1.4.1.1. Use of fish in carcinogenicity studies
Fish have been used in carcinogenicity studies, as biomonitoring species to provide information on the quality of the environment and the health of the population of fish species and as alternative non-mammalian cancer models. Indeed, their high sensitivity to a variety of classes of carcinogens, tumour promoters, low cost of maintenance and short life cycles makes them ideal test species for both environmental monitoring and investigation of molecular mechanisms of tumourigenesis (Small et al, 2010; Bailey et al., 1996). In addition, although the normal structures of some organs are different between mammals and fish, tumours in fish are histopathologically similar to the equivalent tumours in mammals. This allows the same tumour type classification in fish as in mammals and their widespread use in research (Masahito et al., 1988; Grabher and Look, 2006). For instance, rainbow trout (Oncorhynchus mykiss) has been used for biomonitoring of contamination with environmental chemical carcinogens for the last four decades. Their low maintenance cost, ultra-sensitivity at very early stages of life and sensitivity to different classes of carcinogens, in addition to their well characterised tumour pathology, has led to their extensive use in research. As an example, investigations into the cause of high rates of liver tumour incidence in rainbow trout collected from the Pacific Northwest led to the discovery of aflatoxin B1 (AFB1) as a human hepatocarcinogen. Further investigations into AFB1-induced hepatocellular and cholangiocellular carcinomas in trout, revealed that the c-ki-ras gene was mutated in these tumours, similar to rat liver tumours (Bailey et al., 1996). Another example of species of fish used in carcinogenicity studies is medaka (Oryzias latipes). Medaka is commonly used in carcinogenicity studies. The effects of more than 30 common carcinogens and their links with formation of hepatocellular carcinoma are well characterised in this fish species. A number of these carcinogens include AFB1, N-diethylnitrosamine and methylazoxymethanol acetate (Bailey et al., 1996). It was also demonstrated that the functional regions of the
retinoblastoma gene is conserved between humans and medaka and mutations in this gene
can lead to formation of retinoblastoma. These studies as well as highlighting the importance and usefulness of fish species in testing for carcinogenicity, demonstrate the potential use of fish as models for studying cancers in humans (Rotchell et al., 2001b).
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1.4.1.2. Common dab (Limanda limanda) and environmental carcinogenicity studies
Environmentally-induced tumours in fish collected from their natural habitat are mainly observed in bottom-dwelling fish. This has been linked with their high levels of exposure to sediment-associated chemical carcinogens (Masahito et al., 1988). For example an unusually high prevalence of liver tumours, with some sampling sites exceeding 20%, has been reported in the flatfish dab captured from the waters around the UK compared with the frequencies considered to represent a background prevalence of the disease (NMMP, second report, 2004; Lyons et al., 2006). Other bottom-dwelling flatfish with reported high levels of hepatocellular carcinoma and cholangiocarcinoma tumours include English sole (Pleuronectes vetulus) and winter flounder (Pleuronectes americanus) sampled from Boston Harbor, USA. Although tumours are mainly observed in bottom-dwelling flatfish, they are not restricted to these species. For example, tumours have been reported in a wide variety of species including brown bullhead (Ictalurus nebulosus) in the rivers entering Lake Erie and also in white perch (Morone americana) from Chesapeake Bay (Masahito et al., 1988).
As a result, monitoring the disease status of dab and Atlantic cod (Gadus morhua) in offshore waters and European flounder (Platichthys flesus) in inshore regions and estuaries is now established as part of the biomonitoring procedures set up by the International Council for the Exploration of the Seas (ICES). These species are monitored for both internal (i.e. foci of cellular alterations (FCA) and malignant tumours) and external signs of disease (NMMP, second report, 2004). Therefore, the presence of tumours is used as a possible indicator of chemical contaminants at levels which cause adverse health effects and serve as indicators of the health and quality of the marine environment and fish population (NMMP, second report, 2004; Masahito et al., 1988). In addition to chemical induction, it is possible that biological agents such as viruses are causative of tumours. This was shown in the case of viral-induced neurofibromatosis in bicolour damselfish (Stegastes partitus) collected from waters around Florida, USA (Schmale et al., 2002).
