Hepatocellular Fibrillar Inclusions in European flounder (Platichthys flesus): Endocrine Disruption in the Marine Environment?

Abstract

Hepatocellular fibrillar inclusions (HFI) are an unusual pathology of unknown aetiology affecting the flatfish species European flounder (Platichthys flesus) and have previously been observed in UK estuaries impacted by endocrine disrupting chemicals. Using a multidisciplinary approach, this study explores the hypothesis that induction of vitellogenin as a result of exposure to endocrine disrupting chemicals is associated with the development of HFI. Thus HFI could serve as a potential histological biomarker of endocrine disruption in male P. flesus. Liver samples (n= 200) were collected from P. flesus at selected UK estuaries during autumn 2010, processed for histopathology and examined for incidences of HFI. The prevalence of HFI in UK estuaries was ranked in the following order: Mersey (79.3 %) > Tyne (78.6 %) > Thames (63.6 %) > Medway (44.7 %) > Humber > (34.5 %) > Alde (3.7 %). The severity of HFI was evaluated using a qualitative scoring index and revealed that severity appeared to be more pronounced in those estuaries with higher prevalence of HFI (r= 0.98). The data also demonstrated that male fish exhibited a higher prevalence of HFI compared to females (p= <0.001). No association between HFI and P. flesus age was observed (p= 0.648). Electron microscopy examination confirmed that HFI were the result of extensive disorganisation and proliferation of the rough endoplasmic reticulum (RER). The P. flesus from the Mersey and Tyne provided robust biological material for further study and were collectively categorised into biological groups of HFI severity stages. Real-time Polymerase Chain Reaction (qPCR) demonstrated no significant differences between biological groups and transcription of vitellogenin (VtG) (p= 0.3098) and choriogenin (CHR) (p= 0.5317). Plasma VtG concentrations greater than 1 µg ml-1 were only observed in 10 fish during the entire study and 6 of these originated from the Tyne and Mersey (range 1.7-1944.0 µg ml-1). The low level of gene transcription accompanied by near background concentrations of plasma VtG made it difficult to identify relationships between gene transcription, HFI formation and protein translation. Despite this, immunohistochemistry (IHC) successfully identified VtG with an immediate association with HFI. The IHC labelling was clear and distinct exhibiting no unspecific binding of primary antibody. This study was unable to confirm previous reports of microtubules within HFI using IHC incorporating a α/β- tubulin polyclonal primary antibody for zebrafish. Whilst this study was unable to provide conclusive evidence concerning the implication of endocrine disruption on HFI development, the detection of VtG within HFI of male fish does highlight endocrine disruption as a potential avenue for future laboratory studies concerning the pathogenesis of HFI. However, until laboratory experiments are able to establish links, it is important to remember that other mechanisms may still be implicated.