THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THI QUYNH LAM IDENTIFYING THE EFFECT OF EXPOSURE TO DIOXIN AND FURAN ON HUMAN HEALTH LEADING TO DIFFUSE LARGE B LYMPHOMA THROUGH GENE-NETWORK CONSTRUCTION BACHELOR THESIS Study Mode: Full-time Major: Environmental Science and Management Faculty: International Programs Office Batch: 2013 - 2017 Thai Nguyen, December 2017 DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Student name Nguyen Thi Quynh Lam Student ID DTN1353110372 Identifying the effect of exposure to dioxin and furans on Thesis Title human health leading to diffuse large B lymphoma through gene-network construction Supervisor(s) Prof. ChunYu Chuang, Assoc. Tran Thi Thu Ha Abstract: Many studies indicated that exposure to dioxins and dioxins – compounds (e., 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) and polychlorinated dibenzofurans (furans) can induce several outcomes on human and animal in the long term period, and one of them is diffuse large B lymphoma which is considered as the most popular kind of lymphoma. In order to identify the gene expression altered by TCDD and Furans potentially underlying DLBCL development, bioinformatics meta-analysis was applied in this study.
In this study, 10 datasets containing the information of gene expression of DLBCL, TCDD and Furans were obtained from Gene Expression Omnibus (GEO) and Array Express websites, and further analyzed using Cytoscape software and its plugins – ClueGO and CluePedia. As a result, the most differentially expressed genes were found to construct gene- networks of DLBCL, TCDD and furans and hence the potential pathway presented how dioxins could cause the progress of lymphoma. In addition, the analytical result i reported that TCDD and furans have a possibility to induce the receptor AhR which promotes the appearance of protein TWIST1 and enhance the progress of DLBCL. The result of this study has made a great contribution for further dioxins study, and it is also considered as the initial steps of future work for DLBCL diagnosis and treatment.
Keywords: TCDD, Furans, DBLCL, bioinformatics, GEO, Array Express Number of pages: 87 Date of Submission: 20/09/2017 Supervisor’s signature ii ACKNOWLEDGEMENT First of all, I would like to use this opportunity to express my deepest gratitude and special thanks to Prof. Chun-Yu Chuang for her patient to guide and keep me on the correct path and show me many of wonderful things during the time of my internship at the Department of Biomedical Engineering and Environmental Science at National Tsing Hua University. I would like to express my deep thanks to Assoc. Tran Thi Thu Ha for giving me necessary advices and guidance in order to complete my thesis.
My sincere thanks are also given to all the members working in the Department of Biomedical Engineering and Environmental Science for supporting me all the materials and necessities when conducting experiments for my research. Finally, I would like to thank my family and my friends encouraging me and advising me during completion of this thesis. Thai Nguyen, October 2017 Nguyen Thi Quynh Lam iii TABLE OF CONTENTS ACKNOWLEDGEMENT. iii TABLE OF CONTENTS.
iv LIST OF FIGURES. vii LIST OF TABLES. viii LIST OF ABBREVIATIONS. Persistent Organic Compounds (POPs).
Dioxins and dioxin – liked compounds. Lymphoma and non – Hodgkin lymphoma. Diffuse large B lymphoma. SNPs of Diffuse Large B lymphoma.
Gene - network components. Gene network database: Array Express and GEO. Gene Network construction tools. Network Analyst website.
Cytoscape software and plugins: ClueGO and CluePedia Apps. RESULTS AND DISCUSSION. Differentially genes expression. Gene-network construction of DLBCL, TCDD and Furans.
Protein – protein interaction network of DLBCL, TCDD and Furans. Potential pathway showing the relation between TCDD and Furans and Diffuse Large B lymphoma. AhR – mediated key factor of dioxins – like compounds. Key factors of hypoxia response and the risk of MYC – TP53 interaction.
Inhibition of cancer cell apoptosis and tumorigenesis factor in DLBCL. Differentially expressed genes of DLBCL versus normal cell. Differentially expressed genes of exposure to TCDD group and versus control group. Differentially expressed genes of exposure to FURANS group versus control group.
