Gossypetin ameliorates ionizing radiation-induced oxidative stress in mice liver--a molecular approach.

Abstract Radioprotective action of gossypetin (GTIN) against gamma (γ)-radiation-induced oxidative stress in liver was explored in the present article. Our main aim was to evaluate the protective efficacy of GTIN against radiation-induced alteration of liver in murine system. To evaluate the effect of GTIN, it was orally administered to mice at a dose of 30 mg/kg body weight for three consecutive days prior to γ-radiation at a dose of 5 Gy. Radioprotective efficacy of GTIN were evaluated at physiological, cellular, and molecular level using biochemical analysis, comet assay, flow cytometry, histopathology, immunofluorescence, and immunoblotting techniques. Ionizing radiation was responsible for augmentation of hepatic oxidative stress in terms of lipid peroxidation and depletion of endogenous antioxidant enzymes. Immunoblotting and immunofluorescence studies showed that irradiation enhanced the nuclear translocation of nuclear factor kappa B (NF-κB) level, which leads to hepatic inflammation. To investigate further, we found that radiation induced the activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK)-mediated apoptotic pathway and deactivation of the NF-E2-related factor 2 (Nrf2)-mediated redox signaling pathway, whereas GTIN pretreatment ameliorated these radiation-mediated effects. This is the novel report where GTIN rationally validated the molecular mechanism in terms of the modulation of cellular signaling system’ instead of ‘ This is the novel report where GTIN is rationally validated in molecular terms to establish it as promising radioprotective agents. This might be fruitful especially for nuclear workers and defense personnel assuming the possibility of radiation exposure.


Introduction
Gamma ( γ ) radiation exerts its hazardous impacts on living beings through the generation of harmful reactive oxygen species (ROS). It induces cellular DNA damage leading to mutation and chromosomal damage [1,2]. Eventually, this leads to oxidation of proteins, and lipids with subtle and profound biological consequences [3]. γ -radiation creates oxidative stress in body mainly by radiolysis of water. It produces hydroxyl radical (OH • ) which in turn initiates the generation of other ROS-like superoxide anion (O 2 • Ϫ ), hydrogen radical (H • ), hydroperoxyl (HO 2 • ) radical, hydrated electron (e aq Ϫ ), hydronium ion (H 3 O ϩ ), and hydrogen peroxide (H 2 O 2 ) both in the extracellular and intracellular fl uid [4].
Several synthetic compounds like lipoic acid, deoxyspergualin, cysteine, cysteamine, and 2-mercaptopropionylglycine  were tested and recognized to be extraordinarily eff evtive radioprotectors [5 -8]. However, in most of the cases, they exert adverse side eff ects; therefore, the safety of such synthetic radioprotectors has not been much of success. Notwishstanding the above, the cally active organ and should refl ect any systemic derangement upon radiation. The other major aspect of selection of liver is that liver cell regeneration is very slow and thus protection of the liver from any injury would remain a benchmark [16,17].
Thus, the present article is an evidence-based study where GTIN, a potential phytochemical, has prevented the radiation-induced eff ects in physiological as well as molecular levels. Thus, GTIN can be a safe candidate as radioprotectors in clinical uses. However, its application requires further evaluation through clinical trials.

Animals
Swiss albino male mice ( Mus musculus ), 6 -7 weeks old with body weight of 25 Ϯ 2 g, were purchased from Chittaranjan National Cancer Institute, Kolkata, India. They were maintained according to the guidelines set by the Institutional Animal Ethics Committee, maintained under controlled conditions of temperature (23 Ϯ 2 ° C), humidity (50 Ϯ 5%), and a 12-h light -dark cycle. Animals were given standard mice feed and water ad libitum . The care and use of the animals reported in this study was approved by the Institutional Animal Ethics Committee of the University of Calcutta, Kolkata, India (IAEC/proposal/ SD-4/2011SD-4/ dated 4.4.2011.

Irradiation
Mice were irradiated with 60 Co source of γ -radiation at Saha Institute of Nuclear Physics, Kolkata, India. Unanesthetized animals were preserved in well-ventilated perspex boxes and they were exposed to whole-body γ -radiation (5 Gy), at a dose rate of 1 Gy/min and a source-to-surface distance of 77.5 cm.

