Elsevier

Aquatic Toxicology

Volume 169, December 2015, Pages 133-142
Aquatic Toxicology

Estrogenic and anti-estrogenic influences in cultured brown trout hepatocytes: Focus on the expression of some estrogen and peroxisomal related genes and linked phenotypic anchors

https://doi.org/10.1016/j.aquatox.2015.10.010Get rights and content

Highlights

  • Evidence of crosstalk between estrogens and peroxisomal pathways in brown trout.

  • VtgA and ERα mRNA levels increased after 1, 10 and 50 μM of ethinylestradiol (EE2).

  • ERβ-1, catalase and urate oxidase mRNA levels decreased after estrogenic stimuli.

  • Estrogenic effects in VtgA, ERα and Uox mRNA levels were reverted by ICI 182,780.

  • Immunofluorescence/electron microscopy shows smaller peroxisomes after 50 μM of EE2.

Abstract

Estrogens, estrogenic mimics and anti-estrogenic compounds are known to target estrogen receptors (ER) that can modulate other nuclear receptor signaling pathways, such as those controlled by the peroxisome proliferator-activated receptor (PPAR), and alter organelle (inc. peroxisome) morphodynamics. By using primary isolated brown trout (Salmo trutta f. fario) hepatocytes after 72 and 96 h of exposure we evaluated some effects in selected molecular targets and in peroxisomal morphological features caused by: (1) an ER agonist (ethinylestradiol—EE2) at 1, 10 and 50 μM; (2) an ER antagonist (ICI 182,780) at 10 and 50 μM; and (3) mixtures of both (Mix I—10 μM EE2 and 50 μM ICI; Mix II—1 μM EE2 and 10 μM ICI and Mix III—1 μM EE2 and 50 μM ICI). The mRNA levels of the estrogenic targets (ERα, ERβ-1 and vitellogenin AVtgA) and the peroxisome structure/function related genes (catalase, urate oxidase—Uox, 17β-hydroxysteroid dehydrogenase 4—17β-HSD4, peroxin 11α—Pex11α and PPARα) were analyzed by real-time polymerase chain reaction (RT-PCR).

Stereology combined with catalase immunofluorescence revealed a significant reduction in peroxisome volume densities at 50 μM of EE2 exposure. Concomitantly, at the same concentration, electron microscopy showed smaller peroxisome profiles, exacerbated proliferation of rough endoplasmic reticulum, and a generalized cytoplasmic vacuolization of hepatocytes. Catalase and Uox mRNA levels decreased in all estrogenic stimuli conditions. VtgA and ERα mRNA increased after all EE2 treatments, while ERβ-1 had an inverse pattern. The EE2 action was reversed by ICI 182,780 in a concentration-dependent manner, for VtgA, ERα and Uox. Overall, our data show the great value of primary brown trout hepatocytes to study the effects of estrogenic/anti-estrogenic inputs in peroxisome kinetics and in ER and PPARα signaling, backing the still open hypothesis of crosstalk interactions between these pathways and calling for more mechanistic experiments.

Introduction

Synthetic hormonally active compounds, such as xeno-estrogenic substances, have been recognized as prime stressors acting on the endocrine reproductive axis in fish. Numerous studies have documented reproductive impairment, e.g., (Nash et al., 2004, Panter et al., 1998), with still unknown ultimate consequences at the population level (Harris et al., 2011, Kidd et al., 2007). From a classical point of view, exogenous estrogens primarily act via estrogen nuclear receptors (ER) on their transcriptionally regulated pathway in the liver, brain and gonads (key estrogen target organs) in fish and other vertebrates (Nelson and Habibi, 2013), despite the distribution of ER in other organs (Menuet et al., 2002). Nonetheless, evidence exists that estrogens can interfere with pathways regulated by other nuclear receptors, such as those controlled by the peroxisome proliferator-activated receptor (PPAR). This particular aspect has been addressed in mammalian model systems, with strong evidence of interfering interaction between ER and PPAR signaling (e.g., Chu et al., 2014, Zhang et al., 2015, Wang and Kilgore, 2002). Many of these data describe an inhibitory effect of PPAR agonists on estrogen-dependent pathways, e.g., in the uterus (Gunin et al., 2004, Houston et al., 2003). Cancer cell lines have been widely used to study specific interactions between PPARγ and ERα/ERβ (Chu et al., 2014, Wang and Kilgore, 2002), although regulatory effects between PPARα and estrogens have also been suggested (Jeong and Yoon, 2007, Kim et al., 2009).