Hub proteins of DLBCL network. Hub proteins of TCDD network. Hub proteins of FURANS network. 76 vi LIST OF FIGURES Figure 2.1: General molecular structure of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (Source: Pereira, 2004).2: Representative structure of 2,3,7,8-tetrachhlorodibenzo-p-dioxins (TCDD) (Pereira, 2004) .3: A schematic representation of signal transduction after TCDD/AHR interaction (Fracchiolla et al.1: The flowchart of methodology.1: Gene Ontology network showing the relationship of DLBCL, TCDD and Furans .2: Protein – protein interaction network constructed by CluePedia plugin in Cytoscape .3: Flowchart of proteins interaction and involved biological processes .37 vii LIST OF TABLES Table 4.1: Database of DLBCL .2: Database of TCDD and Furans .3: Differentially expressed genes, including up-and down-regulated genes in Diffuse Large B lymphoma compared to normal cells .4: Differentially expressed genes, including up-and down-regulated genes activated by TCDD compared to control group .5: Differentially expressed genes, including up-and down-regulated genes activated by Furans compared to control group .6: Lists of hub proteins containing in DLBCL, TCDD and Furans networks.
34 viii LIST OF ABBREVIATIONS ABC DLBCL activated B – cell like DLBCL AhR Aryl Hydrocarbon Receptor ARNT Aryl Hydrocarbon Receptor nuclear translocator B-NHL B cell non-Hodgkin lymphoma CRE CAMP response element DEG Differentially expressed genes DLBCL Diffuse large B cell lymphoma DNA Deoxyribonucleic acid DNMT1 DNA methyl transferase EGFR Epidermal growth factor receptor FDR False discovery rate GCB DLBCL Germinal center B-cell like DLBCL GEO Gene expression omnibus GO Gene ontology HAHs Halogenate aromatic hydrocarbon MAGE-ML Microarray and Gene Expression Markup Language MAGE-TAB Microarray Gene Expression - Tabular format MIAME Minimum information about microarray experiment MM Mismatch OC Organochlorine PCBs Polychlorinated biphenyls ix PCDD/Fs Polychlorinated dibenzo-p-dioxins/furans PCDDs Polychlorinated dibenzofurans PM Perfect match POPs Persistent organic compounds ROS Reactive oxygen species SNPs Single nucleotide polymorphisms TCDD 2,3,7,8 tetrachlorodibenzo-p-dioxin TNF Tumor necrosis factor XRE Xenobiotic response elements transcription. Research rationale Dioxins and dioxins-like compounds are largely concerned these days due to their permanent impacts on human and animals in the long-term period. TCDD and Furans are representative of dioxins and dioxins-liked compounds, which can influence negatively on human health with a little amount through bio-magnification and food chain. The most significant impact of these chemicals is genetic variation through aryl hydrocarbon receptor (AhR) activation when it passes into nucleus in animal body and hence induces genetic disease and carcinogenesis.
Diffuse large B lymphoma (DLBCL) is the most prevalent B cell non – Hodgkin lymphoma, which occupies 40% of lymphoma diagnoses. The cause of Diffuse Large B lymphoma is exactly unknown, however, many pro-oncogenes and abnormal genes causing lymphoma have been found in previous studies. The identification of biological mechanisms activating those genes, whether they are related to dioxins and dioxins-liked compounds impact or not, is highly essential to be considered. Bioinformatics, including sequence analysis, gene and protein expression, cellular organization analysis, structural bioinformatics, network and system biology and others, has a large contribution to various fields in global scale.
The application of bioinformatics in biomedical has been largely paid attention in many developed countries, by contrast, it is still unpopular in Vietnam. Specifically, many researches indicated that the application of high sequencing and DNA microarray technology has a significant role in attempt to identify genetic/transcriptomic alterations causing DLBCL and prognosis biomarkers for 1 lymphoma treatment. Therefore, the activation of those abnormal genes and the influence of dioxins can be clarified by the application of bioinformatics. In order to clarify diagnosis of lymphoma, the study “Identifying the effect of exposure TCDD and Furans on human health leading to diffuse large B lymphoma through gene- network construction” has been conducted with promotion of Biomedical Engineering and Environmental Science faculty of National Tsing Hua University in Taiwan.
