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Chapter 1 Introduction 1.1 An overview Carcinogenesis is an intricate multistep and multifactorial process in which environment-gene interaction plays an important role (Godderis et al., 2012). It involves sequential genetic mutation resulting in uncontrolled cell proliferation and different homeostatic dysregulation in normal cells. Mechanism of cancer progression is still partially known, which had made it as leading cause of death in the world (Jemal et al., 2011). The concepts of gene-environment interactions and human cancer risk were generated by the synthesis of chemical during the industrialisation in early 20th century. Global epidemiologic studies have identified environmental and occupational exposure of chemicals as a potent carcinogen (Loeb & Harris, 2008). Evidence from epidemiological, occupational and migration studies has consistently pointed to environmental factors as the major cause for cancer (Luch, 2005). In, this regard involvement of ploy aromatic hydrocarbons becomes very important as we are routinely exposed to these arrays of compounds. 1.2 Poly Aromatic Hydrocarbon (PAHs) and its carcinogenicity: Poly Aromatic Hydrocarbon (PAHs) produced naturally by forest fires and volcanoes. Industrialization and change in human life style e.g. burning of coal, wood, petroleum products, tires, polypropylene and motor vehicle exhaust, the level of PAHs has increased in our environment (Cherng et al., 1996).There are approximately 100 different known PAHs in air, soil, foodstuffs, water and diesel exhaust which contains significant amounts of PAHs (Zedeck,1980). Polycyclic aromatic hydrocarbons (PAHs) refer to a ubiquitous group of 1 Chapter 1 Introduction several hundred chemical which are environmentally persistent organic compounds of various structures and varied toxicity. Most of them are formed by a process of thermal breakdown and recombination of organic molecules. Often, PAHs consist of three or more fused benzene rings containing only carbon and hydrogen. PAH-induced carcinogenesis can result in formation of bulky PAH-DNA adduct which is critical for the regulation of cell differentiation or growth. If this DNA aberration remains unrepaired during the cell replication process, it will result in gene mutation which leads to cell transformation or carcinogenesis. The carcinogenicity of certain PAHs is well established in laboratory animals. Researchers have reported increased incidences of skin, lung, bladder, liver, and stomach cancers. Animal studies show that certain PAHs can affect different biological systems likehematopoietic, immune systems, reproductive, neurologic, and developmental system (Dasgupta and Lahiri, 1992; Szczeklik et al., 1994; Zhao, 1990). MCA alkylated derivative of benz[a]anthracene and being widely used all around the world for in vitro cell transformation assay (Peterson et al., 1981; Sakiyama et al., 1986;Miller and Hall,1991) 1.3 Cell Transformation Assay (CTAs) In vitro cell transformation assay has provided powerful tool to study the cellular and molecular mechanisms of chemical carcinogenesis (Fernandez et al., 1980). Two stage cell transformation assay measures the phenotypic conversion of cell from normal to malignant type. CTAs not only provide the powerful tool to study the carcinogenic potential of any chemical compound but it also gives us the molecular mechanism of cancer. When cells are exposed 2 Chapter 1 Introduction to sub-threshold dose of carcinogen (a tumor initiator) and subsequently with a tumor promoter (a typical non-genotoxic carcinogen), results in the formation of transformed foci (Sakai, 2007). Two-stage cell transformation has been performed in many cell systems is regarded as a important method for the screening of chemical as well as valuable approach for the mechanistic study in carcinogenesis. Thus, two-stage cell transformation assay mimics in vivo multistage carcinogenesis (Sakai, 2007). Contact inhibition and anchorage independence are the important characteristic of two-stage cell transformation (Urani et al.,2009 ;Fang et al.,2001).In recent years, cDNA and oligonucleotide microarray technology has allowed researchers to study the effects of chemical induce carcinogenesis in experimental animal and human. Toxicogenomics tools have also been utilized to investigate the chemical-gene interaction to enhance the information on toxicological properties of a particular chemical. Chemical-Gene expression signatures can be used to determine mechanism of unknown test compound. In cancer toxicology this technique can be readily used to identify dysregulated pathways/genes involved in chemical-Gene interaction (Godderis et al., 2012; Luch, 2005). In CTA and as well as in the in vivo carcinogenesis, normal and cancerous cells will be growing in constant communication with surrounding invasive and non invasive cell colonies and are undergoing concomitantly complementary changes in gene expression profile (Priya et al., 2013). 