed by LPO is a highly reactive electrophile, capable of interacting with DNA to form MDA–DNA adducts that induce frame shifts and base-pair substitution mutation, leading to carcinogenesis. In the present study, a significant increase in the levels of LPO was observed in the lung tissue of cancer bearing mice. However, treatment with NRG significantly decreased levels of LPO in pre- and post-treated animals. These observations clearly confirm that NRG significantly inhibits LPO thereby limiting the formation of LPO products such as MDA, suggesting the antioxidant and freeradical scavenging activity of NRG in reducing the toxic effects of B[a]P. 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, GST and GR are critical in combating the reactive oxygen species-induced cell death and tissue injury [32]. SOD catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. CAT breaks down the hydrogen peroxide produced by SOD into water and oxygen molecules. GPx also catalyzes the transformation of hydrogen peroxide and hydroperoxide to non-toxic products, thereby curtailing the quantity of cellular destruction. Thus SOD, CAT and GPx constitute a mutually supportive team of defense against the oxidative insult caused by carcinogens [33]. Glutathione reductase catalyzes the NADPH dependent reduction of glutathione disulfide (GSSG) to glutathione (GSH) thus maintaining the balance between the redox couple. GST is a crucial detoxification enzyme that catalyzes the conjugation of electrophilic compounds with GSH favoring their elimination from the body of the organism. In the present study, analysis of enzymic antioxidants SOD, CAT, GPx, GST and GR in lung tissues of cancer bearing animals showed significantly decreased activities. However, NRG supplementation significantly increased all the above antioxidants enzymes in pre- and post-treated animals which suggest the ability of NRG to scavenge the free radicals and its potential cytoprotective function against B[a]P-induced lung carcinogenesis. GSH and Vit-C comprise the non-enzymic antioxidant system that protects the cells against free radicals. GSH is an important cellular reductant which directly scavenges free radicals by donating a hydrogen

L.N. Bodduluru et al. / International Immunopharmacology 30 (2016) 102–110

atom and thereby neutralizing the hydroxyl radical. Vit C scavenges ROS generated during the metabolism of carcinogen and thus possibly protects the genetic material at the initiation and promotion stages of carcinogenesis [34]. The lowered levels of these non-enzymic antioxidants in B[a]P-induced animals might be due to excessive utilization of this antioxidant for quenching enormous free radicals produced in this condition. NRG supplementation effectively restored the depleted nonenzymic antioxidants which may be due to its potent free radical scavenging activity. Inflammation clearly plays a major role in tumor progression. The proinflammatory cytokines (TNF-α, IL-6 and IL-1β) are the major molecular players involved in the inflammation-to-cancer axis. TNF-α is a pivotal cytokine in inflammatory reactions, however, increasing evidence also suggests that TNF-α is mainly produced by cancers and can act as an endogenous tumor promoter [35]. IL-6 and IL-1β are another major proinflammatory cytokine that participates in carcinogenesis [36]. IL-6 modulates the expression of genes involved in cell cycle progression and inhibition of apoptosis, primarily via the JAK-STAT signaling pathway [37,38]. In the present study, elevated levels of proinflammatory cytokines (TNF-α, IL-6 and IL-1β) was observed in B[a]P-induced lung cancer animals. This increased level of proinflammatory cytokines in lung cancer bearing animals may perhaps be due to enhanced activity of NF-κB during lung carcinogenesis. Treatment (pre and