Experimental design
Swiss albino male mice were chosen from congenital colony and divided into four groups of eight animals each. The dose (30 mg/kg body weight) was selected after the survivality study by applying diff erent doses of GTIN and determining the optimum protective dose. (Supplementary Figure 1 to be found online at http://informahealthcare.com/doi/abs /10.3109/10715762.2015.1053878. ) These groups were Control group: The mice of the control group were given distilled water through oral gavages once a day for three consecutive days.
IR group: Mice were given distilled water for three consecutive days before exposing them to a single dose of 5-Gy 60 Co γ -irradiation.
GTIN group: GTIN was administered in mice (30 mg/kg body weight) orally for three consecutive days.
GTIN ϩ IR group: GTIN was administered in mice (30 mg/kg body weight) orally for three consecutive days. One hour after the administration of the last dose, the animals were exposed to a single dose of 5-Gy γ -irradiation. All the animals were necropsied by cervical dislocation at 6 h of post-irradiation. The liver and serum were collected for the further experiments.

Isolation of hepatocytes from mice liver
Mice were sacrifi ced by cervical dislocation. Single cell of hepatocytes were isolated using the procedure described earlier [15,19] with slight modifi cation. Briefl y, the livers were mechanically disrupted by grinding with a syringe plunger on a cell strainer with 70-μ m nylon mesh. Blood cells were lysed with a hypotonic red blood cell (RBC) lysis buff er (2.42 g Tris and 7.56 g ammonium chloride/L) in sterile distilled water, pH adjusted to 7.2), and a singlecell suspension was made in the complete RPMI 1640 medium supplemented with 100 Unit/mL penicillin and 100 μ g/mL of streptomycin with 10% fetal bovine serum. The cell density used was 2.5 ϫ 10 5 cells/mL; the viability of freshly isolated cells was consistently above 90% as indicated by a Trypan blue exclusion test.

Measurement of intracellular ROS
Intracellular accumulation of ROS level was evaluated after incubation of hepatocytes with a membranepermeable fl uorescent probe, DCFDA [15,20]. Briefl y, the hepatocytes were incubated with PBS with saturating concentrations of DCFDA (1 μ g/mL) in the dark for 30 min at room temperature. The fl uorescence signal was measured using 480-nm excitation and 530-nm emission light on a FACS caliber instrument (BD Bioscience, Mountain View, CA, USA). For each sample, autofl uorescence signal of unstained hepatocytes was measured and used to adjust the fl uorescence intensity of DCFDA-stained hepatocytes. Data were analyzed using Flow Jo software (version 7.6.5) attached with the fl ow cytometer. Analysis of fl uorescence channel-1 (FL-1) fl uorescence was performed with gating on the total unstained hepatocyte population to identify the live hepatocyte population. The same gate was used for all the samples to measure the FL-1 fl uorescence intensity.

Biochemical estimations
Lipid peroxidation . Thiobarbituric acid-reactive substance (TBARS) in the liver tissue homogenate was estimated according to the previous protocol [21].
Superoxide dismutase activity. Superoxide dismutase activity was determined using the modifi ed method [22].
Catalase activity. Catalase activity was evaluated by detecting the decrease in absorbance resulting from the elimination of H 2 O 2 by the action of catalase [23].
Reduced glutathione. Reduced glutathione was determined using the method described earlier [24].

Measurement of serum alkaline phosphatase, serum glutamic oxaloacetic transaminase, and serum glutamic pyruvic transaminase levels
Serum alkaline phosphatase (ALP), serum glutamic oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) levels were measured using kits of Randox Laboratories Ltd by spectrophotometric assay. (Antrim, United Kingdom) according to the manufacturer ' s instructions.

Histological analysis of liver tissue
For histological analysis, a small portion of liver tissue was cleaned and preserved in a fi xative containing 10% buff ered formaldehyde. Liver slices were then processed and embedded in paraffi n wax. Paraffi n blocks were sliced with 5-μ m thickness, processed, and stained with hematoxylin (H) and eosin (E) for histopathological evaluation of liver [27]. The stained slide for each group was observed using a light microscope (Olympus 207444, Tokyo, Japan) at 200 ϫ magnifi cations. The photomicrograph was taken using the Camera Canon Power Shot S70.