Common dab (Figure 1.17) is similar to the other flatfish species living in close proximity to the ocean floor. Due to its diet of sediment-dwelling invertebrates it can be exposed to relatively high levels of sediment-associated chemicals. It is therefore a useful species for monitoring of the bioaccumulation of organic compounds and environmental carcinogenicity research. Liver pathology in dab, including cancer and pre-neoplastic lesions, is used as an
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indicator of the biological effects of contaminants on the marine environment. Recently, attention has focussed on dab caught from UK waters as part of the UK Clean Seas Environmental Monitoring Programme (CSEMP). This is due to the detection of an unusually high prevalence of liver tumours in dab, with some UK sites exceeding 20% (NMMP, second report, 2004; Lyons et al., 2006). Lesions seen in livers of flatfish dab can be separated into 5 main categories; non-specific inflammatory responses, non-neoplastic lesions, FCA, benign tumours and malignant tumours. (NMMP, second report, 2004; Stentiford et al., 2009; Small et al., 2010; Southam et al., 2008; Lyons et al., 2006). Liver is the main organ used for investigation and detection of morphological alterations and detection of tumours in fish collected for biomonitoring. Liver and kidney are the two main organs for xenobiotic metabolism. Based on studies conducted on the livers of rainbow trout, flatfish and zebrafish it has been shown that, similar to humans, fish liver expresses the main phase I and biotransformation enzymes, e.g. cytochrome P450s (CYP), required for activation of the pro- carcinogens and metabolism of compounds and therefore is the main target site for chemically-induced adverse effects. Furthermore, first pass metabolism, in addition to high levels of CYPs, increases the suitability of the liver as a biomonitoring organ. For example, tumours are developed in the liver of rainbow trout in response to exposure to CYP1A- inducing compounds such as 3-methylcholanthrene. Induction of CYP1A is used as a biomarker of exposure to polycyclic aromatic hydrocarbons (PAHs) (Bailey et al., 1996; Bragigand et al., 2006; Sheader et al., 2006). However, different types of tissue can be used for monitoring of different toxicological endpoints. For example skin and gills in fish are extremely useful for monitoring the visible morphological changes as they are directly exposed to the environment. In contrast, biofluids offer a less invasive method for biomonitoring (Lindesjoo and Thulin, 1994; Ward et al., 2006).
However, what is most interesting is that the levels of tumours in dab collected from offshore regions are higher than the levels of tumours detected in its close relative, the European flounder, which is collected from inshore areas. Theoretically, European flounder should be exposed to higher levels of contaminants due to their closer proximity to coastal regions and sources of anthropogenic pollution. So far the causative agents and the molecular mechanisms of these tumours remain unclear, especially in regards to the involvement of environmentally- induced epigenetic changes and their role in dab tumourigenesis. Better understanding of the pathways altered in these tumours and better characterisation of these tumours at molecular
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levels will help to clarify the driving factors behind these tumours. In addition, this may help to identify biomarkers for identification of hazardous environments and early indicators of health problems in fish. As a result, two recent studies by Small et al., (2010) and Southam et al., (2008) attempted to analyse dab liver tumours at the transcriptomic and metabolomic levels, respectively. Both studies provided preliminary data in terms of tumour characterisation and the identification of pathways that are altered in these tumours (i.e. energy pathways, protein synthesis). In the first study, a cDNA microarray designed for the close relative of dab, the European flounder, was used. Although this microarray is limited in the number of the genes presented on this platform (27,648 features –derived from 13,824 clones spotted in duplicate. The clones were derived from a redundant flounder cDNA library and represent approximately 3336 unique sequences), nevertheless the authors showed that genes involved in protein synthesis and vitellogenin, the egg precursor protein, were significantly up-regulated in dab tumours in comparison with surrounding non-tumour liver tissues. As vitellogenin up-regulation in fish is used as a biomarker of exposure to endocrine disrupting chemicals, the authors concluded that these chemicals may be involved in the development of these tumours. However they failed to identify a possible mechanism/evidence for induction of liver tumours by these chemicals in dab.