post) with NRG reduced the proinflammatory cytokine levels in B[a]P intoxicated animals to normal. NF-κB controls the expression of hundreds of genes involved in immunity, inflammation, proliferation, and in defense against apoptosis [39]. The induction of proinflammatory cytokines (TNF-α, IL-6 and IL1β), chemokines, COX-2, iNOS, MMPs and several adhesion molecules are all regulated by the transcription factor NF-κB. Henceforth, NF-κB has been identified as a potential molecular bridge between inflammation and cancer [40]. In the present study, elevated expression of NF-κB was observed in B[a]P-induced lung cancer animals, which could be the reason for pathogenesis of lung cancer due to the generation of inflammatory responses. NRG supplementation significantly down regulated the gene and protein expression of NF-κB in the pre- and post-treated groups. B[a]P is metabolically activated by CYP1A1 to form a highly mutagenic reactive electrophile BPDE, the ultimate carcinogenic metabolite of B[a]P capable of interacting with DNA to form adducts that result in cancer [4]. In this present study a pronounced increase in the expression of CYP1A1 in B[a]P-administered animals denotes carcinogenic processes. Simultaneously, a decrease in CYP1A1 expression was noticed in the NRG pre- and post-treated animals reflecting selective regulation of CYP1A1 possibly via interacting with the aryl hydrocarbon receptor (AhR) pathway. The decrease in CYP1A1 results in the decreased metabolism of PAH and its metabolites. Thus naringenin exerts its chemopreventive properties by reducing the formation of carcinogens through inhibition of CYP1A1 which is known to be involved in carcinogen activation. Literature reports also demonstrated that NRG suppressed 2,3,7,8-tetrachloro- dibenzo-p-dioxin induced 7-ethoxyresorufin Odeethylase activity and CYP1A1 mRNA levels through antagonizing the dioxin response element binding potential of nuclear AhR [41,42]. Moreover, several phytochemicals including kaempferol [43], quercetin [44], galangin [33], curcumin [45], resveratrol [46] and sulforaphane [47] were reported to exert their chemopreventive action based on the inhibition of B[a]P binding to AhR, the binding of the AhR–ARNT– B[a]P complex to xenobiotic response elements, and the inhibition of the formation of CYP1A1-mediated carcinogenic reactive intermediates, notably BPDE. All the reports substantiate that NRG modulates CYP1A1 activity possibly through interacting with the AhR pathway. Cell proliferation is thought to play an important role in several steps of the carcinogenic process. Proliferating cell nuclear antigen (PCNA), a highly conserved nuclear protein of DNA polymerase-delta, has been found to be a useful marker to assess tumor cell proliferation and progression. Elevated expression of PCNA and the number of

109

PCNA positively stained cells in the lung of B[a]P-induced animals indicates the hyperproliferative activity of tumor cells. Decreased expression of PCNA upon NRG treatment depicts the anti-proliferative effect of NRG. The result of this study is in agreement with previous report where NRG diminished PCNA expression in NDEA-induced hepatocarcinogenesis [7]. In addition, the histopathological observations clearly indicate that NRG supplementation reduced the damage in alveolar and bronchiolar regions and showed nearly habitual architecture, which might be due to decrease in tumor formation implying the antitumor action of NRG. Whereas in B[a]P intoxicated mice we observed well differentiated signs of an increased number of hyperchromatic nuclei in the cells of alveolar wall with extensive proliferation of the alveolar epithelium. These histopathological observations were in correlation with biochemical parameters carried out in our study that further support the anticancer