The effects of astaxanthin on liver histopathology and expression of superoxide dismutase in rat aflatoxicosis

Abstract

The metabolism of aflatoxin B₁ (AFB₁) generates reactive oxygen species (ROS) that destroys hepatocytes. Meanwhile, astaxanthin (AX) is known to have stronger antioxidative activity than other carotenoids. This study aimed to investigate hepatoprotective role of AX from AFB₁-induced toxicity in rat by histopathological study and immunohistochemistry of Cu/Zn-SOD (SOD1) which acts as the first enzyme in antioxidative reaction against cell injury from ROS. Twenty Wistar rats were randomly divided into 4 groups. The control and AFB₁ groups were gavaged by water for 7 days followed by a single DMSO and 1 mg/kg AFB₁, respectively. The AXL+ AFB₁ and AXH+ AFB₁ groups were given of 5 mg/kg and 100 mg/kg AX for 7 days before 1 mg/kg AFB₁ administration. The result showed significantly elevated liver weight per 100 g body weight in AFB₁ group. The histopathological finding revealed vacuolar degeneration, necrosis, megalocytosis and binucleation of hepatocytes with bile duct hyperplasia in AFB₁ group. The severities of pathological changes were sequentially reduced in AXL+AFB₁ and AXH+AFB₁ groups. Most rats in AXH+AFB₁ group owned hypertrophic hepatocytes and atypical proliferation of cholangiocytes which are adaptive responses to severe hepatocyte damage. The SOD1 expression was also significantly higher in AXH+AFB₁ group than solely treated AFB₁ and AXL+AFB₁ groups. In conclusion, AX alleviated AFB₁-induced liver damage in rat by stimulating SOD1 expression and transdifferentiation of cholangiocytes in dose dependent manner.

Keywords: aflatoxin B1; astaxanthin; histopathology; immunohistochemistry; superoxide dismutase.

Fig. 1.

The illustration of histopathological findings of rat liver in control group (a) showed normal histology which some megalocytes, binucleated hepatocytes and apoptotic cells could be noticed (b). Liver tissue from AFB₁ group (c) revealed massive vacuolar degeneration in all lobules and bile duct hyperplasia in portal area. Megalocytes, binucleated hepatocytes and their degenerated forms as well as necrotic cells that underwent karyorrhexis were extensively found in this group (d). The vacuolar degeneration of hepatocytes was gradually reduced from AXL+AFB₁ group to AXH+AFB₁ group (e and g). The appearance of megalocytes and binucleated cells was still high in AXL+AFB₁ group (f). Necrotic and apoptotic cells were slightly observed in AXL+AFB₁ and AXH+AFB₁ groups (h). Thick arrow; Megalocyte, Black arrowhead; Binucleated hepatocyte, Black thin arrow; Degenerative binucleated hepatocyte, White thin arrow; Necrotic cell (karyorrhexis), White arrowhead; Apoptotic cell. AFB₁: aflatoxin B₁, AXL: low dose astaxanthin, AXH: high dose astaxanthin. H&E. Bar in a, c, e, g=200 µm, Bar in b, d, f, h=50 µm.

Fig. 2.

Hypertrophic hepatocytes associated with proliferation of cholangiocytes (gray arrows) revealed in some rats of AXL+AFB₁ and AXH+AFB₁ groups (this picture is representative fom AXH+AFB₁ group). Few hepatocytes showed abnormal large sized nucleus (megalocytes; black arrows) but most cells possessed increasing cellular content but normal nuclear size. H&E. AXL: low dose astaxanthin, AXH: high dose astaxanthin, AFB₁: aflatoxin B₁. Bar=50 µm.

Fig. 3.

Superoxide dismutase 1 (Cu/Zn-SOD: SOD1) Immunoreactivity demonstrated an expression in hepatocytes of (a) control, (b) AFB₁, (c) AXL+AFB₁ and (d) AXH+AFB₁ groups. The other types of cell, such as Kupffer cells and endothelial cells, were stained blue from hematoxylin dye. AFB₁: aflatoxin B₁, AXL: low dose astaxanthin, AXH: high dose astaxanthin. Bar=50 µm.

Fig. 4.

Percentage of superoxide dismutase 1 (Cu/Zn-SOD: SOD1) immunostained area from control, AFB₁, AXL+AFB₁ and AXH+AFB₁ groups. AFB₁: aflatoxin B₁, AXL: low dose astaxanthin, AXH: high dose astaxanthin. *,**,*** indicate significant difference among groups.

Fig. 5.

Western blot analysis of liver extract containing 25 µg of protein in each lane to detect superoxide dismutase 1 (Cu/Zn-SOD: SOD1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) band density. Normalized densitometric values of SOD1/GAPDH, presented as mean ± SD, indicated that the SOD1 expression level in AXH+AFB₁ group was significantly higher than that in AFB₁ group (P<0.05). AFB₁: aflatoxin B₁, AXL: low dose astaxanthin, AXH: high dose astaxanthin. a and b indicate significant difference among groups.