Drug preparation and animals
THJ-2201 was purchased from Cerilliant Corporation, Round Rock, Texas, USA. The drug was initially dissolved in dimethyl sulfoxide (DMSO) (final concentration was 2%) and brought to the final volume with corn oil. DMSO and corn oil was also used as the vehicle control.
Swiss albino male mice, aged 6 weeks and weighing 20–25 g, were obtained from the Experimental Animal Care Center, College of Pharmacy, King Saud University. All experimental mice were provided with free access to water and commercial pelleted diet (Saudi Grains Organization, Riyadh, Saudi Arabia). The animals were maintained under controlled conditions of temperature with constant humidity. The room was controlled with cycles of 12 h of light and 12 h of darkness. The mice were adapted to the laboratory environment for 1 week before starting the experiment.
LD50 and acute toxicity symptoms
The single-dose study was carried out according to OECD 423 guidelines for testing of chemicals (OECD 2017). A total of 30 mice were randomly distributed into five groups, including one control group. Each group consisted of three males and three females.
In acute toxicity, five groups of mice, with each group comprising six animals, were orally gavaged with THJ-2201 in doses of 5, 50, 300, and 2000 mg/kg body weight (bw) in a final volume of 0.25 mL. A control group of six animals received only vehicle control.
The mice were individually observed for their general behavior at 1, 2, 3, 5, and 24 h after treatment. The number of deaths within this period was recorded, and LD50 (the dose that kills 50% of animals) was determined according to the probit method (method of least squares) using the [Software Stat Plus] (Ver. 2015 Build 22.214.171.124 ©2015).
Necropsy was carried out on all animals, and renal as well as hepatic tissues was preserved in 10% buffered formalin for evaluation of histopathological alterations.
At the end of the test duration, the mice had been added into the anesthetic chamber and sedated with 5% diethyl ether for 1 min. Blood from every mouse was collected from the heart to determine the hematological indices and was stored in tubes containing the anticoagulant ethylenediaminetetraacetic acid (EDTA). An automated hematology analyzer was used to investigate hematological parameters which include the subsequent: white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).
The formalin preserved hepatic and renal tissue samples of THJ-2201-dosed rats, and the control group were processed in an automated tissue processor (Tissue–tek VIP-5, from SAKURA). The tissues were then processed by routine procedures to tissues in paraffin wax blocks. Paraffin sections (4–5 μm) were stained with hematoxylin and eosin, the conventional staining technique.
Stained sections were examined for necrosis, apoptosis, inflammation, and vascular modifications in renal tissue.
The hepatic tissue was evaluated for any change in the structure, portal or lobular inflammation, sinusoidal dilatation, and congestion together with the presence of granulomas, degeneration, necrosis, and fatty change.
The significance of variations between means was compared at every time point the use of Duncan’s multiple range test (DMRT) after ANOVA for one-way classified data (Snedecor and Cochran, 1989).