Materials

All chemicals were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) unless otherwise noted.

Animal model and experimental groups

This research protocol complied with the regulations regarding the care and use of experimental animals published by the National Institutes of Health and was approved by the Institutional Animal Use and Care Committee of the University of Pittsburgh Medical School. Male C57BL/6 mice weighing 20 to 25 g (Jackson Laboratories, Bar Harbor, ME, USA) were used in this study. The animals were maintained at the University of Pittsburgh Animal Research Center with a 12-hour light-dark cycle and free access to standard laboratory food and water. The animals were fasted over night prior to the experiments.

Experiment A

ALI was induced by a single dose of APAP (350 mg/kg dissolved in 1 mL sterile saline) administered by intraperitoneal injection. APAP-challenged mice were then randomized into the NAC (n = 7) group or the saline group (n = 7). Six mice injected with saline not containing APAP served as a control group. Two hours after APAP administration, each group was given the following treatments every 12 hours for a total of 72 hours: 100 mg/kg NAC dissolved in 0.6 mL sterile saline for the NAC group, and 0.6 mL saline for the saline group and the control group. Seventy-two hours after APAP injection, all surviving mice in each group were anaesthetized with sodium pentobarbital (90 mg/kg intraperitoneally), and the following procedures were performed: blood was aspirated from the heart for the subsequent measurements of ALT and AST; the left lobe of the liver was harvested for pathology (H&E staining); and the right lobe of the liver was harvested and frozen for measurement of hepatic NF-κB DNA binding by electrophoretic mobility shift assays (EMSA) and hepatic tissue cyclin D1 expression by western blot.

Experiment B

Three separate groups of mice were treated as described above with the exception that the treatment period was only 24 hours (n = 6 for each group).

Plasma aminotransferase measurements

Plasma levels of AST and ALT were measured at 37°C with a commercially available kit (Sigma Diagnostic, St Louis, MO, USA).

Histological analysis

Consecutive sections (5 μm) of paraffin-embedded liver were prepared for H&E staining. The percentage of necrosis was estimated by evaluating the number of microscopic fields with necrosis compared with the entire cross-section. In general, necrosis was estimated at low power (×100) and questionable areas were evaluated at higher magnification (×200 or ×400). The pathologist (XH) evaluated all histological sections in a blinded fashion. Inflammatory cell infiltration results were scored semi-quantitatively by averaging the number of inflammatory cells per microscopic field at a magnification of 200×. Five fields were evaluated per tissue sample, and six animals in each group were examined.

Tissue myeloperoxidase

Neutrophil infiltration was measured at 72 hours by determining myeloperoxidase (MPO) activity in liver tissue homogenates and was used as an index of neutrophil infiltration in all groups. At the time of sample determination, liver tissue samples (400 mg) were harvested and snap frozen immediately in liquid nitrogen and stored at -70°C until analysis was performed. Samples were homogenized in suspension buffer (50 mmol/L potassium phosphate/0.5% hexadecyltrimethylammonium bromide; pH 6.0), sonicated on ice, freeze-thawed twice, resonicated and then centrifuged for 15 minutes as 20,000 g at 4°C. Liver supernatants were heated for two hours at 60°C, and recentrifuged for 15 minutes at 20,000 g at 4°C.

To determine the MPO activity, 100 μL of the supernatants was incubated for three minutes at 37°C in a reaction solution containing 40% PBS, 8% N,N-dimethylformamide, 1.6 mmol/L 3,3', 5,5'-tetramethylbenzidine, 0.3 mmol/L hydrogen peroxide and 80 mmol/L sodium phosphate (pH 5.4). The reaction was stopped by adding ice-cold 800 mmol/L acetic acid solution (pH 3.0) and by placing the assay tubes on ice. Samples were read at 655 nm and total MPO activity calculated as the change in absorbency per minute per gram of tissue multiplied by the dilution factor. The levels were expressed as units per gram of tissue (U/g) [17].

EMSA

NF-κB activation was determined by EMSA as previously described [18]. The gels were dried and exposed to Biomax film (Kodak, Rochester, NY, USA) at -70°C overnight with use of an intensifying screen.

Western blot

Liver protein was extracted as previously described [19]. Equivalent amounts of protein were boiled in sample buffer and separated on 7.5% pre-cast SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to nylon membranes. Membranes were then probed with a specific antibody against cyclin D1 (Cell signaling Technology, Lexington, KY, USA) protein, visualized with an Enhanced Chemiluminescence substrate (ECL, Amersham Pharmacia Biotech, Piscataway, NJ, USA) and exposed to x-ray film according to the manufacturer's instructions.

Statistical methods

Results are presented as means ± standard error of the mean (SEM). Continuous data were analyzed using student's t-test or analysis of variance followed by Fisher's least significant difference test. P values less than 0.05 were considered significant. Summary statistics are presented for densitometry results from studies using western blot for cyclin D1 expression but these results were not subjected to statistical analysis because the method employed was only semi-quantitative (n = 6).