In the second paper (Southam et al., 2008) the authors used one-dimensional 1H NMR spectroscopy approach for identification of the metabolites whose concentrations were significantly altered in dab tumours. One of their findings was that concentrations of two metabolites corresponding to choline and betaine were altered in dab tumours. As these metabolites are involved in the one-carbon cycle; therefore, they concluded that possibly the DNA methylation pathway is disrupted in these tumours. However, this work required further investigation of the key metabolites in the one-carbon pathway (SAM and SAH) and required evidence of altered DNA methylation at gene levels.
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Furthermore, both studies mainly focused on characterisation of the tumours and were limited to tumours and apparently healthy tissues surrounding these tumours. Including completely healthy liver tissues from these fish species as a third category may help to identify the involvement of environmental factors and the possibility of higher susceptibility of the tumour bearing fish to chemicals. However, considering the challenges of working with non- model, un-sequenced species; both studies have provided significant insight into the pathways that are altered in these tumours.
Figure 1.17. A picture of the flatfish dab (Limanda limanda).
1.4.1.3. Zebrafish (Danio rerio) as a laboratory cancer model
The initial use of zebrafish in developmental biology dates back to a few decades ago. The transparency and ex-utero development of zebrafish embryos made this species an ideal model for studies on developmental biology. Its use has now expanded to different aspects of biosciences, including cancer biology and it is now being recognised as a convenient model for studying human tumourigenesis alongside more traditionally used models, such as mice, rats and nonhuman primates (Feitsma and Cuppen, 2008; Lawrence et al., 2009). Several factors make zebrafish a suitable model for cancer research. These include the sequenced genome, sensitivity to a variety of carcinogens, low cost of maintenance, short reproductive cycle, possibility of field studies and portability, potential for lifetime bioassays, transplantation of fluorescent mammalian cells and ease of forward and reverse screening (Bailey et al., 1996; Spitsbergen et al., 2000; Feitsma and Cuppen, 2008). In addition, fundamental concepts in development of tumours in humans, such as genome instability, tumour invasion and progression, presence of cancer stem cells, tumour suppressor genes and
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oncogenes have also been recognised in the processes that lead to development of tumours in zebrafish (Feitsma and Cuppen, 2008).
In addition to the significant histopathological similarities between zebrafish tumours and corresponding human tumours (Lam and Gong, 2006), recently it has been shown that transcription profiles of the tumours between the two phylogenetically distant species are also similar (Lam et al., 2006). Indeed, gene expression signatures in chemically-induced liver tumours in zebrafish show clear similarities to those in human tumours. Thus, genes involved in cell cycle/proliferation, DNA replication and repair, apoptosis and genes with liver-specific function were found to be deregulated in both human and zebrafish liver tumours. Furthermore, pathways such as Wnt-β-catenin and Ras-MAPK which are commonly distorted in human liver cancers, especially in hepatocellular carcinoma (HCC), are also altered in zebrafish liver tumours (Cha and DeMatteo, 2005; Lam et al., 2006).
Finally, although there are many benefits in the use of zebrafish for cancer research, there are still many unknown factors associated with the use of zebrafish (Feitsma and Cuppen, 2008) or any other fish species both as human cancer models and for environmental carcinogenicity studies. The role of epigenetics in fish tumourigenesis and environmentally-induced changes is one of these factors. So far no research has been conducted to investigate the involvement of epigenetic mechanisms in the development of tumours in fish. Indeed, consideration of epigenetic factors as one of the key components involved in human tumourigenesis cannot be disregarded in any model that it is currently in use for studying neoplasia in humans. This further highlights the need for studying epigenetic mechanisms in these species.