Histology, Bioenergetics and Oxidative Stress in Mouse Liver Exposed to N-Diethylnitrosamine

Materials and Methods

Animals and experimental conditions. All experimental procedures were developed according to ordinance no. 1005/92, and EU Directive 2010/63/EU on the use of laboratory animals.

Four- to five-week-old male ICR mice were purchased from the Harlan-Interfauna company (Barcelona, Spain). The subsequent quarantine period lasted for one week. The animals were kept under controlled conditions at 23±2°C, with 12 h light/12 h dark and controlled ventilation. They did not receive any treatment prior to the start of the study. Animals were randomly divided into groups, kept on hardwood bedding and received a standard laboratory maintenance diet (Harlan Global Diet 2014, Barcelona, Spain) and water ad libitum throughout the experiment. Maintenance and cleaning were carried out twice a week. Drinking water was replaced weekly or earlier if necessary. Consumption of food and water was measured at the same time as animals' body weight was determined. The animals were observed daily to assess their general health.

Experimental procedure. A total of 20 male ICR mice were divided into two groups consisting of 10 animals each: a control group which did not receive any treatment and a DEN-treated group subjected to treatment with the carcinogen. DEN was acquired from Sigma-Aldrich Company (Portuguese affiliated Office, Sintra, Portugal). The carcinogenic solution was administered via intraperitoneal route, once a week, for eight consecutive weeks, at a dose of 3.5 μl/g bodyweight per mouse. DEN solution was prepared at 1% concentration (99 ml of normal saline – NaCl 0.9% to which was added 1 ml of concentrated DEN solution (0.01 μg/μl)).

Animals were checked daily for clinical signs of toxicity and mortality. Their ponderal homogeneity index _iPH=2W_l/(W_l+W_h) and ponderal gain PG=W₂-W₁/W₂×100 were calculated, with W_l being the lowest average animal weight, W_h the highest average animal weight, W₁ initial body weight and W₂ final body weight.

Sample collection. Fifteen weeks after the start of the experiment, the animals were euthanased by means of a lethal-dose intraperitoneal injection of pentobarbital sodium. Whole blood samples were collected via intracardiac punction and placed in appropriately sized heparinized tubes. At this time, blood samples were also collected into heparinized capillary tubes for microhematocrit procedure. Samples were centrifuged in a Labofuge I (Burladingen, Germany) at 656 ×g for 15 min to obtain plasma and kept at −20°C until used. Complete necropsies were subsequently performed; the heart, lungs, liver, kidneys, urinary bladder and spleen were collected, weighed and examined macroscopically. Relative organ weights (liver, kidney, spleen, lungs, heart and urinary bladder) were calculated as the ratio of the organ weight to the mouse's total bodyweight.

Liver function tests. Automated standardized procedures (RX Daytona, Randox Laboratories, Dublin, Ireland) were used to measure and assess liver function, alanine transaminase and aspartate aminotransferase plasma activity, and plasma levels of total bilirubin and albumin.

Haematology. The capillaries were centrifuged in a Hermle Z320® unit (Gosheim, Germany) at 6000 ×g for 5 min, and the hematocrit was measured on a hematocrit ruler.

Histological evaluation. Representative fragments of each organ were fixed in 10% buffered formalin and embedded in paraffin wax. Tissue sections of 2 μm were processed and stained with haematoxylin and eosin (H&E). The livers were sectioned for reticulin and Masson's trichrome staining, according to the techniques described by Jones (12). Liver samples were analyzed via optical microscopy to identify lesions. The following parameters were evaluated: architecture, fibrosis, bile ductal hyperplasia, cholestasis, hepatocyte necrosis, the presence of inflammatory cells, hydropic degeneration, hyperplasia, anisokaryosis, binucleated cells, pseudo-nucleoli, apoptosis, focal hepatic necrosis and mitosis. As far as reticulin expression and Masson's trichrome staining were concerned, the following parameters were evaluated: liver parenchyma, portal space, centrolobular space, portal space and the presence of bridges in adjacent centrolobular veins.

Isolation of liver mitochondria. Mitochondrial isolation was performed by conventional methods (13), with minor modifications. The homogenization medium contained 0.25 M sucrose, 5 mM Hepes (pH 7.4), 0.2 mM ethyleneglycol-bis(-aminoethyl ether) N, N, N, N-tetraacetic acid (EGTA), and 0.1% fatty acid-free bovine serum albumin (BSA). Ethylenediaminetetra-acetic acid (EDTA), EGTA, and BSA were omitted from the final washing medium, which was adjusted to pH 7.2. The final concentration of the mitochondrial protein was determined by the Biuret method (14), using BSA as standard protein.