effect of NRG. Although, NRG exerts a wide spectrum of pharmacological activities including antioxidant, anti-inflammatory and anticancer effects, naringenin's low oral bioavailability has remained a major hurdle. NRG, like other flavonoids possess low aqueous solubility and instability in physiological medium resulting in poor bioavailability, poor permeability and extensive first pass metabolism before reaching the systemic circulation. To overcome these disadvantages and improve the efficacy and bioavailability, approaches such as the delivery of NRG using novel nanodelivery systems have to be developed [48,49]. 5. Conclusion The present study reports for the first time that NRG ameliorates B[a]P mediated oxidative stress in the lung tissues which was evidenced by the decline in lipid peroxidation, proinflammatory cytokines and enhanced antioxidant defense enzymes. Moreover, NRG exerts antiinitiating, anti-proliferative and anti-inflammatory activities through down regulation the CYP1A1, PCNA and NF-κB in the lung of B[a]P induced mice as evidenced from RT-PCR and Western blot data. The histological and immunostaining analysis made on the lung tissues substantiate that NRG inhibits cell proliferation and protects the lung architecture from B[a]P mediated oxidative damage exemplifying the protective nature of NRG. Thus the results demonstrate that NRG exerts chemopreventive effect and protects the lung from B[a]P induced toxicity. Conflict of interest The authors declare no conflicts of interest. Acknowledgements We would like to thank the Department of Pharmaceuticals, Ministry of Chemical and Fertilizers, Government of India for financial support. All authors are extremely grateful to Mr. Veera Ganesh Yerra and Prashanth Komirishetti (Research scholars, NIPER—Hyderabad) for their assistance in the completion of this work. Also, the first author expresses sincere thanks to Institutional Biotech Hub of NIPER—Guwahati for providing the laboratory facilities. References [1] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, et al., Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Cancer 136 (2015) E359–E386. [2] Howlader N, Noone AM, Krapcho M, Garshell J, Miller D, Altekruse SF et al., SEER Cancer Statistics Review, 1975–2012, National Cancer Institute. Bethesda, MD, (http://seer.cancer.gov/csr/1975_2012/) [3] C.S. Dela Cruz, L.T. Tanoue, R.A. Matthay, Lung cancer: epidemiology, etiology, and prevention, Clin. Chest Med. 32 (2011) 605–644. [4] E.R. Kasala, L.N. Bodduluru, C.C. Barua, C.S. Sriram, R. Gogoi, Benzo(a)pyrene induced lung cancer: role of dietary phytochemicals in chemoprevention, Pharmacol. Rep. 67 (5) (2015) 996–1009.

110

L.N. Bodduluru et al. / International Immunopharmacology 30 (2016) 102–110

[5] H.V. Gelboin, Benzo[alpha]pyrene metabolism, activation and carcinogenesis: role and regulation of mixed-function oxidases and related enzymes, Physiol. Rev. 60 (1980) 1107–1166. [6] L.N. Bodduluru, E.R. Kasala, N. Thota, C.C. Barua, R. Sistla, Chemopreventive and therapeutic effects of nimbolide in cancer: the underlying mechanisms, Toxicol. in Vitro 28 (2014) 1026–1035. [7] E.R. Kasala, L.N. Bodduluru, R.M. Madana, V. AK, R. Gogoi, C.C. Barua, Chemopreventive and therapeutic potential of chrysin in cancer: mechanistic perspectives, Toxicol. Lett. 233 (2015) 214–225. [8] I.A. Mir, A.B. Tiku, Chemopreventive and therapeutic potential of “naringenin”, a flavanone present in citrus fruits, Nutr. Cancer 67 (2015) 27–42. [9] Patel K, Singh GK, Patel DK. A review on pharmacological and analytical aspects of naringenin. Chin. J. Integr. Med. DOI http://dx.doi.org/10.1007/s11655-014-1960-x [10] P. Subramanian, D. Arul, Attenuation of NDEA-induced hepatocarcinogenesis by naringenin in rats, Cell Biochem. Funct. 