Silveira

GROUP LEADER NAME (as it appears in publications):

Silveira TR (Themis Reverbel da Silveira)

AFFILIATION:

Universidade Federal do Rio Grande do Sul – Hospital de Clínicas de Porto Alegre

TEL:*

55 51 3359 8847

FAX:*

55 51 3359 8761

EMAIL:*

themis.silveira@gmail.com

WEB:

GROUP NAME / RESEARCH INTEREST IN FEW WORDS:

Laboratório de Hepatologia e Gastroenterologia Experimental

SHORT SUMMARY OF RESEARCH INTEREST (MAX. 200 WORDS):

Note: please attach at the end of the form a longer description, with a maximum of 2000 words.

LIST OF UP TO FIVE RELEVANT PUBLICATIONS:

1 - Schneider, ACR. ; Schaefer PG ; Porawski, M. ; Maurer RL ; Hammes, TO. ; Pedroso, G. L. ; Silveira, TR . Alcoholic Steatosis in Zebrafish: a New Model to an Old Disease. 2010. (5 th Aquatic Animal Models dor Human Disease. (Oral presentation).

2 - Taurine effect in hepatic steatosis induced by thioacetamide in Zebrafish (Danio rerio). 7th European Zebrafish Meeting 2011. (Poster presentation)

3 - Evaluation of Lactobacillus rhamnosus GG in Zebrafish intestines after ethanol exposure. ). 7th European Zebrafish Meeting 2011. (Poster presentation)

4 - The effect of Taurine on hepatic steatosis induced by thioacetamide in Zebrafish (Danio rerio). Submitted in Digestive Disease and Science.

GROUP MEMBERS (NAME, POSITION, EMAIL):*

1- Ana Cláudia dos Reis Schneider; Doctoral Student; aschneider@hcpa.ufrgs.br

2- Thais Ortiz Hammes; Masters Student; thaisortizhammes@hcpa.ufrgs.br

3- Gabriela Lima Pedroso; Masters Student; glimapedroso@gmail.com

4- Djuli Hermes; Masters Student; dhermes@hcpa.ufrgs.br

5- Laisa Beduschi Fracasso; Graduate student; laisabeduschi@hotmail.com

FISH FACILITIES (TYPE OF FISH SYSTEM/TANKS, CAPACITY, ETC.)*

The laboratory has eleven aquariums com biological filters with the following capacities:

- Two aquariums with 50 liters

- Two aquariums with 35 liters

- Four aquariums with 10 liters

- One aquarium with 17, 8 and 5 liters each.

FISH LINES KEPT IN STOCK:*

OTHER EQUIPMENT RELATED TO ZEBRAFISH RESEARCH*

LAB EXPERTISE AND TECHNICAL CAPABILITIES (RELATED TO ZEBRAFISH RESEARCH)*

- Optimized technique of Blood Collection

- Biochemical Analysis standardized to small blood quantities

- qRT-PCR

- Models of the hepatic steatosis (alcoholic and non-alcoholic)

- Analysis of intestinal microbiota

- Histological Analysis

OTHER RELEVANT INFORMATION:*

Longer description:

Biomedical research depends, in many aspects, on the use of animal models to understand the etiopathogenesis of several human diseases and for developing and testing new therapies. Animal models are required to help us understand the signaling pathways underlying several disorders. The liver is an essential organ in the body and perform a number of vital activities including metabolism of endogenous and exogenous substances, detoxification and homestasis. The very important metabolic role of the liver is related to its unique position within the systemic circulation. The liver of fishes can store large amounts of substances, like lipids and glycogen heterotopically distributed in the organ as a response to endogenous or exogenous changes. Research over the past two decades has demonstrated that the liver of ZF contain many of the same metabolic pathways and enzymes known for human livers however differences exist. One is the absence of a functional metabolic zonation.

Liver diseases including Hepatitis B and C , cirrhosis, hepatocellular carcinoma and the spectrum of disorders related to alcoholism are worldwide health problems. As early as the 1930s the Zebrafish (ZF) was being used as a model for understanding human diseases. The ZF has become a particularly useful preparation to analyse how vertebrate development is regulated at the cellular and molecular levels because both genetic and experimental embryological methods can be easily applied to this model. The identification of thousands of early developmental mutants established the ZF as an important model in developmental biology.