'Bioavailability of GTIN by High-performance liquid chromatography High-performance liquid chromatography
To estimate the GTIN concentration in mice liver, highperformance liquid chromatography (HPLC) was carried out using liver homogenate according to the previous method [16]. For this analysis, mice were orally administered with GTIN (30 mg/kg body weight) for three consecutive days. Animals were sacrifi ced after 1 and 7 h of GTIN administrations (as in this study, mice were irradiated 1 h after the last dose of GTIN administration and sacrifi ced 6 h after irradiation). Liver tissues were collected and homogenized with 10 mM sodium phosphate buff er (pH: 7.4) containing 10 mM MgCl 2 in ice. Liver homogenate (1.5 mL) was mixed with 555 μ L of antioxidant solution (containing 0.2 g/ml ascorbic acid and 1 mg/ml EDTA) and 30 μ l of o-phosphoric acid. The mixture was vortexed, centrifuged, and supernatant was collected for HPLC. Solvent A consisted of 4% acetic acid in water and solvent B composed of acetic acid/methanol/water (1:25:25). These solutions were eluted as follows: 0 -1.5 min, 100% A; 1.5 -10 min, 100% A to A:B (50:50); 10 -12 min, A:B (50:50) to 100% B. The retention time for GTIN was about 30 min at 276 nm.

Alkaline single-cell gel electrophoresis (comet assay)
Radiation-induced DNA double strand breakage in hepatocytes was evaluated using single-cell gel electrophoresis based on the method of Singh et al [28]. In brief, frosted slides were covered with 1% NMA in PBS and allowed to solidify. It was followed by the addition of a second layer of LMA containing approximately 2 ϫ 10 5 cells at 37 ° C and immediately cover slips were placed on the slides. After solidifi cation of the LMA, the cover slips were removed and chilled lysing solution (containing 2.5 M sodium chloride (NaCl), 100 mM disodium EDTA (Na 2 -EDTA), 10 mM Tris -HCl at pH of 10, 1% DMSO, and 1% Triton X100) was applied for overnight at 4 ° C. After removal of the slides from the lysing solution, they were placed on a horizontal electrophoresis tank fi lled with freshly prepared alkaline buff er (300 mM NaOH, 1 mM Na 2 -EDTA, and 0.2% DMSO, at pH Ն 13 . 0). The slides were equilibrated with the same buff er for 20 min before electrophoresis at 25 V, 180mA for 20 min. After electrophoresis, the slides were washed with 0.4 M Tris -HCl buff er, pH: 7.4, to remove the alkali. Slides were stained with 50 μ l of EtBr (20 μ g/ml) and observed under microscope with bright fi eld phase contrast and epifluorescence facility (Leica DC 300 FX, Wetzler, Germany) using 400 ϫ magnifi cations. The quantifi cation of the DNA strand breaks of the images were done using the Comet Score software by which % DNA in tail, tail length, tail moment, and Olive tail moment were determined.

Measurement of cytokine levels
The levels of murine serum tumor necrosis factor alpha (TNF-α ) and interleukin 6 (IL-6) were measured using a sandwich ELISA Kit purchased from Endogen Inc. (Rockford, IL, USA) [17].

Immunofl uorescence
Immunofl uorescence was performed according to the method described by Das et al. [29]. After fi xing of liver tissue in 4% buff ered formalin, the tissue was embedded in paraffi n, serially sectioned at 5 μ M. Immunohistochemical staining for nuclear factor kappa B (NF-κ B) (p65) was carried out on paraffi n sections with anti-NF-κ B (p65) antibody (Imgenex, San Diego, CA, USA). Briefl y, sections were deparaffi nized in xylene and made penetrable by treating with 0.1% Triton X100. Thereafter, antigens were unmasked by heating the sections at 90 ° C for 10 min in 10 mM citrate buff er, pH: 6. The sections were then allowed to cool at room temperature for 20 min. Next, sections were incubated with diluted primary antibodies overnight at 4 ° C and washed with PBS after incubation. The bound primary antibodies were incubated with fl uorescein isothiocyanate (FITC)-tagged secondary antibody. Nuclei were stained using 4 ′ , 6-diamidino-2-phenylindole (DAPI). Fluorescent signals were observed under a microscope (Olympus IX 81) and if there is any nuclear translocation of NF-κ B, the color of FITC was observed into the DAPI-stained nuclei. Quantifi cation of NF-κ B nuclear translocation was done by evaluating the color intensity using " Image J software. "

