Hepatocellular adenoma in a European flatfish (Limanda limanda): Genetic alterations in laser-capture micro-dissected tissue and global transcriptomic approach
Introduction
Liver pathologies of flatfish dab (Limanda limanda) English sole (Parophrys vetulus) and European flounder (Platichthys flesus) have been used internationally to monitor the effects of exposure to marine pollution since the 1980s (Malins et al., 1985, Vethaak and Rheinallt, 1992, Stentiford et al., 2003, Lyons et al., 2015). Such lesions have been associated with exposure to anthropogenic contaminants such as polycyclic aromatic hydrocarbons (PAHs) (Malins et al., 1985, Vethaak and Rheinallt, 1992). Dab possess both a similar histopathological tumour profile to humans (Stern and Zon, 2003) and homologs of human cancer genes such as ras and retinoblastoma (Rb), including mutational alterations of the Rb gene in tumour tissues (Du Corbier et al., 2005). In this respect, we have previously proposed that the dab hepatocellular tumour development could act as surrogate for cancer and the tumourigenesis process in human populations (Rotchell et al., 2009). Studies using this species also facilitate a better understanding of chemically-induced carcinogenesis in wild animals (Stentiford et al., 2005, Ward et al., 2006, Southam et al., 2008).
The histopathology of tumours and pre-tumours in dab liver are routinely diagnosed using a quality assured process involving histological tissue sections generated from wax-embedded samples (Feist et al., 2004). This diagnostic approach has recently been coupled with molecular analyses of tumour and surrounding normal tissue (Small et al., 2010). In addition, gross lesions and apparently normal tissues have been resected from the dab liver for molecular investigations such as genetic alterations of neoplastic associated genes (Du Corbier et al., 2005, Rotchell et al., 2009, Lerebours et al., 2014), transcriptomic (Small et al., 2010), proteomic (Stentiford et al., 2005) and metabolomic studies (Stentiford et al., 2005, Southam et al., 2008). Traditional microdissection and molecular analysis of populations of cells presents a challenge in that cellular heterogeneity within tissues samples may result in misleading findings (Cole et al., 1999, Sluka et al., 2008). Laser Capture-Microdissection (LCM) allows the ability to view and dissect target cells microscopically, thereby providing a direct link between a specific histopathological lesion and the molecular profile of that lesion (Gillespie et al., 2001). LCM has previously been applied to pathological studies in aquatic organisms including fish (Vinas and Piferrer, 2008, Jorgensen et al., 2009, Kitahashi et al., 2009, Lerebours et al., 2013, Nowak et al., 2013, Leguen et al., 2015, Schaeck et al., 2016), crustaceans (Small et al., 2008), and cnidarians (Wiebring et al., 2010).
The study applies the LCM technique to facilitate an investigation of the Rb allele status in hepatocellular adenoma (HCA) and normal hepatocytes populations from North Sea sampled dab. Rb was selected on the basis that Rb mutations have previously been reported in dab liver tumour samples (Du Corbier et al., 2005, Lerebours et al., 2014). In parallel, a global transcriptomic approach using the suppressive subtractive hybridisation (SSH) method was used to analyse the differentially expressed genes between HCA-bearing liver and normal liver samples.
Section snippets
Sample collection
L. limanda were captured at UK Clean Seas Environmental Monitoring Programme (CSEMP) sites (Nicolaus et al., 2016) at the Dogger Bank, North Sea during July 2008 and 2009 using 30 min tows of a standard Granton trawl aboard the RV Cefas Endeavour. Upon landing, fish were immediately removed from the catch and placed into flow-through tanks containing aerated seawater. The sex, size (total length), weight (g), and Fulton index Condition (FC) (FC = BW / L3 × 100), and presence of external signs of
Fish biometric data
There were no significant differences of sex (p = 0.11), length (p = 0.198), weight (p = 0.348) or FC (p = 0.168) between the (presumptively) normal fish and those with HCA (Table 2). In addition, no significant effect of the sex was found on the biometric parameters in the LCM-derived samples (length: p = 0.08; weight: p = 0.19; FC: p = 0.26) and the SSH (length: p = 0.10; weight: p = 0.10; FC: p = 0.74).
Rb mutational profile characterisation
Rb genetic profiles were characterised in LCM-derived normal and HCA samples of North Sea dab. Four
Discussion
Herein, we adopted two parallel approaches, Rb allele frequencies and global differential gene expression, to characterise molecular-level hallmarks of HCA development. The biometric parameters used showed that no significant difference occurred between the apparently normal and HCA phenotype samples analysed. This is consistent with a previous comprehensive study, using 165 dabs and 6 sites, whereby the HSI was not significantly different between sites with high prevalence of tumour-bearing
Acknowledgements
This work was conducted under the auspices of the Clean Seas Environmental Monitoring Programme (CSEMP) and funded by the Department for Environment, Food and Rural Affairs (Defra) under project SLA22G.
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