Perfluorooctanoic acid-induced cellular and subcellular alterations in fish hepatocytes
Introduction
The assessment of effects of water quality on organisms is critical, since most aquatic ecosystems worldwide are affected by anthropogenic pressure, especially chemical pollution (Vörösmarty et al., 2010). The past 20 years have seen increasing focus on per- and polyfluoroalkyl substances (PFAS), due to their ubiquity, persistence, and adverse effects on biota, especially that of aquatic environments (Ahrens and Bundschuh, 2014). The global distribution and high number of emission sources of PFAS is the result of the exploitation of their physical and chemical properties for a plethora of applications including fluoropolymer production, photographic film, metal plating, aqueous film-forming foams, paper, textiles, insecticides, and household products (Prevedouros et al., 2006). Perfluorooctane sulfonate and perfluorooctanoic acid (PFOA) represent the PFAS most frequently found in the environment and in biological matrices, and are the most studied (Stahl et al., 2011). They have been shown to be hepatotoxic, immunotoxic, carcinogenic, and to cause endocrine alterations potentially harmful to reproduction and development (Lee et al., 2020).
Perfluorooctanoic acid is listed as substance of very high concern in the EU under Registration, Evaluation, Authorisation, and Restriction of Chemicals, and, together with its salts and related compounds, was added to the Stockholm Convention list of persistent organic pollutants, Annex A, in 2019. However, these chemicals exhibit bioaccumulation potential and resistance to degradation and are expected to remain in aquatic ecosystems for years.
Among aquatic animals, fish are key indicator organisms, and a high number of species have been used for toxicology and ecotoxicology research in laboratory and field studies (Rašković and Poleksić, 2017). Fish represent organisms crucial to investigation of the impact of pollutants, as they occur in almost all aquatic habitats at or near the top of the food web, and are a food source for humans. Their responses can be studied at several levels employing a wide range of biomarkers and approaches (Van der Oost et al., 2003). The liver is commonly studied to assess fish health with respect to exposure to chemicals that typically accumulate and exert their effects in hepatic tissue, including PFAS (Falk et al., 2015; Filgo et al., 2015; Hoff et al., 2003).
Research on PFAS has expanded in recent years, however there is still a lack of knowledge of the mechanism of PFOA toxicity (Li et al., 2017), and there is a dearth of documentation of its ultrastructure effects, especially in fish.
The present study of common carp experimentally exposed to PFOA, focuses on morphology of hepatocytes as revealed by light and electron microscopy and box-counting fractal image analysis. In previous research using the same experimental fish model that evaluated image analysis techniques for biomedical and environmental research, liver structure analysis was demonstrated to discriminate among experimental groups (Manera et al., 2017), while grayscale differential box-counting fractal analysis gave a reliable measure of liver structure complexity, associated with ‘cloudy swelling’ as a consequence of PFOA exposure (Manera et al., 2019). This represented an initial attempt to characterize PFOA-induced modification of common carp Cyprinus carpio L. hepatocyte ultrastructure (Manera et al., 2019), but subcellular alterations due to PFOA exposure have not been extensively studied in fish.
The aim of the present study was to elucidate liver PFOA pathophysiology in common carp and its corresponding morphofunctional characteristics at a cellular and subcellular level. Box-counting fractal analysis of ultrathin sections was evaluated as an objective reproducible method to quantify the effect of PFOA exposure on cell structure complexity and heterogeneity by assessing fractal dimension and lacunarity, respectively. As an element of novelty, emphasis was given to a systemic approach and to consider possible functional relationships between changes in cell structure complexity (especially at the interface of cytoplasm portions) and ultrastructural signs of mitochondrial energy imbalance. Fractal analysis is a reliable tool for this purpose. Fractal dimension measures the complexity, the roughness of an object, while lacunarity refers to its ‘gappiness,’ or heterogeneity (Mandelbrot, 1982). Fractal analysis can be employed to assess complexity modifications related to pathological changes in biological systems (Cross and Cotton, 1994; Varela et al., 2010) and, as a consequence, to evaluate possible adaptive versus disadaptive response related to cell morphological variation (Manera et al., 2019).
Section snippets
Materials and methods
This research was conducted on fish utilized in a previous study (Giari et al., 2016). The experimental design, fish biometrics, and analytical techniques were as previously described (Giari et al., 2015, 2016; Guerranti et al., 2013; Manera et al., 2019). In brief:
Light microscopy
Light microscopy of ultrathin liver sections showed compartmentation typical of hepatocyte cytoplasm. In control fish, most of the cytoplasm was visible as a pale area, indicating abundant glycogen, and a darker portion forming a thin layer around the nucleus and along the inner surface of the cell membrane, containing cell organelles (Fig. 1A). Lipid droplets were primarily seen in the darker portion of the cytoplasm. The ratio of the cytoplasm components varied in both PFOA exposures, with a
Discussion
Integration of light and electron microscopy is recommended for study of the effects of pollutants on fish (Rašković and Poleksić, 2017). Alterations in organelle morphology, cell compartmentation, and the ratio of storage products are important markers indicating possible impaired cell structure or function (Schramm et al., 1998).
Perfluorooctanoic acid altered cell structure and ultrastructure even at the lowest tested concentration. It was not detected in the liver of low exposure (200 ng L−1
Conclusions
PFOA affects cell structure and ultrastructure even at a low, environmentally relevant, concentration that did not accumulate at detectable levels in the liver. The following PFOA pathophysiological processes may be suggested with respect to the hepatocyte: PFOA affected oxidative phosphorylation, leading to ROS production. As a consequence, mitochondria were altered, endoplasmic reticulum stress developed, and β-oxidation disturbance occurred, either by direct PFOA action or indirectly due to
Funding
This work was partially supported by the University of Teramo (Fund ID: FARDIB 2019 – BIRI 00162).
CRediT authorship contribution statement
Maurizio Manera: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition. Giuseppe Castaldelli: Formal analysis, Investigation, Resources, Writing - review & editing, Funding acquisition. Elisa A. Fano: Formal analysis, Investigation, Resources, Writing - review & editing. Luisa Giari: Conceptualization,
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
Thanks are due to The Lucidus Consultancy for English correction and suggestions provided.
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