Although ZF has been a popular subject of research in the past three decades its liver and biliary function have not been well studied compared to others laboratory model animals (like the mouse and the rat). In recent years, however, ZF is increasingly being used to study liver diseases and now has a prominent position in the field of hepatology’s research. They are a powerful vertebrate system for modeling human disorders and have been employed in studies concerning a great diversity of aspects of liver diseases and embryonic development. The ZF has become a particularly useful preparation to analyse how vertebrate development is regulated at the cellular and molecular levels. Genes essential for differentiation of hepatocytes and bile duct cells were described. Jagged 1,2,3 and notch 1a,1b,2 and 5 are expressed in liver at 48 hpf and 72 hpf, precisely the time when bile ducts are formed. The absence of jagged and notch genes affects the biliary development causing a phenotype similar to the human Alagille Syndrome ( Ductular hypoplasia). Very recently Matthews and colleagues proposed a epigenetic activation of Interferon-gama signalling as a common etiologic mechanism of intrahepatic duct defects in Extra-hepatic Biliary Atresia – a progressive fibroinflammatory disorder of bile ducts - based in studies of the DNA-methylation of ZF larvae.

Carcinogenesis and investigation of molecular mechanisms of disease and toxicity are increasingly studied in ZF. ZF develop several malignant tumors in response to mutagens, carcinogens and spontaneously as well as on loss of tumor suppressor function like p53. One well- known example of induced tumorigenesis in ZF is Lymphoid leukemia resulting from overexpression of an oncogene (Tg(rag2-c-Myc). Recently liver tumors were generated in ZF by treating the fish with carcinogens and the authors obtained the gene set for liver tumors via microarray hybridization establishing the ZF as a model system to study liver tumorogenesis.

Considering the DNA sequence homologies found between ZF genes and other vertebrate genes (for example those of mammals) complex human diseases like fatty liver disorders may be investigated. Hepatic steatosis or accumulation of fat in hepatocytes, is a common histologic finding. The lower limit of steatosis to be considered “normal” is not established in ZF. It is a lesion common in, but no restricted to obesity, alcoholic liver disease, viral hepatitis C and consequence of several drugs. It was already showed that thiocetamide, hexachlorocyclohexanen and alcohol can induce fatty liver. The fish is a very good model for studying the molecular mechanism behind hepatic steatosis. Several mutants have been shown this: the foie-gras mutant, the gonzo mutant and mutations in gene S-adenosyl-homocysteine hydrolase increasing TNF-α expression cause hepatic steatosis phenotype in the ducttrip mutant. Many factors are involved in the pathogenesis and progression of steatosis to steatohepatitis, such as oxidative stress, insulin resistance and inflammatory cascade. A two-hit theory postulates that steatosis, the first hit, increases the sensitivity to oxidative stress and to proinflammatory cytokines, which characterizes the second hit. Recently a third hit – hepatocyte death and lack of repair – was included to explain the progression.

Obesity is a common and increasingly prevalent disorder in the developed and developing world. It is an important national health problem. A third of adults in Brazil are overweight and almost another third are obese. The children/adolescents are getting fatter every year. Obesity should be viewed as a condition multi-systemic rather than a specific disease with a single cause. Probably it is a heterogeneous condition that is influenced by genetics and environmental factors. Determination of the factors that influence the development of obesity remains a high priority if prevention is to be undertaken. Great part of total body energy is stored in the form of triglycerides and adipocyte fat comprises nearly all the energy stores under normal physiological conditions. One of the most common metabolic complications of obesity is fatty liver disease in both animals and humans. An increasing number of patients requiring liver transplantation appear to have steatosis-related cirrhosis, without evidence of alcoholic liver disease, toxicity of other drugs, or viral hepatitis. Although there are studies with ZF mutated in cytokines and receptors in the setting of obesity, research using obese wild ZF are restrict. Recently a diet-induced obesity model in ZF was established, and validated, that shared common pathophysiological pathways with mammalian condition. ZF can, then, be used to identify putative pharmacological targets and to test novel drugs for the treatment of human obesity.

Ethanol is a well-known toxicant to mammals and alcoholism is one of the biggest and costliest diseases whose mechanisms are not well understood. Four decades ago Charles Lieber and colleagues published a seminal article showing that alcohol itself is the primary cause for the higher prevalence of liver disease seen in alcoholic patients and not the dietary deficiencies and malnutrition that often exist in alcoholism. Alcoholic liver diseases encompasses a varied of clinical and histological spectrum. On one end is fatty liver (steatosis) which used to be reversible with abstinence and alcoholic hepatitis which may or may not improve with abstinence; on the other end of the spectrum are cirrhosis that have a poor prognosis . In summary, from the human perspective, chronic alcohol abuse commonly causes three types of liver disease: fatty liver, alcoholic hepatitis and fibrosis/cirrhosis. In addition alcohol causes hepatocellular carcinoma and contributes to liver damage in patients with other liver diseases, especially viral hepatitis C.

