Basic Study
Copyright ©The Author(s) 2017. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Hepatol. Mar 18, 2017; 9(8): 418-426
Published online Mar 18, 2017. doi: 10.4254/wjh.v9.i8.418
Chronic exposure to ethanol causes steatosis and inflammation in zebrafish liver
Ana Claudia Reis Schneider, Cleandra Gregório, Carolina Uribe-Cruz, Ranieli Guizzo, Tais Malysz, Maria Cristina Faccioni-Heuser, Larisse Longo, Themis Reverbel da Silveira
Ana Claudia Reis Schneider, Carolina Uribe-Cruz, Ranieli Guizzo, Larisse Longo, Themis Reverbel da Silveira, Laboratório Experimental de Hepatologia e Gastroenterologia, Hospital de Clínicas de Porto Alegre, Porto Alegre, 90035-903 RS, Brazil
Ana Claudia Reis Schneider, Carolina Uribe-Cruz, Larisse Longo, Themis Reverbel da Silveira, Programa de Pós-Graduação em Ciências Médicas, Gastroenterologia e Hepatologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, 90035-003 RS, Brazil
Cleandra Gregório, Programa de Pós-Graduação em Genética e Biologia Molecular (UFRGS), Porto Alegre, 90035-003 RS, Brazil
Tais Malysz, Instituto de Ciências Básicas da Saúde (UFRGS), Porto Alegre, 90035-190 RS, Brazil
Maria Cristina Faccioni-Heuser, Departamento de Ciências Morfológicas (UFRGS), Porto Alegre, 90035-190 RS, Brazil
Maria Cristina Faccioni-Heuser, Centro de Microscopia Eletrônica (UFRGS), Porto Alegre, 91501-970 RS, Brazil
Author contributions: Schneider ACR and da Silveira TR conceived the project and designed the experiments; Gregório C, Guizzo R and Longo L carried out genetic assays and zebrafish liver histology; Malysz T and Faccioni-Heuser MC performed ultrastructural analysis; Schneider ACR and Uribe-Cruz C analysed the data; the manuscript was written by the first author Schneider ACR and reviewed by an English grammar professor; all authors contributed clarifications and guidance on the manuscript; all authors were involved in editing the manuscript; all authors read and approved the final manuscript.
Institutional animal care and use committee statement: The protocols were approved by the Research Ethics Committee of Hospital de Clínicas de Porto Alegre, Brazil (No. 10.0327). The protocols were conducted in accordance with international guidelines for the care and use of laboratory animals. Animal care is described in the manuscript.
Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at schneiderac@gmail.com. Participants gave informed consent for data sharing. No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Ana Claudia Reis Schneider, PhD, Laboratório Experimental de Hepatologia e Gastroenterologia, Hospital de Clínicas de Porto Alegre, 2350/sala 12214, Porto Alegre, 90035-903 RS, Brazil. schneiderac@gmail.com
Telephone: +55-51-33598847 Fax: +55-51-33598760
Received: July 12, 2016
Peer-review started: July 13, 2016
First decision: October 10, 2016
Revised: October 28, 2016
Accepted: January 11, 2017
Article in press: January 14, 2017
Published online: March 18, 2017
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: Ethanol, Hepatic steatosis, Inflammation, Zebrafish, Alcoholic fatty liver

Core tip: Excessive alcohol consumption remains one of the most important causes of liver disease worldwide. Alcoholic steatosis results from the deposition of fat in liver cells and is the earliest stage of alcohol-related liver disease. Usually inflammation is associated with steatohepatitis, however our results demonstrate that chronic ethanol exposure increased the expression of the inflammatory gene interleukin-1b. Paradoxically the expression of adiponectin receptor-2 and sirtuin-1 also increased for attenuating the liver injury. Ultrastructural abnormalities were observed showing early alterations in liver cells. Knowledge of alcohol injury mechanisms will contribute to the development of novel therapies in the treatment of alcoholic liver disease.