31 (2013) 511–517. [11] G. Ekambaram, P. Rajendran, V. Magesh, D. Sakthisekaran, Naringenin reduces tumor size and weight lost in N-methyl-N′-nitro-N-nitrosoguanidine-induced gastric carcinogenesis in rats, Nutr. Res. 28 (2008) 106–112. [12] E. Ganapathy, R. Peramaiyan, D. Rajasekaran, M. Venkataraman, S. Dhanapal, Modulatory effect of naringenin on N-methyl-N′-nitro-N-nitrosoguanidine and saturated sodium chloride-induced gastric carcinogenesis in male Wistar rats, Clin. Exp. Pharmacol. Physiol. 35 (2008) 1190–1196. [13] T. Leonardi, J. Vanamala, S.S. Taddeo, L.A. Davidson, M.E. Murphy, B.S. Patil, et al., Apigenin and naringenin suppress colon carcinogenesis through the aberrant crypt stage in azoxymethane-treated rats, Exp. Biol. Med. 235 (2010) 710–717. [14] D. Sabarinathan, P. Mahalakshmi, A.J. Vanisree, Naringenin, a flavanone inhibits the proliferation of cerebrally implanted C6 glioma cells in rats, Chem. Biol. Interact. 189 (2011) 26–36. [15] K. Anand, A. Sarkar, A. Kumar, R.K. Ambasta, P. Kumar, Combinatorial antitumor effect of naringenin and curcumin elicit angioinhibitory activities in vivo, Nutr. Cancer 64 (2012) 714–724. [16] L.N. Bodduluru, E.R. Kasala, C.C. Barua, K.C. Karnam, V. Dahiya, M. Ellutla, Antiproliferative and antioxidant potential of hesperetin against benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice, Chem. Biol. Interact. 242 (2015) 345–352. [17] E.R. Kasala, L.N. Bodduluru, C.C. Barua, R.M. Madhana, V. Dahiya, M.K. Budhani, et al. Chemopreventive effect of chrysin, a dietary flavone against benzo(a)pyrene induced lung carcinogenesis in Swiss albino mice. Pharmacol. Rep. doi:http://dx.doi. org/10.1016/j.pharep.2015.08.014. [18] H. Ohkawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [19] G.L. Ellman, Tissue sulphydryl groups, Arch. Biochem. Biophys. 82 (1959) 70–77. [20] K.A. Sinha, Colorimetric assay of catalase, Anal. Biochem. 47 (1972) 389–394. [21] J.T. Rotruck, A.L. Pope, H.E. Ganther, A.B. Swanson, D.G. Hafeman, W.G. Hoekstra, Selenium: biochemical role as a component of glutathione peroxidase, Science 179 (1973) 588–590. [22] I. Carlberg, B. Mannervik, Glutathione reductase, Methods Enzymol. 113 (1985) 484–490. [23] W.H. Habig, M.J. Pabst, W.B. Jakoby, G. S-transferases, The first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130–7139. [24] S.T. Omaye, J.D. Turnbull, H.E. Sauberlich, Selected methods for the determination of ascorbic acid in animal cells, tissues, and fluids, Methods Enzymol. 62 (1979) 3–11. [25] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248–254. [26] D. Sabarinathan, A.J. Vanisree, Plausible role of naringenin against cerebrally implanted C6 glioma cells in rats, Mol. Cell. Biochem. 375 (1–2) (2013) 171–178. [27] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [28] G. Ramakrishnan, C.M. Elinos-Báez, S. Jagan, T.A. Augustine, S. Kamaraj, P. Anandakumar, et al., Silymarin downregulates COX-2 expression and attenuates hyperlipidemia during NDEA-induced rat hepatocellular carcinoma, Mol. Cell. Biochem. 313 (2008) 53–61. [29] V. Magesh, J.P. Singh, K. Selvendiran, G. Ekambaram, D. Sakthisekaran, Antitumour activity of crocetin in accordance to tumor incidence, antioxidant status, drug metabolizing enzymes and histopathological studies, Mol. Cell. Biochem. 287 (2006) 127–135.

