Genistein Ameliorates Cyclophosphamide - Induced Hepatotoxicity by Modulation of Oxidative Stress and Inflammatory Mediators

Abstract

Aim: The present study investigated the protective effect of the phytoestrogen, genistein (GEN), against (CP)-induced acute hepatotoxicity in rats.

Material and methods: Male adult rats were randomly assigned into five groups. Normal control group received the vehicles; CP group received a single dose of CP (200 mg/kg, i.p). The other three groups received subcutaneous GEN at doses of 0.5, 1 and 2 mg/kg/day, respectively, for 15 consecutive days prior CP injection. Sera and liver tissues were collected forty-eight hours after CP injection for assessment of liver function enzymes (ALT and AST) in rat sera, the hepatic oxidative/nitrosative biomarkers (GSH, MDA and NOx), hepatic interleukin-1β, and myeloperoxidase activity. Immunohistochemistry of cyclooxygenase-2 and histopathological examination of liver tissues were also conducted.

Results: The CP-induced acute liver damage was evidenced by elevated serum ALT and AST accompanied by increased hepatic oxidative stress and inflammatory biomarkers. Immunohistochemical outcomes revealed hepatic cyclooxygenase-2 expression in CP group with distortion of liver architecture. GEN-pretreatment significantly ameliorated the deterioration of liver function and exerted significant anti-oxidant and anti-inflammatory activity with a marked decline in hepatic cyclooxygenase-2 expression in a dose dependent-manner.

Conclusion: The present study demonstrated that the antioxidant and anti-inflammatory activities of GEN might contribute to its protective effects against CP-induced liver damage.

Keywords: Cyclophosphamide; Myeloperoxidase; Oxidative stress; genistein; interleukin-1β.

Figure 1

Effects of genistein on serum alanine transaminase (ALT) and aspartate transaminase (AST) in cyclophosphamide-induced hepatotoxicity in rats. Rats of the normal control group received s.c. Injections of DMSO. Acute hepatotoxicity was induced in the remaining four groups by single intraperitoneal injection of cyclophosphamide (CP; 200 mg/kg) on the 15th day of treatment. Group 2 received only saline s.c. for 15 days and served as CP control group. Groups 3, 4 and 5 received genistein (GEN; 0.5, 1 and two mg/kg/day, s.c.) respectively for 15 days before CP treatment. All animals were sacrificed 48 h after CP injection, blood samples were collected, and sera were separated. Data are presented as mean ± SEM (n = 8). a, Significantly different from Normal control group at p < 0.05 (Tukey’s post hoc test). b, significantly different from CP control group at p < 0.05 (Tukey’s post hoc test). c, Significantly different from CP+GEN (0.5 mg/kg) group at p < 0.05 (Tukey ’s post hoc test). d Significantly different from CP+GEN (l mg/kg) group at p < 0.05 (Tukey’s post hoc test)

Figure 2

A. Photomicrograph of liver sections prepared from a normal control rat showing negative. COX-2 immunostaining of the kupffer cells and the hepatocytes (COX-2 immunohistochemistry H&E X 80). B. Photomicrograph of liver sections prepared from a cyclophosphamide-control rat showing severe COX-2 immunostaining of the kupffer cells and the hepatocytes (COX-2 immunohistochemistry H&E X 80). C. Photomicrograph of liver sections prepared from a rat treated with genistein (0.5 mg/kg/day, s.c.) for 15 days before CP treatment showing moderate COX-2 immunostaining of the kupffer cells and the hepatocytes (COX-2 immunohistochemistry H&E X 80). D. Photomicrograph of liver sections prepared from a rat treated with genistein (1 m///lkg/day, s.c.) for 15 days before CP treatment showing mild COX-2 immunostaining of the kupffer cells and the hepatocytes (COX-2 immunohistochemistry H&E X 80). E. Photomicrograph of liver sections prepared from a rat treated with genistein (2 m/l:g/day, s.c.) for 15 days before CP treatment showing negative COX-2 immunostaining of the kupffer cells and the hepatocytes (COX-2 immunohistochemistry H&E X 80)

Figure 3

Photomicrograph of a liver section of a rat from a normal control group (H&E X 40) showing no histopathological alteration and the normal histological structure of the central vein and surrounding hepatocytes were recorded (Figure 3A). Photomicrograph of a liver section of a rat from the cyclophosphamide-control group (H&E X 40) showing severe congestion and dilatation of the central and portal veins associated with degeneration in the hepatocytes surrounding the central vein and oedema in the portal area (Figure 3B). Photomicrograph of a liver section of a rat that received genistein (0.5 mg/kg/day, s.c.) for 15 days before CP treatment (H&E X 40) showing mild improvement in the overall histopathological picture. Inflammatory cells infiltration was apparent in the portal area while the hepatocytes showed degenerative change (Figure 3C). Photomicrograph of a liver section of a rat that received genistein (1 mg/kg/day, s.c.) for 15 days before CP treatment (H&E X 40) showing minimal improvement in the overall histopathological picture. Dilatation and congestion in the central veins associated with degeneration in the surrounding adjacent hepatocytes were apparent (Figure 3D). Photomicrograph of a liver section of a rat that received genistein (2 mg/kg/day, s.c.) for 15 days before CP treatment (H&E X 40) showing moderate improvement in the overall histopathological picture. Congestion in the central and portal veins along with degeneration in the surrounding hepatocytes was also apparent (Figure 3E)