Research objectives The objectives of this research are: - To investigate respectively the differentially expressed genes for diffuse large B lymphoma (DLBCL) tissues and dioxin exposure of human cell lines; - To construct the gene-network for exploring number whether exposure to dioxin can induce DLBCL; - To identify the potential pathway exposure to dioxin corresponding to DLBCL. Persistent Organic Compounds (POPs) Persistent organic compounds include a variety of lipophilic compounds that relate to environmental degradation. Amongst various kinds of POPs, for example, Organochlorine (OC) pesticides or industrial chemicals of by products, the category containing Cl atom has a great ability to cause the most deleterious effects and as a consequently, they have been banned and strictly regulated in many countries. Despite of that regulation, POPs exposure sustains in general population due to the consumption of fatty acid derived from animals.
The concentration of POPs has a tendency of increasing which corresponds to the level of food webs in order to perform biomagnification, as a results, the POPs concentration accumulating in human bodies might be higher compared to the external environment (Fisher et al. In addition, POPs accumulated in adipose tissue in life is considered as one route of chronic exposure since they are continuously released from adipose tissue to the circulation and vital organs with lipid content (La Merrill et al. POPs consist of these main properties. The first property is mentioned as a combination of lipophilic compounds that accumulate mainly in lipid – containing tissues like adipose tissue and move within the body bound to lipids (Lewis et al.
In addition, POPs are always presented as chemical mixtures in the external environment due to mixing in the environment, food web, long – term retention of fat tissues (Kortenkamp et al., 2008) Therefore, these distinct groups of OC pesticides, polychlorinated biphenyls (PCBs) and dioxins are classified referring to chemical mixtures of each POPs subclasses. Dioxins and dioxin – liked compounds Polychlorinated dibenzo-p-dioxins/furans (PCDD/Fs) is classified as ubiquitous POPs. PCDD/Fs is two of the three subclasses of the halogenated aromatic hydrocacbon and two of them are referred as dioxins and dioxin-liked-compounds respectively (see Figure 2.1: General molecular structure of polychlorinated dibenzo-p-dioxins (PCDD) and dibenzofurans (PCDF) (Source: Pereira, 2004). They are widespread in almost area in the environment, especially there is no exception for the remote area.
Dioxins and dioxin-liked-compounds tend to be persistent and lipophilic in the external environment so that they can be bio- accumulated through food chains and potentially cause potential effects on human health biota and even human. PCDD/Fs are two of subclasses of the halogenate aromatic hydrocarbon (HAHs), which are specified by the basic aromatic structure of a benzene ring, a hexagonal carbon structure with conjugated double bonds connecting to the carbon. The difference of both dioxins and dioxins like compounds depends on the number of oxygen rings in their structure; are 2 and 1 rings respectively. The biological impacts of PCDDs/Fs are activated by the production of 4 similar spectrum of toxic effects through binding of dioxins and dioxins-liked- compounds to a receptor protein – Aryl Hydrocarbon Receptor (AHR).
The molecular planar shape facilities binding to the receptor and its relative potency depends to a large degree on its persistence and how well it fits to the receptor. PCDDs/Fs and one component of PCDDs – tetrachlorodibenzo-p-dioxins (TCDD) have a high affinity to AHR and fit very well on that receptor, actively. PCDD/Fs are derived from 4 main sources, including (1) combustion, (2) meta – smelting, refining and processing, and (3) biological and photochemical process (US National Research Council, 2006). PCDD/Fs has a potential to cause cancer, birth effect, reproductive disorders, immunotoxicity, and other potential toxic end points, including liver diseases, thyroid dysfunction, lipid disorders, neurotoxicity, cardiovascular disease, and metabolic disorders, such as diabetes (US National Research Council, 2006).
* 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD): According Pereira (2004) 2,3,7,8-tetrachhlorodibenzo-p-dioxins (TCDD) is structured as below (see Figure 2.2: Representative structure of 2,3,7,8-tetrachhlorodibenzo-p-dioxins (TCDD) (Source: Pereira, 2004). 5 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic members of the family of polychlorinated dibenzodioxin (PCDDs) and represents a nearly ubiquitous environmental contaminant (Pesatori et al.