[30] N. Ravichandran, G. Suresh, B. Ramesh, G.V. Siva, Fisetin, a novel flavonol attenuates benzo(a)pyrene-induced lung carcinogenesis in Swiss albino mice, Food Chem. Toxicol. 49 (2011) 1141–1147. [31] H.S. Kim, S.J. Kwack, B.M. Lee, Lipid peroxidation, antioxidant enzymes, and benzo[a]pyrene-Quinones in the blood of rats treated with benzo[a]pyrene, Chem. Biol. Interact. 127 (2000) 139–150. [32] C. Naveenkumar, S. Raghunandhakumar, S. Asokkumar, T. Devaki, Baicalein abrogates reactive oxygen species (ROS)-mediated mitochondrial dysfunction during experimental pulmonary carcinogenesis in vivo, Basic Clin. Pharmacol. Toxicol. 112 (2013) 270–281. [33] D. Devadoss, M. Ramar, A. Chinnasamy, Galangin, a dietary flavonol inhibits tumor initiation during experimental pulmonary tumorigenesis by modulating xenobiotic enzymes and antioxidant status, Arch. Pharm. Res. (2014), http://dx.doi.org/10. 1007/s12272-014-0330-8. [34] E. Karaoz, F. Gultekin, M. Akdogan, M. Oncu, A. Gokcimen, Protective role of melatonin and a combination of vitamin C and vitamin E on lung toxicity induced by chlorpyrifos-ethyl in rats, Exp. Toxicol. Pathol. 54 (2002) 97–108. [35] F. Balkwill, C. Joffroy, TNF: a tumor-suppressing factor or a tumor-promoting factor? Future Oncol. 6 (2010) 1833–1836. [36] S. Rose-John, H. Schooltink, Cytokines are a therapeutic target for the prevention of inflammation-induced cancers, Recent Results Cancer Res. 174 (2007) 57–66. [37] P. Anandakumar, S. Kamaraj, S. Jagan, G. Ramakrishnan, S. Asokkumar, C. Naveenkumar, et al., Capsaicin inhibits benzo(a)pyrene-induced lung carcinogenesis in an in vivo mouse model, Inflamm. Res. 61 (2012) 1169–1175. [38] H. Kai, Y. Kitadai, M. Kodama, S. Cho, T. Kuroda, M. Ito, et al., Involvement of proinflammatory cytokines IL-1β and IL-6 in progression of human gastric carcinoma, Anticancer Res. 25 (2005) 709–713. [39] N. Chandrashekar, A. Selvamani, R. Subramanian, A. Pandi, D. Thiruvengadam, Baicalein inhibits pulmonary carcinogenesis-associated inflammation and interferes with COX-2, MMP-2 and MMP-9 expressions in-vivo, Toxicol. Appl. Pharmacol. 261 (2012) 10–21. [40] E. Pikarsky, R.M. Porat, I. Stein, R. Abramovitch, S. Amit, S. Kasem, et al., NF-κB functions as a tumor promoter in inflammation-associated cancer, Nature 431 (2004) 461–466. [41] J.Y. Kim, E.H. Han, D.W. Shin, T.C. Jeong, E.S. Lee, E.R. Woo, et al., Suppression of CYP1A1 expression by naringenin in murine hepa-1c1c7 cells, Arch. Pharm. Res. 27 (2004) 857–862. [42] S.W. Allen, L. Mueller, S.N. Williams, L.C. Quattrochi, J. Raucy, The use of a highvolume screening procedure to assess the effects of dietary flavonoids on human cyp1a1 expression, Drug Metab. Dispos. 29 (2001) 1074–1079. [43] D. Puppala, C.G. Gairola, H.I. Swanson, Identification of kaempferol as an inhibitor of cigarette smoke-induced activation of the aryl hydrocarbon receptor and cell transformation, Carcinogenesis 28 (2007) 639–647. [44] N.Z. Jin, Y.P. Zhu, J.W. Zhou, L. Mao, R.C. Zhao, T.H. Fang, et al., Preventive effects of quercetin against benzo[a]pyrene-induced DNA damages and pulmonary precancerous pathologic changes in mice, Basic Clin. Pharmacol. Toxicol. 98 (2006) 593–598. [45] A. Sehgal, M. Kumar, M. Jain, D.K. Dhawan, Modulatory effects of curcumin in conjunction with piperine on benzo(a)pyrene-mediated DNA adducts and biotransformation enzymes, Nutr. Cancer 65 (2013) 885–890. [46] A. Revel, H. Raanani, E. Younglai, J. Xu, I. Rogers, R. Han, et al., Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects lung from DNA damage and apoptosis caused by benzo[a]pyrene, J. Appl. Toxicol. 23 (2003) 255–261. [47] D. Kalpana Deepa Priya, R. Gayathri, D. Sakthisekaran, Role of sulforaphane in the anti-initiating mechanism of lung carcinogenesis in vivo by modulating the metabolic activation and detoxification of benzo(a)pyrene, Biomed. Pharmacother. 65 (2011) 9–16. [48] F.L. Yen, T.H. Wu, L.T. Lin, T.M. Cham, C.C. Lin, Naringenin-loaded nanoparticles improve the physicochemical properties and the hepatoprotective effects of naringenin in orally-administered rats with CCl(4)-induced acute liver failure, Pharm. Res. 26 (2009) 893–902. [49] B.V. Bonifácio, P.B. da Silva, M.A. dos Santos Ramos, K.M.S. Negri, T.M. Bauab, M. Chorilli, Nanotechnology-based drug delivery systems and herbal medicines: a review, Int. J. Nanomedicine 9 (2014) 1–15.