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Frequency of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from the Douro River estuary, Portugal

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Abstract

Liver lesions in wild fish have been associated with xenobiotic exposure. Facing reports of pollution in the Douro River estuary (north of Portugal), we have been making field surveys using fishes and targeting histopathological biomarkers of exposure and effect. Herein, we intended to better characterize and report the rate of one poorly understood lesion—hepatocellular fibrillar inclusions (HFI)—found in European flounder (Platichthys flesus). With this report, we aimed to establish sound baseline data that could be viewed as a starting point for future biomonitoring, while offering the world's second only pool of field data on such a liver toxicopathic lesion, which could be compared with data available from the UK estuaries. Sampling was done in the Douro River estuary over 1 year. A total of 72 animals were fished with nets, in spring–summer (SS) and autumn–winter (AW) campaigns. Livers were processed for histopathology and both routine and special staining procedures (alcian blue, periodic acid Schiff (PAS), tetrazonium coupling reaction). Immunohistochemistry targeted AE1/AE3 (pan cytokeratins). The severity of the HFI extent was graded using a system with four levels, varying from 0 (absence of HFI) to 3 (high relative density of cells with HFI). Cells (isolated/groups) with HFI appeared in 35 % or more of the fish, in the total samples of each season, and over 40 % in more homogeneous sub-samples. There were no significant differences when comparing samples versus sub-samples or SS versus AW. When merging the data sets from the two seasons, the frequency of fish with HFI was ≈36 % for the total sample and ≈49 % for the sub-sample. The extreme group (biggest and smallest fish) revealed a HFI frequency of only 16 %, which differed significantly from the total and sub-sampled groups. Immunostaining and PAS were negative for the HFI, and alcian blue could, at times, faintly stain the inclusions. These were positive with the tetrazonium reaction. We showed the presence of HFI in European flounder from the Douro River estuary, proving that they are essentially protein in nature, that no seasonal changes existed in the HFI frequency, and that it was rarer in the smallest and biggest fish groups. Within the ranges of weight/size of our total sample, we estimate that the frequency of HFI in the local flounder is ≈35 %. That rate stands as a baseline value for future assessments, namely for biomonitoring purposes targeting correlations with the estuary pollution status.

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Acknowledgments

This work was partially supported by the European Regional Development Fund (ERDF) through the Competitiveness and Trade Expansion Program (COMPETE) and by National Funds provided by Fundação para a Ciência e a Tecnologia (FCT) via the research projects PTDC/MAR/70436/2006 and PEst-C/MAR/LA0015/2011, and the PhD grant SFRH/BD/25746/2005. We are grateful to Mrs. Nádia S. Santos for performing the histochemistry and immunohistochemistry procedures in selected liver sections.

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Authors and Affiliations

  1. CITAB—Centre for the Research and Technology of Agro-Environmental and Biological Sciences, ECVA—School of Life and Environmental Sciences, UTAD—University of Trás-os-Montes and Alto Douro, Quinta de Prados, Apartado 1013, 5001-801, Vila Real, Portugal

    João Carrola & António Fontaínhas-Fernandes

  2. ECVA—School of Agriculture and Veterinary Sciences, UTAD—University of Trás-os-Montes and Alto Douro, Quinta de Prados, Apartado 1013, 5001-801, Vila Real, Portugal

    Maria João Pires

  3. ICBAS—Institute of Biomedical Sciences Abel Salazar, Laboratory of Histology and Embryology, UPorto—University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal

    Eduardo Rocha

  4. CIIMAR—Interdisciplinary Centre of Marine and Environmental Research, CIMAR Associate Laboratory, Laboratory of Cellular, Molecular and Analytical Studies, UPorto—University of Porto, Rua dos Bragas 289, 4050-123, Porto, Portugal

    Eduardo Rocha

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  1. João Carrola
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  4. Eduardo Rocha
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Correspondence to Eduardo Rocha.

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Carrola, J., Fontaínhas-Fernandes, A., Pires, M.J. et al. Frequency of hepatocellular fibrillar inclusions in European flounder (Platichthys flesus) from the Douro River estuary, Portugal. Environ Sci Pollut Res 21, 3116–3125 (2014). https://doi.org/10.1007/s11356-013-2248-y

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