Cytoprotective effects of amifostine, ascorbic acid and N-acetylcysteine against methotrexate-induced hepatotoxicity in rats

Abstract

Aim: To investigate the potential role of oxidative stress and the possible therapeutic effects of N-acetyl cysteine (NAC), amifostine (AMF) and ascorbic acid (ASC) in methotrexate (MTX)-induced hepatotoxicity.

Methods: An MTX-induced hepatotoxicity model was established in 44 male Sprague Dawley rats by administration of a single intraperitoneal injection of 20 mg/kg MTX. Eleven of the rats were left untreated (Model group; n = 11), and the remaining rats were treated with a 7-d course of 50 mg/kg per day NAC (MTX + NAC group; n = 11), 50 mg/kg per single dose AMF (MTX + AMF group; n = 11), or 10 mg/kg per day ASC (MTX + ASC group; n = 11). Eleven rats that received no MTX and no treatments served as the negative control group. Structural and functional changes related to MTX- and the various treatments were assessed by histopathological analysis of liver tissues and biochemical assays of malondialdehyde (MDA), superoxide dismutase (SOD), catalase, glutathione (GSH) and xanthine oxidase activities and of serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and total bilirubin.

Results: Exposure to MTX caused structural and functional hepatotoxicity, as evidenced by significantly worse histopathological scores [median (range) injury score: control group: 1 (0-3) vs 7 (6-9), P = 0.001] and significantly higher MDA activity [409 (352-466) nmol/g vs 455.5 (419-516) nmol/g, P < 0.05]. The extent of MTX-induced perturbation of both parameters was reduced by all three cytoprotective agents, but only the reduction in hepatotoxicity scores reached statistical significance [4 (3-6) for NAC, 4.5 (3-5) for AMF and 6 (5-6) for ASC; P = 0.001, P = 0.001 and P < 0.005 vs model group respectively]. Exposure to MTX also caused a significant reduction in the activities of GSH and SOD antioxidants in liver tissues [control group: 3.02 (2.85-3.43) μmol/g and 71.78 (61.88-97.81) U/g vs model group: 2.52 (2.07-3.34) μmol/g and 61.46 (58.27-67.75) U/g, P < 0.05]; however, only the NAC treatment provided significant increases in these antioxidant enzyme activities [3.22 (2.54-3.62) μmol/g and 69.22 (61.13-100.88) U/g, P < 0.05 and P < 0.01 vs model group respectively].

Conclusion: MTX-induced structural and functional damage to hepatic tissues in rats may involve oxidative stress, and cytoprotective agents (NAC > AMF > ASC) may alleviate MTX hepatotoxicity.

Keywords: Amifostine; Ascorbic acid; Hepatotoxicity; Methotrexate; N-acetyl cysteine; Oxidative stress.

Figure 1

Methotrexate-induced effects on hepatic structure in rats. Photomicrographs of representative liver tissues are shown for the control group (A) and model group (B-D). A: Normal histological appearence of H-E stained control liver tissues, showing vena centralis (VC) at × 40; B: Abnormal histological appearance of H-E stained model liver tissues, showing hepatocytes with eosinophilic cytoplasm around the portal area (arrows) at × 40; C: Inflammatory cells in the portal area (arrows) and congestion at × 40 (arrowhead); and D: Hydropic degeneration (vacuolization/cellular swelling) in hepatocytes at × 40.

Figure 2

Methotrexate-induced effects on glycogen storage in hepatocytes. Photomicrographs of representative liver tissues are shown for the control group (A), model group (B, C), and antioxidant treatment groups (D-F). Normal histological appearence of PAS stained control liver tissue showin glycogen within the hepatocytes (magenta color) at × 20 (A). Decreased glycogen storage in hepatocytes is shown in PAS stained model liver tissues at × 10 (B) and × 20 (C) . Differential effects on MTX-reduced glycogen storage are shown in PAS stained liver tissues from the MTX + NAC group at × 20 (D), MTX + AMF group at × 20 (E), and MTX + ASC group at × 20 (F). VC: Vena centralis; MTX: Methotrexate; NAC: N-acetyl cysteine; AMF: Amifostine; ASC: Ascorbic acid; PAS: Periodic acid-Schiff.

Figure 3

Methotrexate-induced structural damage to hepatic tissues in rats treated with antioxidant agents. A: Photomicrographs of representative H-E stained liver tissues are shown at × 40; B:The MTX + NAC group showed radial hepatocytes in the region from the central vein to the periphery; C: The MTX + AMF group showed sinusoidal dilatation (arrows) that was similar to that observed in the untreated model group. VC: Vena centralis; H-E: Hematoxylin-eosin; MTX: Methotrexate; NAC: N-acetyl cysteine; AMF: Amifostine.