Enzymatic activity of mitochondrial complexes. Complex I or nicotinamide adenine dinucleotide (NADH)-dehydrogenase activity was measured spectrophotometrically at 25°C by following the oxidation of NADH (reduced nicotinamide dinucleotide) into NAD+ (oxidized nicotinamide dinucleotide), and this decrease in the concentration of NADH was read at 340 nm. The assay was performed in a buffer containing 50 mM potassium phosphate, pH 7.4, 2 mM KCN, 5 mM MgCl₂, 2.5 mg/ml BSA, 2 μM antimycin, 100 μM decylubiquinone, and 0.3 mM K₂NADH; 15-25 μg of the mitochondrial sample were used to initiate the reaction. The enzymatic activity was measured for 3 min and values were recorded 30 s after the initiation of the reaction. Specific activities were determined by calculating the slope of the reaction in the linear range in the presence or absence of 1 μM rotenone (complex I inhibitor) and expressed in nmol NADH/min/mg of protein (15). Succinate dehydrogenase activity was measured spectrophotometrically by the reduction of 2,6-dichlorophenolindophenol at 600 nm in the presence of phenazine methasulphate (16). The reaction was performed in 1 ml of the standard reaction medium supplemented with 5 mM succinate, 2 M rotenone, 0.1 g antimycin A, 1 mM KCN, 0.025% Triton X-100 at 25°C, and 0.5 mg protein of disrupted mitochondria (two cycles of freezing and thawing). Cytochrome c oxidase activity was measured polarographically (16) at 25°C in 1 ml of the standard reaction medium supplemented with 5 mM succinate, 2 M rotenone, 10 M cytochrome c, and 0.5 mg protein broken mitochondria. The reaction was initiated by the addition of 5 mM ascorbate plus 0.25 mM N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD). F₀-F₁ -ATPase activity was determined by monitoring the pH change associated with ATP hydrolysis (16). The reaction was carried out in 2 ml of a medium containing 130 mM sucrose, 50 mM KCl, 5 mM MgCl, and 0.5 mM Hepes (pH 7.2), supplemented with 2 M rotenone and mitochondria (1 mg protein of disrupted mitochondria). The reaction was initiated by the addition of 2 mM Mg-ATP and was completely inhibited by the addition of oligomycin (2 g/mg mitochondrial protein). Proton production was again calculated 2 min after starting the reaction. The final concentration of the mitochondrial protein was determined by the Biuret method (14) using BSA as standard.

Oxidative stress evaluation. Liver homogenization: Livers were placed in freezing medium consisting of 0.2 M mannitol, 0.07 M sucrose, 20% of Dimethyl sulfoxide (DMSO) and subsequently frozen at −70°C for subsequent homogenization. Livers were weighed and added to 10% (w/v) 50 mM phosphate buffer and were homogenized. The homogenate obtained was centrifuged at 16,000 ×g for 20 minutes at 4°C, the protein supernatant was used to measure antioxidant enzyme activities and protein thiol content.

Determination of protein thiol content: Total protein thiols expressed as reduced glutathione (GSH) were measured as described by Peixoto et al. (17). Absorption was measured at 412 nm, using GSH for calibration.

Determination of antioxidant enzymes: Superoxide dismutase (SOD) activity was determined according to the method of Paya et al. (18), using the xanthine–xanthine oxidase system. One unit of SOD activity was defined as the amount of SOD inhibiting the reduction rate of nitroblue tetrazolium chloride (NBT) by 50%. Catalase (CAT) was assayed as described by Aebi (19) and the activity was determined without disruption of mitochondria in the presence of 0.5% Triton X-100. Glutathione reductase (GR) activity was performed according to Smith et al. (20). The rate was calculated from the linear portion of the curve and expressed as nmol NADPH/min/mg protein. The activity of glutathione S-transferase (GST) was assayed at 25°C and 340 nm due to the conjugation of GSH to 1-chloro-2,4-dinitrobenzene (CDNB). The reaction mixture contained 650 μl of sodium phosphate buffer (100 mM, pH 7.0), 10 μl CDNB (50 mM), and 25 μl GSH (50 mM). The absorbance was measured at 340 nm, and enzyme activity expressed in μmol CDNB conjugated/min/mg of protein (ε=9.6×10³/M cm) (21).

Statistical analysis. Continuous data were expressed as mean±standard deviation (SD), with comparisons of continuous data made using a one-way ANOVA, Bonferroni and Tukey's multiple comparison test at the 5% significant level (p<0.05). All tests were performed using the GraphPad Prism, version 5.01 (GraphPad Software, Inc., La Jolla, CA, USA).