Immunoblot assay
Liver was homogenized using tissue homogenizer (Sono Plus, Germany) in ice-cold 0.2 mM phosphate buff er (pH: 7.4) containing protease inhibitors (0.1 mM EDTA, 1.0 mM phenylmethylsulfonyl fl uoride (PMSF), 1 mM dithiothreitol (DTT), 0.1 mM ethylene glycol tetraacetic acid (EGTA), 0.3% NP-40, and 1 g/mL pepstatin A) to obtain a 10% tissue homogenate. The tissue homogenate (1.5 mL) was centrifuged at 12,000g (30 min at 4 ° C) in a cold centrifuge (Sorvall, USA). The supernatant was separated and centrifuged again at 15,000g (30 min at 4 ° C). The supernatant was taken for analysis of cytoplasmic fractions. The pellet procured after the fi rst spin (12,000g) was washed thrice with PBS at 900g (10 min, 4 ° C) and resuspended in 0.5 mL PBS containing protease inhibitors. The suspension was centrifuged at 100,000g for 1h at 4 ° C using an ultracentrifuge (Hitachi, Japan). The separated supernatant was used for analysis of nuclear protein. Protein concentration was determined using Lowry method [30]. Equal amounts of protein (50 μ g) in each lane were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% SDS-PAGE) and transferred to a nitrocellulose membrane. After completion of gel electrophoresis, proteins were electrotransferred (100 V, 1 h) to a nitrocellulose membrane using a mini-trans blot assembly (Bio-Rad, USA). The nitrocellulose membrane was blocked using blocking solution (3%, w/v, BSA in TBS) for 2 h at room temperature. Protein expressions were analyzed by probing with the respective mouse monoclonal primary antibodies (1:1000 dilutions) against the former (Imgenex, San Diego, USA). Following three washes of 15 min each in washing buff er (TBS, 0.2% Tween 20), membranes were incubated in TBS containing 1:10,000 dilutions of goat anti-mouse IgG alkaline phosphate-conjugated secondary antibodies. The membranes were again washed (three times each for 15 min) with washing buff er and then treated with nitro-blue tetrazolium and 5-bromo-4 -chloro-3-indolyl-phosphate (NBT-BCIP) reagent for 20 min. The protein bands obtained were further subjected to densitometric analysis using Gel Documentation system (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis
The values were given as mean Ϯ standard error of the mean (SEM). One-way analysis of variance (ANOVA) with Tukey ' s post hoc test was done for statistical evaluation of the data and for the determination of the level of signifi cance in various groups. In all cases, a value of p Ͻ 0.05 was considered signifi cant.

GTIN scavenged the radiation-induced intracellular ROS
To determine whether GTIN can protect the liver cells from γ-radiation-mediated oxidative stress by altering the level of intracellular ROS, hepatocytes were incubated with DCFDA and then the intensity of oxidized fl uorescent product of DCFDA was monitored by fl ow cytometric analysis ( Figure 2). The analysis showed that in the IR group, intracellular ROS was higher as revealed from the fl uorescence intensity which was signifi cantly higher than non-irradiated control. In case of GTIN ϩ IR group, the ROS level reduced signifi cantly by 55% as the intensity reduced than the IR group ( p Ͻ 0.05). These observations indicated that γ -irradiation enhanced the intracellular ROS level and GTIN pretreatment scavenged these ROS in murine hepatocytes.

Catalase activity
The catalase activity liver homogenate from control and IR group were 31.08±0.61 Unit and 17.29±0.33 Unit. The unit of catalase activity was defi ned as μmol H2O2 reduced/ mg protein'-is inserted instead of deleted word. Therefore, irradiation caused the 44% diminution of catalase activity than control groups. In GTIN ϩ IR group activity was 27.25 Ϯ 0.38 Unit. Thus, in GTIN ϩ IR catalase group, the catalase activity was signifi cantly superior (57%) to irradiated mice ( Figure 3C).