We have previously hypothesized an interference scenario between estrogens and PPARs in teleost fish (Batista-Pinto et al., 2009). Such crosstalk would lead to an array of peroxisomal and lipid profiling impacts that, if disrupted, could compromise hepatic function, ovary differentiation and normal egg development. A set of morphological experiments using brown trout (Salmo trutta f. fario) as a model revealed a negative correlation between estrogens titers and peroxisome size during gonadal maturation (Rocha et al., 1999). Further, the activities of target peroxisomal enzymes were reduced under similar conditions (Resende et al., 2005, Rocha et al., 2004). In a second set of tests, lower PPARα expression in vitellogenic females was noted in parallel to higher estradiol levels (Batista-Pinto et al., 2009). With this background, a crosstalk between estrogens and PPAR-dependent pathways in brown trout was proposed (Batista-Pinto et al., 2009).

To further clarify these questions we established an in vitro assay using primary hepatocytes isolated from brown trout, which may be used in future applications as a tool to understand in vivo endocrine disruption processes in this species. As metabolically active cells, primary fish hepatocytes have been successfully used to test distinct xenobiotic inputs, including estrogenic ones, within various research purposes, e.g., (Hultman et al., 2015, Maradonna et al., 2013, Sovadinová et al., 2014). Our goal was to address the impacts of a model ER agonist (ethinylestradiol—EE2), at 1, 10 and 50 μM, an antagonist of ER (ICI 182,780—ICI), at 10 and 50 μM, and their combined action (Mix I—10 μM EE2 and 50 μM ICI; Mix II—1 μM EE2 and 10 μM ICI and Mix III—1 μM EE2 and 50 μM ICI), in brown trout hepatocytic peroxisomes, regarding morphological qualitative and quantitative aspects. In addition, we were also interested in measuring the mRNA expression levels of genes involved in biological pathways potentially related to estrogen signaling and peroxisomal activity and function. For estrogenic response, we selected ERα, ERβ-1 and the gene encoding female egg yolk protein—vitellogenin A (VtgA). As peroxisomal endpoints, we analyzed the following targets: catalase, the product of which is a peroxisomal enzyme used as an environmental indicator of stress (Orbea et al., 2002); urate oxidase—Uox, the product of which degrades urate to allantoin (Hayashi et al., 2000); PPARα, mainly expressed in the liver, with a product that plays a major role in governing fatty acid oxidation (Grygiel-Gorniak, 2014); peroxin 11α—Pex11α, the product of which is involved in peroxisome division (Fagarasanu et al., 2007); and 17β-hydroxysteroid dehydrogenase 4—17β-HSD4, with a product which is as an active participant on peroxisomal fatty acid β-oxidation and in the conversion of estradiol to estrone (Breitling et al., 2001).

The aim of the study was to elucidate estrogenic/anti-estrogenic effects, not only in direct estrogenic signaling, but also on PPARα-mediated pathways, some of them directly influencing the hepatocytic structure and function. Our investigation aims to shed new light on the regulatory processes involved in PPAR and ER interactions in fish, which will be crucial to decode crosstalk aspects between the two nuclear pathways.

Section snippets

Animals

One-year-old juveniles of brown trout (S. trutta f. fario) were obtained from a state facility for repopulation purposes (Aquaculture Station of Torno, Portugal), and maintained under controlled conditions with a natural photoperiod (13–15 h daylight hours), at approximately 16 °C. Animals were allowed to acclimate to laboratory conditions for 4 weeks before the experiments. All the animal procedures were performed according to the Portuguese Decree-Law No. 113/2013 implementing EU Directive No.

Cell culture viability

We were able to maintain the isolated hepatocytes up to 96 h after exposure with a viability ranging from 82% to 89%, in experiment I, and from 81% to 87% in experiment II.

Qualitative morphological analysis: catalase cytochemistry for electron microscopy

The ultrastructure of the hepatocytes in both solvent control groups (from experiment I and II) was quite similar to the general histology previously reported in this fish species (Rocha et al., 2010). The use of catalase cytochemistry allowed an efficient recognition of peroxisomes. These organelles typically have a spherical

Discussion

In fish, the glycolipophosphoprotein Vtg (an ER-regulated gene) responds promptly to estrogenic stimulus with the increase of its hepatic synthesis for subsequent accumulation in yolky oocytes, hence its common recognition as a biomarker of estrogenic exposure (Beitel et al., 2015, Petersen and Tollefsen, 2011, Sumpter and Jobling, 1995). Thus, it is not surprising that herein the metabolic active primary hepatocytes have promoted a markedly increase in the VtgA expression after the single EE2

Acknowledgments

This study was supported through FCT—Foundation for Science and Technology, primarily under project “PTDC/CVT/115618/2009”, post-doc grant SFRH/BPD/97139/2013 (to T.V.M) and additionally by project “PEst-C/MAR/LA0015/2013”. The research was also supported by the Strategic Funding UID/Multi/04423/2013, through national funds provided by FCT, European Regional Development Fund (ERDF), in the framework of the programme PT2020. Finally, the electron microscopy studies benefited from the project

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