1.4 Redox regulation by GSH in cancer Glutathione is a tripeptide (L-γ-glutamyl-L-cysteinyl-glycine) which has important functions in eukaryotic cells. It is an antioxidant which carry active thiol group. It protects cell by interacting with free radical, reactive oxygen 3 Chapter 1 Introduction species (ROS), and reactive nitrogen species (RNS) because of its reactivity, high intracellular concentration (10mM in the liver cell and malignant cells) and high redox potential. GSH is synthesis is a two step process catalyzied by L-glumate: L-cysteine γ-ligase and glutathione synthase (Estrela et al., 2006). Glutathione in cancer cells is particularly relevant in the regulation of carcinogenic mechanisms; sensitivity against cytotoxic drugs, ionizing radiations, some cytokines, DNA synthesis, cell proliferation and death (Ortega et al., 2011). The intra and extra cellular GSH levels are responsible for the cell homeostasis. It can be determined by the balance between its production, consumption and transportation. Imbalance of GSH is observed in a wide range of pathologies, including cancer, neurodegenerative disorders, cystic fibrosis (CF), HIV, and aging. The elevated level of GSH inside a cell, disturb the cell homeostasis which causes cell transformation. Maintaining proper GSH levels and oxidation state are important for cell function and their disruptions are observed in many human diseases. GSH deficiency leads to an increased susceptibility to oxidative stress and, thus, progression of many disease states. On the other hand, elevated GSH levels increase antioxidant capacity and resistance to oxidative stress and this is observed in many types of cancer (Abdalla, 2011). While GSH is important in the detoxification of carcinogens, Increase in GSH level is already reported in different human carcinoma cell lines e.g. A549 cell (human lung carcinoma) and HepG2 cell (heptocellular carcinoma) which may increase resistance or alters the cytotoxicity of many chemotherapy drugs or radiation (Vojislav et al., 2001; Balendiran et al., 2004).This increase in GSH may be an important factor in chemo- or radiotherapy resistance seen in these cells (Goodwin and Baylin, 4 Chapter 1 Introduction 1982; Carney et al., 1983; Huang et al., 2000Yu and Brown, 1984; Guichard et al., 1986). Therefore, GSH depletion is a common strategy used by the pharmacologist as a possible target for cancer prevention. Manipulation of intracellular GSH using chemicals such as L-buthionine-(S,R)-sulfoximine (BSO), Diethyl maleate(DEM), Phorone(PHO) and t-butyl hydro peroxide have been found to reduce GSH level (Griffith et al., 1979,Anderson,1998).These chemicals has been used to increase the sensitivity of different tumour cell lines to therapy and showed that selective differential chemotherapy responses of normal versus tumour cells is possible (Griffith et al., 1979; Williamson et al., 1982). However, it’s important to note that different cells respond differently to oxidative stress inducing therapies (Mattson et al., 2009).The relationship between GSH depletion, chemotherapy, and/or the radiation response has been examined in many tumor cells after treatment with different drugs, including BSO, diethylmaleate, 2-oxothiazolidine-4- carboxylate, and different radio sensitizing agents (Rappa et al 1997;Mistry and Harrap,1991;Estrela et al.,2006;Bump and Brown,1990;Meister,1991). Moreover, GSH depletion only appears to be therapeutically effective when very low levels of this tripeptide can be achieved within the cancer cells (Estrela et al., 2006). Thus, achievement of selective tumour GSH depletion under in vivo or in vitro conditions appears as a remarkable pharmacological challenge. In fact, since the molecular background is firmly established, the potential benefits of GSH depletion for cancer therapy have remained in the shadow for two decades. Nevertheless, recently, new research has offered some light on how to make GSH depletion a useful tool in cancer therapy (Ortega et al., 2011). 5 Chapter 1 Introduction The strategy of GSH depletion as a chemo-therapeutic tool for cancer prevention is already used by other researchers but the genomic profile responsible for the GSH depletion, using a GSH depletor, is still unknown and never been evaluated earlier. In light of these facts, we proposed following aims and objectives of our study. 1.5 Aims and objective Study regulatory significance of cellular GSH depletion in experimental carcinogenesis. Elucidate its mechanism using microarray approach and validate altered gene expression using qPCR. Identify functional relevance of critically altered genes/pathway for their regulatory role in toxicity-carcinogenesis. 1.6 Study plan 1.6.1 Chemical induced Cell Transformation Assay (CTAs) in Fibroblast cell lines: We determined the cell transforming dose of MCA and transformed C3H10T1/2 cells using MCA (initiator) +TPA (promoter), in two-stage cell transformation assay. Cell transformation was characterized by soft agar assay, which measures acquisition of the anchorage independent growth. In addition to Giemsa staining, CytoselectTM based method was used to quantify colonies formed in the soft agar. Altered genomic profile of transformed cell was investigated using microarray approach and validation of altered gene expression was done by qPCR. Results of this study (C3H10T1/2 cells) were 6 Chapter 1 Introduction also validated in MCA +TPA treated BALB/c cells using RT-PCR (Chapter 3). 1.6.2 DEM exposure to MCA+TPA transformed cell lines We determined the non-cytotoxic dose of DEM (Diethyl malate) in MC+TPA transformed C3H10T1/2 cells was identified. GSH content, ROS generation, Cell cycle, TUNEL assay was performed to measure the changes due to DEM exposure in both the transformed cells (C3H10T1/2 and BALB/c). Altered genomic profile of DEM treated transformed cell was investigated using microarray approach and validation of altered gene expression was quantified by quantitative PCR (qRT-PCR). Result of this study (C3H10T1/2 cells) were also validated in DEM treated transformed BALB/c cells using RTPCR. CytoselectTM based method was used to quantify colonies formed in the soft agar by DEM exposure (Chapter 4). 1.6.3 PHO exposure to MCA+TPA transformed cell lines In this plan, we have investigated the alteration in global gene expression profile of MCA+TPA transformed C3H10T1/2 after GSH depletion by exposure to PHO; and the similar set of investigations were conducted in BALB/c 3T3 cell line and validated using qPCR (Chapter 5). 7