Superoxide dismutase activity
The liver homogenate of control group showed superoxide dismutase (SOD) activity of 2.99 Ϯ 0.17 unit/mg of protein. After exposure of mice with 5-Gy γ -radiation it showed a signifi cant 23% reduction of (2.29 Ϯ 0.11 unit/ mg protein) of SOD level in comparison to control. SOD activity was restored (2.77 Ϯ 0.03 unit/mg protein) in the GTIN ϩ IR group which was signifi cant ( p Ͻ 0.05) compared with IR group ( Figure 3D). Therefore, GTIN was able to conserve the SOD activity of mice liver homogenate even after the radiation exposure.

GTIN prevented gamma-radiation-mediated hepatic toxicity
The systemic toxicity after γ -radiation was assessed by liver toxicity markers. The liver function status was assessed by estimating the serum level of liver enzymes GPT, GOT, and ALP (Table I). It was observed that γ -radiation elevated SGPT, SGOT, and serum ALP levels. In case of GPT it was observed that there was signifi cant diff erence between control (9.45 Ϯ 0.27 IU/L) and IR (19.26 Ϯ 0.24 IU/L) group. A signifi cant diff erence existed between IR and GTIN ϩ IR (11.74 Ϯ 0.22 IU/L) values.
In case of SGOT, the level signifi cantly increased in IR group (40.5 Ϯ 0.89 IU/L) than control (28.78 Ϯ 0.91 IU/L), whereas in GTIN ϩ IR group (27.38 Ϯ 1.09 IU/L) the level signifi cantly decreased than IR group. The serum ALP elevated signifi cantly in IR group (12.36 Ϯ 0.52 KA unit) than control (8.64 Ϯ 0.11 KA unit). The ALP level remained signifi cantly low in GTIN ϩ IR group (8.52 Ϯ 0.41 KA unit) than IR group.

GTIN pretreatment ameliorated gamma-radiationmediated hepatic alterations
The histological investigations of the liver sections in IR group showed that radiation exposure resulted in signifi cant morphological changes characteristic of infl ammation. Compared with control, these morphological changes include infl ammatory cell infi ltration, intensive infl ammatory response around the central vein, and hematoxylin-rich nuclei. Some of the hepatocytes were found to be swelled and membranes appeared disrupted resulting in necrosis and sinusoidal space augmentation' is inserted. These were the indications of liver damage as a result of infl ammation. GTIN treatment prior to radiation restored the radiation-mediated hepatic alterations (Figure 4).

Bioavaibility of GTIN
The native form of GTIN in liver was determined from HPLC analysis using GTIN standard. After 1st hour and 6th hour of GTIN administration, liver GTIN concentrations were 2.26 mg/g of liver tissue and 7.14 mg/g of liver tissue, respectively ( Figure 5A and B). The percentages of bio-absorption of free GTIN were 8% after 1 h and 24% after 6 h. It was also observed that after 24 h, no free GTIN was available in liver. Therefore, free GTIN was available to combat the oxidative stress during these time periods. On the basis of this, the mice were irradiated 1 h after last dose of GTIN administration and sacrifi ced 6 h after irradiation, so that during this time period free GTIN remains available in liver. Thus, based on the above experiments, we further designed the time of administration of GTIN and the sacrifi ce time of animal after radiation.
GTIN ameliorated radiation-mediated DNA damage γ radiation exposure (5 Gy) signifi cantly increased all the comet parameters, that is, tail length, % DNA

GTIN prevented the gamma-radiation-mediated nuclear translocation of NF-κ B (p65)
Maximum nuclear localisation of NF-κ B was observed in radiation-exposed mice. The nuclear entry of p65 was lessened in GTIN Ϯ IR than in IR. From the immuno blot data it was revealed that the expression of cytosolic NF-κ B (p65) protein in irradiated group was signifi cantly ( p Ͻ 0.05) declined than that in control. In case of GTIN ϩ IR, the expression of cytosolic NF-κ B (p65) significantly augmented than IR group. In case of nuclear NF-κ B (p65) expressions of the proteins were reciprocally present in the cytosol and nucleus (Figure 8). The immunofl uorescence study confi rmed the NF-κ B localization in the cytoplasm of the control hepatocytes as well as radiation-induced nuclear translocation of NF-κ B. In case of IR, 47 nuclei were p65 positive out of 60 nuclei in the selected region ( Figure 9) whereas 14 nuclei were p65 positive out of 60 nuclei in GTIN ϩ IR slide. Therefore, in the GTIN ϩ IR group, nuclear translocation of p65 was less in comparison to the IR group.

