Low dose of chronic ethanol exposure in adult zebrafish induces hepatic steatosis and injury

Abstract

Chronic alcohol consumption is a major cause of chronic liver disease worldwide. Adult zebrafish have emerged as a new vertebrate model of alcoholic liver disease. In previous research, a high dose of chronic ethanol treatment induced characteristic features of steatosis and hepatic injury in adult zebrafish, yet the ethanol concentration in that study was significantly higher than the lethal dose in humans. In the current study, we examined whether a low dose of chronic ethanol exposure in adult zebrafish induced the metabolic and pathological features seen in alcoholic liver disease. We found that chronic ethanol treatment at 0.2% ethanol (v/v) concentration for 4 weeks induced a significant elevation of serum glucose and triacylglycerol in adult zebrafish. In addition, serum alanine aminotransferase activity was significantly elevated after ethanol treatment. Histological analysis revealed steatosis and hepatocyte ballooning phenotype. Gene expression analysis using quantitative real-time PCR suggested that ethanol treatment induced inflammation, apoptosis, and fibrosis. In addition, we found significant increases in gene expression involved in glucose and lipid metabolism as well as mitochondrial biogenesis and function. Importantly, expression of genes involved in oxidative and endoplasmic reticulum stress, two major stress signaling pathways underlying hepatic injury in alcoholic liver disease, were highly upregulated in the livers of adult zebrafish after chronic ethanol treatment. In conclusion, we found that 4 weeks of low dose ethanol exposure leads to typical ethanol-induced liver disease, with pathological and gene expression patterns.

Keywords: Ethanol; Hepatic injury; Liver; Steatosis; Zebrafish.

Figure 1.

Chronic ethanol treatment induced hepatic injury, inflammation in the liver and elevation of serum glucose and triglycerides. Adult zebrafish were exposed to 0.2% ethanol for 4 weeks. (A) Hematoxylin and eosin (H&E) staining and Picrosirius Red staining in the liver of untreated and 0.2% ethanol treated adult zebrafish (top and middle). Control livers showed normal hepatocytes (n=8/10) and ethanol treated siblings showed hepatic injury (n=9/10). Control livers showed thin layer of picrosirius red positive staining surrounding blood vessels (n=6/6), ethanol exposed group showed intense staining of fibrotic tissue in some cases (n=2/5) Scale bar=50μm (top, bottom), 25 μm (middle). (B) Alanine aminotransferase assay from blood samples of control, 0.2 % ethanol exposed zebrafish. (C) Relative mRNA expression of genes associated with inflammation and liver injury. RNAs extracted from three to five siblings were used for analysis. (D) Quantification of serum glucose in control and ethanol treated zebrafish. (E) Quantification of serum triglycerides in control and ethanol treated zebrafish. Error bars indicate standard deviation of the mean. * p<0.05, ** p<0.005.

Figure 2.

Chronic ethanol treatment induced steatosis in the liver of adult zebrafish. (A) The relative mRNA expression of genes associated with lipid metabolism include lipogenic transcription factors, lipid synthesis, lipid uptake, and fatty acid transporting into the mitochondria. (B) Oil red O staining the section of liver in untreated control (left) and 0.2% ethanol treated wild type zebrafish. Scale bar=50 μm. (C) The quantification of triglycerides in the liver of control and ethanol treated zebrafish. Error bars indicate standard deviation of the mean. * p<0.05, ** p<0.005.

Figure 3.

Ethanol treatment in adult zebrafish activated genes associated with superoxide, hydrogen peroxide generation and regulation of redox balance in the liver. (A) Ethanol exposure increased relative mRNA expression in cyp2p6, cyp2y3, nox1, nox2 and nox5 in the liver. (B) Relative mRNA expression of nrf2, sod2 and genes associated with redox balance. (C) ROS quantification, (D) GSH level and (E) GSH/GSSG ratio were measured in the liver of control and 0.2% ethanol exposed groups. (F) Relative mRNA expression of genes associated with GSH synthesis including glutathione synthase (gss), glutamate-cysteine ligase, catalytic subunit (gclc), glutamate-cysteine ligase, modifier subunit (gclm), cystathionine b-synthase a (cbsa), betaine--homocysteine S-methyltransferase (bhmt). Error bars indicate standard deviation of the mean. * p<0.05, ** p<0.005.

Figure 4.

Low dose of ethanol treatment induced genes in UPR/ER stress in the liver of zebrafish. Error bars indicate standard deviation of the mean. * p<0.05, ** p<0.005.

Figure 5.

Ethanol treatment induced transcriptional activation of genes in gluconeogenesis, glycolysis, and pentose phosphate pathway in the liver of zebrafish. Relative mRNA expression of phosphoenolpyruvate carboxykinase 1 (pck1), glucose-6-phosphatase catalytic subunit (g6pc), glucokinase (gck), pyruvate kinase L/R (pklr) and glucose-6-phosphate dehydrogenase (g6pd). Error bars indicate standard deviation of the mean. * p<0.05, ** p<0.005.