L-glutathione (GSH), oxidized glutathione (GSSG), 1-chloro-2,4-dinitrobenzene (CDNB), nicotinamide adenine dinucleotide phosphate in the reduced form (NADPH), 4-nitro blue tetrazolium chloride (NBT), sodium azide (NaN3), hydrogen peroxide (H2O2), glutathione reductase (GR) from the baker’s yeast (S. cerevisiae), IGEPAL CA-630, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA) and all other reagents were from SIGMA-Aldrich (St. Louis, Mo, USA). Bradford protein assay reagent (Bio-Rad). Protease inhibitor cocktail for use with mammalian cell and tissue extract from SIGMA-Aldrich (St. Louis, Mo, USA).
Fourteen week old female CD1 mice (Envigo) were kept in the Interdepartmental Service Centre—Station for Animal Technology, University of Rome “Tor Vergata” (Italy) and housed at a constant temperature of 20 ± 2 °C, relative humidity of 50 ± 10%, on a 12/12 h light/dark cycle and ventilation 10–15 times/hours. Standard laboratory rodent pellet diet (4RF18; Mucedola srl, Italy) and water were provided to the animals ad libitum. All animal procedures were approved by Ethical Committee, conducted in accordance with national and international laws and policies (Italian Legislative Decree n. 116/92, now Italian Legislative Decree n. 26 4/3/14). Before all procedures mice were fully anesthetized with intraperitoneal injection of tiletamine/zolazepam (40 mg/kg) (Zoletil 100, Virbac, Italy) and xylazine (15 mg/kg) (Rompun, Bayer, Italy). Livers were explanted, divided in four parts, re-suspended in physiological solution and irradiated (Fig. 3a). For in vivo irradiation four animals in total were used. Mice were positioned on the left side and they have been irradiated for 3.5 min at 8 Gy and 2 Gy. After 16 h animals were sacrificed, the liver has been removed, divided in four parts and re-suspended in physiological solution (Fig. 3b). In the last experiment four mice were sacrificed and livers were explanted for homogenates production (Fig. 3c).
The irradiation of biological tissue was performed at room temperature, using a linear accelerator (Elekta Precise®), with 6 MV photons at a dose rate of 6 Gy/min. Single fraction of radiation doses of 2, 4, 8, 16, 24, and 32 Gy respectively were delivered to the biological tissue depending on the experiment. For irradiation, test specimens were immersed in an aqueous gel cube. A planning computed tomography (CT) was obtained in order to calculate the dose distribution within the test specimens (the enzymes in hydrosaline solution and liver homogenates). The treatment plan consisted of 2 opposing fields and was calculated on treatment planning system Pinnacle version 9.8 (Philips Medical System, Andover, MA). The radiation dose homogeneity was estimated at 3%. For mouse irradiation, each mouse underwent a planning CT to delineate the target volume (the whole liver) and a treatment plan was developed to deliver a dose (Fig. S2).
In the two experiments of irradiation: (a) irradiation of explanted mouse liver and (b) irradiation of the upper abdomen in anesthetized mice followed by liver explant, about 0.5 g of explanted liver unirradiated or irradiated with 2, 4 or 8 Gy was homogenized. The liver was put in 5 ml of 0.1 M phosphate buffer, pH 7.4 containing protease inhibitor cocktail and immediately after, 1 ml of homogenized liver was centrifuged at 13,300 rpm for 3 min. The supernatants were transferred in tubes on ice ready for enzymatic activity measurements. In the case of irradiation experiments on mouse liver homogenates, about 1 g of mouse liver was homogenized in 12 ml of physiologic solution with 0.9% sodium chloride containing protease inhibitor cocktail and 2 ml centrifuged at 13,300 rpm for 5 min. The supernatant was transferred in four vials on ice ready for irradiation experiments at 2, 4, 8, 16, 24 and 32 Gy.
Glutathione peroxidase activity
Activity of GPx in liver was determined with a spectrophotometric assay at 340 nm (25 °C): 2 μl of homogenate was incubated in 1 ml of 0.1 M phosphate buffer, pH 7.4 (EDTA 0.1 mM) with 0.1 mM of NaN3, 1 mM of GSH, 1 µL of GR and 0.1 mM of NADPH. After linearity was reached, immediately 0.1 mM of H2O2 was added12. Each activity was normalized to the amount of total protein content determined by Bradford assay13.
Activity of GST in liver was determined spectrophotometrically as described previously14. Briefly, 5 µl of homogenate was diluted in 1 ml of 0.1 M phosphate buffer, pH 6.5 and incubated with 1 mM GSH and 1 mM of CDNB. The enzymatic activity was followed at 340 nm (25 °C). Each spectrophotometric determinations were subtracted by the spontaneous reaction of the two co-substrates (GSH and CDNB). Activity determinations were normalized to the amount of total protein content determined for each sample by Bradford assay13.
Glutathione reductase activity
Activity of GR in liver was determined spectrophotometrically at 340 nm (25 °C) diluting 10 μl of homogenate in 0.1 M phosphate buffer, pH 7.4 with 0.1 mM NADPH and, after the reaction reached linearity, suddenly 1 mM of GSSG was added15. Activity measurements were normalized to the amount of total protein content determined for each sample by Bradford assay13.
Activity of CAT in liver was determined with a spectrophotometric assay at 240 nm (25 °C): 10 μl of homogenate was diluted in 1 ml of 0.05 M phosphate buffer, pH 7.0 (EDTA 0.1 mM) with 1 mM of H2O2 according to the standard procedure described previously16. Each activity was normalized to the amount of total protein content determined by Bradford assay13.
Preparation of cell lysates
Cell pellets were resuspended in lysis buffer containing 10 mM Tris-HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, 0.5% IGEPAL CA-630 and protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA). After 30 min incubation on ice, cell lysates were centrifuged at 17,000 rpm for 15 min. Then supernatants were used to measure the SOD1 activity.
Measurement of superoxide dismutase-1
SOD1 activity was evidenced on non-denaturing 7.5% polyacrylamide gels by loading 50 µg of total protein extracts. After electrophoresis, the gel was incubated in NBT solution (2.5 mM) for 30 min in the dark with gentle shaking, followed by 30 min incubation with a solution containing 30 mM tetramethylenediamine and 10 µg/ml riboflavin. SOD1 activity was detected as the achromatic band on the violet-colored gel, obtained after light exposure17. Density of immunoreactive bands was calculated using the software Quantity one (Bio-Rad). Catalase or β-tubulin was used as loading control. Proteins were assayed by the method of Lowry18.
The literature search was performed taking into account the following conditions: mouse as animal model, liver as target organ, whole body or extirpated liver irradiated, enzymatic activity limited in particular for the antioxidant enzymes GPx, GST, GR, CAT and SOD. Animal models for tumor(s), radiation resistant, and treated with natural and synthetic drugs were excluded. Only in few analysis of mRNAS25,S26 and “adaptive response” enzyme activityS21,S23,S24 results from rat liver were included. Experimental data from different authors were obtained digitalizing histograms and graphs and then calculated the percentage values respect the control group in each study. The digitalization was performed using GetData Graph Digitizer software (v2.24). The graphic and results visualization were obtained by GraphPad Prism (La Jolla, CA, USA). Statistical analysis was performed between data pairs with a t-test; n.s. in Table S1 indicates that the observed difference between irradiated samples and controls are not statistically significant. P < 0.05 defines statistical significant differences (GraphPad InStat (La Jolla, CA, USA)).