MATERIALS and METHODS
The study was conducted with the approval of the Ethics Committee of İnönü University Experimental Animals (protocol No: 2019/A-20), and all experimental procedures adhered to the ARRIVE guidelines (Colak et al., 2012). A total of 32 female Wistar Albino breed rats aged 4-6 months were obtained from İnönü University Experimental Animal Production and Research Center and were housed in rooms maintained at 60-65% humidity, constant heat of 25±2°C, and ventilation. The rats were exposed to 12 hours of light and 12 hours of darkness and were provided with standard commercial rat feed (pellets) and tap water ad libitum .
The rats were randomly assigned to the following groups:
• Control group: the experiment was terminated on the 5th day after administering 0.5 ml intraperitoneal (i.p.) saline.
• MTX group: the experiment was terminated on the 5th day after administering 20 mg/kg MTX i.p.
• RAN+MTX group: RAN 100 mg/kg/day per oral (p.o.) was administered for 7 days, and a single dose of MTX 20 mg/kg i.p. was administered on the 2nd day after RAN administration. The trial was terminated on the 7th day.
• MTX+RAN group: RAN 100 mg/kg/day p.o. was administered for 5 days starting from the 5th day after administering a single dose of MTX 20 mg/kg i.p. The trial was terminated on the 10th day.
Prior to the experiment’s termination, the rats were weighed using a highly sensitive weighing instrument. At the end of the experiment, the rats were anesthetized with i.p. 5 mg/kg xylazine and 75 mg/kg ketamine before being sacrificed. To conduct serum biochemical analysis [alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH)], 4-6 mL of blood was taken from the heart using a sterile syringe, centrifuged for 10 minutes at 3000 rpm, and stored at -80°C. The midline was cut, and weighing was performed after total hepatic resection. For histopathological examinations, the lobus hepatis dexter was separated and fixed with 10% formaldehyde-containing tubes. The lobus hepatis sinister was wrapped in aluminum paper and held at -80°C for the measurement of tissue biochemical parameters [superoxide dismutase (SOD), catalase (CAT), reduced glutathione (tGSH), and malonyldialdehyde (MDA)].
The biochemical analysis involved determining AST, ALT, ALP, and LDH levels from the serum samples obtained after centrifugation, using the spectrophotometric method in an Architect C16000 device (Abbott, Chicago, IL, USA) based on the manufacturer’s instructions (Vardi et al., 2010).