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Predictive Toxicogenomics for the Identification of Chemical Carcinogens : Application to Human Hepatic Cell Lines

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Predictive Toxicogenomics for the Identification of Chemical Carcinogens : Application to Human Hepatic Cell Lines Predictive toxicogenomics for the identification of chemical carcinogens : application to human hepatic cell lines Citation for published version (APA): Christina Magkoufopoulou, C. (2011). Predictive toxicogenomics for the identification of chemical carcinogens : application to human hepatic cell lines. Datawyse / Universitaire Pers Maastricht. https://doi.org/10.26481/dis.20111208cc Document status and date: Published: 01/01/2011 DOI: 10.26481/dis.20111208cc Document Version: Publisher's PDF, also known as Version of record Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. 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If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: www.umlib.nl/taverne-license Take down policy If you believe that this document breaches copyright please contact us at: [email protected] providing details and we will investigate your claim. Download date: 30 Sep. 2021 Predictive toxicogenomics for the identification of chemical carcinogens: Application to human hepatic cell lines Christina Magkoufopoulou Christina Magkoufopoulou, Maastricht 2011 ISBN: 978 94 6159 096 1 Layout: Lia Rozou Printing: Datawyse/Universitaire Pers Maastricht, The Netherlands Predictive toxicogenomics for the identification of chemical carcinogens: Application to human hepatic cell lines DISSERTATION to obtain the degree of Doctor at Maastricht University, on the authority of the Rector Magnificus, Prof. dr. G.P.M.F Mols, in accordance with the decision of the Board of Deans, to be defended in public on Thursday 8th December 2011, at 10:00 by Christina Magkoufopoulou UMP UNIVERSITAIRE PERS MAASTRICHT Supervisor Prof. dr. J.C.S. Kleinjans Co-supervisor Dr. J. van Delft Assessment Committee Prof. dr. F.C.S. Ramaekers (chairman) Prof. dr. D. Kirkland Prof. dr. P. Lambin Prof. dr. L.H.F. Mullenders Prof. dr. H. Schmidt This research project was supported by grants from the Dutch Technology Foundation STW, applied science division of NWO and the Technology Program of the Ministry of Economic Affairs (STW MFA6809). Contents Chapter 1 General introduction 7 Chapter 2 Comparison of HepG2 and HepaRG by whole-genome gene expression 31 analysis for the purpose of chemical hazard identification Chapter 3 Comparison of phenotypic and transcriptomic effects of false positive 53 genotoxins, true genotoxins and non-genotoxins using HepG2 cells Chapter 4 A transcriptomics-based in vitro assay for predicting chemical 77 genotoxicity in vivo Chapter 5 Characterization of the importance of NR0B2 as a classifying gene for 97 genotoxicity prediction: a silencing approach prior and after genotoxic treatment Chapter 6 Transcriptomics-based evaluation in HepG2 cells of human 119 carcinogenic potential of known rodent non-genotoxic carcinogens Chapter 7 Summary and general discussion 143 Chapter 8 Samenvatting en algemene discussie 157 Acknowledgements 173 Curriculum vitae and list of publications 177 Chapter 1 General introduction 7 8 Chapter 1 Molecular biology of cancer With cancer being one of the leading causes of death in the western world, accounting for approximately 25% of total deaths (1, 2), a lot of effort is made for the understanding of the molecular events that precede carcinogenesis. The main hypothesis for the explanation of cancer development refers to the occurrence of gene mutations. Whereas several types of cancer have been associated with the presence of specific gene mutations, these are not suf- ficient or required to induce cancer. For instance, although the inheritance of specific mutations of the BRCA1 or BRCA2 genes predispose for breast cancer; different studies result in different average risk for breast cancer in BRCA1 mutation carriers varying from 57% to 90% (3). In a similar manner, whereas mutations in the APC gene are responsible for the development of familial adenomatous polyposis, a hereditary disease leading to colorectal cancer, a significant number of patients diagnosed with this disease do not carry these mutations (4). In general, hereditary cancers account for only 5%-10% of all diagnosed cancers, whereas the remaining 90%-95% of cancer incidences, which are characterized as sporadic, are due to environmental exposures, dietary factors, hormones, normal aging and other influences (5, 6). Independently from the type of cancer, whether it is inherited or sporadic, it is generally acknowledged that carcinogenesis is a very complicated process that comprises several alterations in the cells required for the progressive conversion of normal cells into cancer cells (7, 8). These alterations, at the molecular level, mainly refer to changes in the genome of a particular cell and include point mutations leading to an altered protein product; chro- mosomal imbalance or instability resulting in an altered expression of a particular gene; chromosomal breakage and rearrangement resulting to loss of a gene or its fusion with an- other gene leading to the production of a chimeric protein with altered function; and epige- netic modifications to DNA causing gene silencing. Occurrence of mutations to oncogenes or tumor suppressor genes is considered the initial event of the process of carcinogenesis. Oncogenes are defined as genes whose products positively regulate cell proliferation. Mu- tations to these genes usually result to an increased activity of the proteins that they code for. On the contrary, tumor suppressor genes code for proteins that negatively regulate cell proliferation and, in most cases, mutations to these genes inactivate the protein function (6). Usually, a mutation to one copy of the oncogene is sufficient to alter the function of the protein, whereas for the tumor suppressor genes both copies need to be mutated for the function of the protein to change. Therefore, mutations in oncogenes are called dominant, while mutations in tumor suppressor genes are called recessive (9). At the cellular level, the alterations that occur provide the cells with growth advan- tages and can be divided into 8 different types: sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, reprogramming energy metabolism, evading immune destruction, inducing angiogenesis and activating invasion and metastasis. Besides the genomic instability described above, another cause that can lead to the acquisition of these properties from the cancer cells is the presence of inflammatory cells in the tumor microenvironment, as inflammatory cells can release General introduction 9 chemicals that are mutagenic for the nearby cancer cells and therefore can accelerate their genomic instability (10). Chemical carcinogenesis Although the alterations occurring in a cell that can lead to its transformation into a tumor cell as described above, can happen spontaneously, many times these alterations are caused by exogenous factors, such as chemical compounds. Exposure to these com- pounds may lead to carcinogenesis. This process of chemical carcinogenesis has been di- vided in three distinct stages: initiation, promotion and progression (Figure 1) (8). Figure 1: Chemical Carcinogenesis. After exposure to chemicals the process of chemical carcinogenesis is initiated. The cells can repair the induced DNA damage and return to the normal state or proliferate with damaged DNA entering the promotion phase of chemical carcinogenesis. The cells with DNA adducts as well as the initiated cells may either induce apoptotic mechanisms or mechanisms of toxicity that will lead to cell death or further proliferate entering the progression phase of chemical carcinogenesis. The cells that are in the progression phase will be transformed to can- cer cells. [Figure modified from (8)] Initiation is the first stage of carcinogenesis where the cells accumulate genomic changes caused by the chemical agents. These genomic changes are due to the direct in- teraction of the chemical or its metabolites with the DNA, via the formation of DNA-adducts (11). At this stage, the cells may avoid tumor transformation by either repairing the DNA- damage or by activating apoptotic mechanisms (12, 13). However, if the cells proceed to cell division while carrying these genomic changes, these changes are irreversible and trans-
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