The effects of ethanol on ZF have been extensively studied from a development biology perspective in ZF. Pregnant women who consume large amounts of ethanol often give birth to children exhibiting phenotypic abnormalities (Fetal alcohol syndrome) and ZF have been proposed as a model of this disease. There are several well conducted studies into the effects of ethanol on the developing nervous system, eye development and behavioral changes related to effects of acute and chronic exposure. The genes and pathways necessary for alcohol metabolism are highly conserved in ZF. A model of acute alcoholic liver disease was developed and the role of Unfolded Protein Response (UPR) studied. Exposing 4 dpf larvae to 2% alcohol for 32 hours causes morphologicalchanges that include pericardial edema, lordosis and hepatic steatosis with mitochondrial swelling. The role of UPR was demonstrated to be a central pathway of this disease. Gerlai and colleagues showed the similarity of behavioral effects of acute and chronic ethanol treatment in the ZF brain with the human response. They also provide evidence for adaptation to ethanol in adult ZF. After two week long chronic treatment with ethanol, ZF that receive the same ethanol dose during testing essentially behave the same way as fish that never exposed to ethanol. However, not much is known about the results of the alcohol chronic exposure on the liver function and histology of adult, wild, ZF. The pathogenesis of Alcohol liver disease is multifactorial. Alcohol and its toxic metabolites can damage key liver cells through the excessive generation of free radicals. In addition to its direct effect on the liver alcohol can increase the “leakiness” of the intestine cell wall. Concerning the alcoholic liver disease in humans the bowel-liver interactions are well described. Alcohol abuse is associated with increased gut permeability and with high frequency of endotoxemia. Endotoxin (lipopolysaccharide) leaking from the gut is recognized by the Toll-like receptors, interacts with hepatic cells which causes secretion of cytokines that promotes a wide range of inflammatory responses which lead to the production of additional cytokines. This complex chain of events plays a key role in the development and progression of liver disease. It is necessary to examine whether the ZF model could also be used to study the intestine-liver axis in alcohol chronic abuse. The modulation of the bowel flora may play a role in the pathogenesis and treatment of patients with this severe condition.

In our laboratory (at Centro de Pesquisas do Hospital de Clínicas de Porto Alegre) the present interest is the study of ZF liver steatosis related to different agents. We are particularly interested in the hallmark of early alcohol-induced liver injury that is steatosis. Although many hypothesis have been postulated for the toxic effects of ethanol to the adult ZF liver the mechanisms is unknown. Steatosis due to chronic abuse is largely attributed to the oxidative stress (which results in mitochondrial damage and reduced lipid oxidation) and the activation of sterol response element binding protein (SREBP) of the lipid synthesis induced. In part, the hepatotoxicity of ethanol, a byproduct of glucose metabolism by yeast, is believed to be associated with ethanol-mediated induction of cytochrome P4502EI (CYP2EI) and a resulting shift in ethanol metabolism to include a greater contribution by CYP2EI vs. the dominant pathway (alcohol and acetaldehyde dehydrogenases). CIP2EI is a relatively leaky P450 and substancial O2 and H2O2 are generated as a result of its activity, which can thereby contribute to oxidative stress in the liver during ethanol chronic exposure.

We are looking for the alterations of biochemical biomarkers as initial response from alcoholic liver disorders. In a condition that spans the spectrum from mild fatty change to inflammation, necrosis, apoptosis and fibrosis there are numerous alterations in normal biochemical pathways and physiologic processes. Although the pathogenesis of hepatic steatosis still remains poorly understood it is known that free fatty acids and adipocytokines, such as TNF-α and adiponectin, have a central role in its development. It is well known that there is no consensus about therapies for non alcoholic fatty liver disease (NAFLD) and, actually, many pharmacological agents are being tested for this purpose. In order to search for new therapies for NAFLD we did a study evaluating whether taurine treatment has a protective effect on hepatic steatosis induced by thiacetamide in ZF. Although there are some studies that demonstrated the antioxidant role of taurine we have not found any one that has analysed the effect on oxidative stress in the adult ZF liver. In our opinion taurine seems to improve steatosis by reducing oxidative stress and increasing SIRT1 ( Sirtuin 1) expression. Based on our results, taurine may be a promising therapy for treating hepatic steatosis.