Eff ect of GTIN on radiation-induced expression of Cdc42, phosphorylated stress-activated protein kinase/c-Jun NH2-terminal kinase, and Bax protein in liver
Our data revealed that Bax-and Cdc42-phosphorylated SAPK/JNK expressions were signifi cantly activated in IR group than that in control. GTIN reduced radiationmediated Cdc42 activation followed by SAPK/JNK phosphorylation and Bax activation ( Figure 10). This suggested that GTIN can modulate radiation-induced mitochondrialdependent apoptotic pathways.

GTIN activated Nrf2-driven Mn-SOD via PI3K and Akt phosphorylation
As shown in Figure 11, exposure to radiation decreased the phosphorylation of Akt and PI3K in IR-treated groups than that in control groups signifi cantly. Inhibition of the PI3K/Akt pathway by IR reduced the NF-E2-related factor 2 (Nrf2) translocation and Mn-SOD protein levels. In our present study, we observed that GTIN pretreatment signifi cantly upregulated PI3K and Akt phosphorylation leading to Nrf2 nuclear translocation and Mn-SOD activation when compared with IR group.

Discussion
In the present study, the strategies were to fi nd whether IR perturbs the metabolic oxidative balance. Thus in vivo model for IR induced stress was developed. Liver is the major metabolic organ. If liver experiences stress and strain, then it will lead to serious physiological consequences in the entire organism. The present analysis involved the assessment of damage using key signaling molecules of cellular metabolism as well as infl ammatory development. With the purpose of establishing an agent that potentially prevents the physiological stresses after radiation exposure, we hypothesized the GTIN is an eff ective compound. Due to its unique chemical structure, it eff ectively combats the reactive species after radiation. Moreover, due to its bioavailability for wide period of post absorption, it can mediate its eff ects in the metabolic balances.
In our study, it was shown that 5-Gy γ -radiation diminished the endogenous antioxidant system [31]. Increased level of LPO confi rmed the radiation-mediated membrane damage of liver. Furthermore, radiation caused enhanced leakage of liver function enzymes like GPT, GOT, and ALP in IR groups. These enzyme markers are the indicators of liver toxicity [32]. The underlying mechanism of these eff ects leading to liver toxicity is that radiation generates free radicals, damaging the cell membrane and thereby releasing the cytosolic enzymes [16,33]. Overall tissue damage in IR groups was evident from our histological study. The enhancement of the comet parameters in IR group in indicated radiation-mediated DNA damage, which may be the cause of the overall architectural damage of the liver tissue.
From immunofl uorescence and Immuno blot analysis, we demonstrated that γ -radiation augmented the nuclear translocation of NF-κ B (p65) in murine hepatocytes. However, NF-kB pathway is considered as a prototypical proinfl ammatory signaling pathway. Ionizing rays would not be an exception to activate redox-sensitive transcription factor NF-κ B due to the generation of ROS [34]. Moreover, NF-kB is responsible for expression of proinfl ammatory genes including cytokines, chemokines, and adhesion molecule [35] which was supported by the enhanced TNF-α and IL-6 expression in IR groups in our study. It was reported that oxidative stress and SAPK/JNK maintain a balance and modulate NF-κ B activity, which determines stress response and other infl ammatory phenomena [36]. Activated Cdc42 triggered JNK pathway resulting in initiation of apoptosis regulating the activities of preexisting Bcl-2 family proteins [37,38,39]. In our present study, it was evident that radiation enhanced intracellular ROS which leads to activation of Cdc42 and further activation of SAPK/JNK. Results also revealed the increased level of Bax protein in the IR-treated group, indicating that the Bax caused the outer mitochondrial membrane to become leaky, which helped in releasing cytochrome-c into the cytosol thereby triggering apoptosis [36].
