Chronic exposure to ethanol causes steatosis and inflammation in zebrafish liver

Abstract

Aim: To evaluate the effects of chronic exposure to ethanol in the liver and the expression of inflammatory genes in zebrafish.

Methods: Zebrafish (n = 104), wild type, adult, male and female, were divided into two groups: Control and ethanol (0.05 v/v). The ethanol was directly added into water; tanks water were changed every two days and the ethanol replaced. The animals were fed twice a day with fish food until satiety. After two and four weeks of trial, livers were dissected, histological analysis (hematoxilin-eosin and Oil Red staining) and gene expression assessment of adiponectin, adiponectin receptor 2 (adipor2), sirtuin-1 (sirt-1), tumor necrosis factor-alpha (tnf-a), interleukin-1b (il-1b) and interleukin-10 (il-10) were performed. Ultrastructural evaluations were conducted at fourth week.

Results: Exposing zebrafish to 0.5% ethanol developed intense liver steatosis after four weeks, as demonstrated by oil red staining. In ethanol-treated animals, the main ultrastructural changes were related to cytoplasmic lipid particles and droplets, increased number of rough endoplasmic reticulum cisterns and glycogen particles. Between two and four weeks, hepatic mRNA expression of il-1b, sirt-1 and adipor2 were upregulated, indicating that ethanol triggered signaling molecules which are key elements in both hepatic inflammatory and protective responses. Adiponectin was not detected in the liver of animals exposed and not exposed to ethanol, and il-10 did not show significant difference.

Conclusion: Data suggest that inflammatory signaling and ultrastructural alterations play a significant role during hepatic steatosis in zebrafish chronically exposed to ethanol.

Keywords: Alcoholic fatty liver; Ethanol; Hepatic steatosis; Inflammation; Zebrafish.

Figure 1

Hematoxylin-eosin staining of liver sections from zebrafish. A: C group (2 wk), the hepatocytes are aligned in cords, absence of fat droplets; B: E group (2 wk), without apparent changes compared with the C group; C: E group (4 wk), enlarged hepatocytes due to fatty infiltration. Magnification: 400 ×.

Figure 2

Oil red staining sections of zebrafish liver. A: C group (4 wk), absence of lipid droplets; B: E group (2 wk), mild presence of lipid droplets; C: E group (4 wk), intense lipid accumulation induced by ethanol in hepatocytes. Magnification: 400 ×.

Figure 3

Electron micrographs of liver sections of control (A, C and E) and ethanol exposed groups (B, D and F). A: Polygonal hepatocytes (H), spherical nucleus (n) sinusoid (s), endothelial cell (ec); B: Presence of large amount of glycogen (g) and lipid droplets (ld) in the hepatocytes cytoplasm; C: Intracellular canaliculus with large number of microvilli (a) within; E: It is noted the parallel arrangement of rough endoplasmic reticulum (rer) around the core; D: Myelin figure (mf) inside an intracellular canaliculus; F: Rough endoplasmic reticulum (rer) composed by 8-12 parallel cisterns; H: Hepatocytes; ec: Endothelial cell.

Figure 4

Effect of ethanol on mRNA liver expression of tumor necrosis factor-alpha and interleukin-1b. tnf-a was decreased significantly in E group compared to C at 2^nd week (P = 0.018) and increased along time up 4^th week (P < 0.001), reaching C group levels. Il1-b expression increased between 2 and 4 wk (P = 0.001) and at 4^th week there was a significant difference between C and E groups (P = 0.024). Statistical data were determined by the Kruskal-Wallis test and Dunn as post hoc test. Values significantly different where indicated: ^aSignificant statistical difference between 2 and 4 wk; ^bSignificant statistical difference between C and E groups. P < 0.05 was considered. tnf-a: Tumor necrosis factor-alpha; il: Interleukin.

Figure 5

Effect of ethanol on mRNA liver expression of adiponectin receptor 2 and sirtuin-1 of zebrafish. adipor2 and sirt-1 expression increased in E group between 2 and 4 wk; P < 0.0001 and P = 0.001, respectively. At 4^th week adipor2 of E group was increased compared to C, P = 0.006. ^aSignificant statistical difference between C and E groups; ^bSignificant statistical difference between 2 and 4 wk. P < 0.05 was considered significant. adipor2: Adiponectin receptor 2; sirt-1: Sirtuin-1.