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).