One major system that reacts with oxidative stress to restore the redox balance involves genes coordinately   In all these cases statistical comparison: * control vs IR; ^IR vs GTIN ϩ IR. p < 0.05 was considered signifi cant. Figure 9. NF-κ B (p65) activation in mice was determined by immunofl uorescence. Control: without any treatment, IR: mice irradiated with 5-Gy γ -radiation, GTIN: mice treated with GTIN (30 mg/kg body weight) for three days, respectively, GTIN ϩ IR: GTIN treated plus irradiated. NF-κ B positive nuclei were estimated from the fi xed region. regulated by transcription through the antioxidant response element (ARE). This is activated primarily by binding of the transcription factor Nrf2 [40]. We observed that the irradiation caused the signifi cant reduction of Nrf2 nuclear translocation. Mn-SOD is a mitochondrial matrix enzyme that scavenges ROS and protects the cell against the insults of oxidative stress. It mediates a key role in cell survival and is necessary for the maintenance of mitochondrial integrity in cells exposed to oxidative stress [40,41]. Therefore, radiation-induced downregulation of nuclear translocation of Nrf2 caused the reduction in Mn-SOD level also, which was confi rmed by our Western blot data. The molecular mechanism of perturbation leading to mitochondrial damage was further confi rmed by showing several signaling pathways, including PI3K/ AKT which was involved in the induction of Nrf2/AREdriven gene [37,39].
In our study, GTIN treatment prior to radiation was found to protect liver from radiation-induced liver infl ammation by inhibiting the nuclear translocation of NF-kB and reducing lipid peroxidation. Thus, the membranes remained protected from leaking liver function enzymes into the extracellular matrix. The maintenance of the endogenous antioxidant level in GTIN ϩ IR-treated groups can cause to combat the liver cell against radiationmediated oxidative stress. GTIN prevented apoptosis induction in GTIN ϩ IR groups by reducing intracellular ROS and inhibition of the upstream SAPK/JNK, recruitment of Cdc42, reduced expression of Bax, and promoting survival of the liver against radiation. Protective action of GTIN against radiation-mediated DNA damage was crucial to protect the liver from apoptosis because DNA damage is one of the major indicators of cellular apoptosis.
It was revealed that GTIN upregulated Mn-SOD expression by increasing the nuclear translocation of Nrf2 which were attenuated by radiation. The possible mechanisms leading to nuclear translocation of Nrf2 include its release from Keap1 into the cytosol and enhancing ARE-binding activity of Nrf2. Our results demonstrated that GTIN increased the nuclear translocation of Nrf2, suggesting that GTIN may indirectly enhance the Mn-SOD in this way. It is known that several signaling pathways, including PI3K/AKT are involved in the induction of Nrf2/AREdriven gene [39]. The activation of the PI3K/Akt pathway is a key step in diverse biological processes, including cell proliferation, growth, and survival. Therefore, our results demonstrated that the Nrf2-mediated increase in Mn-SOD protein induced by GTIN is dependent on the activation of PI3K/Akt pathway.
Thus, GTIN prevented radiation-induced systemic and cellular oxidative stress. The protective action may depend on either the ability of the native GTIN to act as a scavenger of radiation-induced reactive species or by interacting with cell-signaling cascades either by itself or its metabolized form ( Figure 12).
In conclusion, here we report for the fi rst time that GTIN mediated a vital role in protecting the liver from radiation hazards. It was responsible for preventing the radiation-mediated oxidative stress. Therefore, our present result confers the evidence to the usefulness of GTIN-rich diet in preventing higher levels of radiation-induced infl ammation, lipid peroxidation, and oxidative stress. It may remain an important justifi cation for nuclear workers or defense personnel assuming possibility of nuclear exposure. However, further studies are warranted to fi nd out its dose and toxicity profi le and its protective eff ects on normal cells vis-a-vis tumor cell protection in radiation exposures being put into clinical use.  Radioprotective action of gossypetin 1185 sish Bandhopadhyay of CGCRI, Kolkata for his editorial comments on manuscripts.

Declaration of interest
The authors report no confl icts of interest. The authors alone are responsible for the content and writing of the paper.