Effects of the pharmaceuticals diclofenac and metoprolol on gene expression levels of enzymes of biotransformation, excretion pathways and estrogenicity in primary hepatocytes of Nile tilapia (Oreochromis niloticus)

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Abstract

The expression levels of key enzymes of the xenobiotic metabolism and excretion pathways concerning biotransformation phases I (cytochrome P4501A), II (glutathione S-transferase) and III (multidrug resistance protein) and of the estrogenic biomarker vitellogenin (vtg) were investigated in primary hepatocytes isolated from male Nile tilapia (Oreochromis niloticus) after exposure to diclofenac and metoprolol, two pharmaceuticals prevalent in the aquatic environment worldwide. The lowest test concentration (4 × 10 9 M) was chosen to reflect an environmentally relevant exposure situation. Furthermore concentration dependent effects were investigated. Therefore a series of concentrations higher than the environmentally relevant range were used (10- and 100-fold). Diclofenac significantly induced all chosen biomarkers already at the environmentally relevant concentration indicating that biotransformation and elimination occur via the pathways under investigation. Estrogenic potential of this substance was demonstrated by VTG up-regulation as well. Metoprolol was either less effective than diclofenac or metabolized using different pathways. Key enzymes of the xenobiotic metabolism were less (CYP1A, GST) or not (MDRP) induced and a mild increase in vtg mRNA was detected only for 4 × 10 8 M. No concentration-dependency for metoprolol was found.

Introduction

Pharmaceutically active compounds (PhACs) are considered as a relatively new environmental concern among a large number of chemical substances contaminating aquatic environments worldwide. They are used in human and veterinary medicine, as well as for agriculture and aquaculture purposes (Dietrich et al., 2002). PhACs are often not or only partly removed during wastewater treatment and are therefore found in the range of nanograms (ng) to micrograms (μg) per liter (L 1) in the aquatic environment (Corcoran et al., 2010).

Non-target organisms, such as fish, are exposed to the PhACs and their metabolites and knowledge is needed to understand interactions with the detoxification mechanisms in these taxa (Wassmur et al., 2013). This is of special interest since different authors found the lowest observed effect concentration in fish to be around 1 μg L 1, hence in the environmentally relevant range (Triebskorn et al., 2004, Triebskorn et al., 2007, Hong et al., 2007).

Diclofenac (DCF) is a non-steroidal anti-inflammatory drug sold in many countries at high quantities annually (Heberer, 2002). DCF has a high persistence in the environment and is found in concentrations up to several μg L 1 in waste waters and surface waters (Heberer, 2002, Ashton et al., 2004, Montagner and Jardim, 2011). Recent studies about the effects of environmentally relevant concentrations of DCF are limited to few fish species, mainly rainbow trout (Oncorhynchus mykiss) and Japanese medaka (Oryzias latipes; Schwaiger et al., 2004, Triebskorn et al., 2004, Triebskorn et al., 2007, Hong et al., 2007, Cuklev et al., 2011).

Metoprolol (MTP) is a ß-blocker used to treat cardiovascular diseases (Regårdh et al., 1974). Since fish, like all vertebrates, possess ß-receptors in the heart, liver and gonads, there is a high probability that they will be affected from exposure to ß-blockers (Santos et al., 2010). Concentrations as high as 2.2 μg L 1 were already detected in surface waters (Ternes, 1998). Studies on the impacts of MTP on fish are scarce. Triebskorn et al. (2007) found ultrastructural effects in trout liver caused by MTP concentrations as low as 1 μg L 1.

Here, we investigated in vitro the effects of DCF and MTP on primary hepatocytes of male Oreochromis niloticus with regard to xenobiotic metabolism and excretion pathways as well as endocrine disruption potential using the estrogenic biomarker vitellogenin (VTG). O. niloticus is an economically important aquaculture species, frequently used as a model for toxicological research (Costa et al., 2012). Primary hepatocytes are considered as the closest available in vitro experimental model in terms of normal fish liver physiology (Schmid et al., 2000, Zaja et al., 2008). The isolated hepatocytes possess all benefits of intact cells such as functional organelles and enzyme interactions (Segner, 1998) but in comparison to in vivo assays fewer animals are needed for toxicity testing (Schirmer, 2006).

The biotransformation pathway is mainly located in the liver and typically consists of three major phases. Phase I and II lead to more easily excretable hydrophilic metabolites of the chemicals by activation and conjugation whereas phase III is used for immediate transportation and final excretion. Cytochrome-P450-monooxygenases are the key enzymes of phase I activation. The isoform CYP1A is widely used as a biomarker in ecotoxicological studies due to its inducibility by many different environmental pollutants (Van der Oost et al., 2003). Furthermore, it is used as an indicator of cellular toxicity caused by chemicals (Sarasquete and Segner, 2000). Glutathione-S-transferases (GST) are a major group of detoxification enzymes of phase II (Sheehan et al., 2001). GSTα, as used in this study, is one of the most expressed classes of cytosolic GSTs in mammals (Fu and Xie, 2006, Li et al., 2010). Finally, different efflux pumps such as multidrug resistance proteins (MDRP; ABCC) are involved in the cellular defense by regulating accumulation and excretion (Leslie et al., 2005). Depending on the transported compounds, ABC transporters function also in phase 0 of the biotransformation pathway, especially if they act as a first line of defense preventing unmodified compounds from accumulating in the cell (Bard, 2000, Sturm and Segner, 2005). Knowledge of the presence and function of ABC efflux transporters in fish is relatively modest (Zaja et al., 2008). For Nile tilapia, the MDRP was first described recently by Ziková et al. (2010).

Additionally, primary hepatocytes can provide initial information on the endocrine activity of test substances (Kloas et al., 1999, Navas and Segner, 2006). VTG genes coding for the synthesis of egg yolk protein precursor are expressed in the liver. The induction of VTG in male fish is an indicator of exposure to estrogen or estrogen-like chemicals (Sumpter and Jobling, 1995). Many pharmaceuticals may disrupt the endocrine systems of non-target organisms (Kloas et al., 2009, Massarsky et al., 2011).

In this study, primary hepatocytes of O. niloticus were exposed to increasing concentrations of DCF and MTP, respectively, where the lowest concentrations are in the range of the concentrations found in surface waters worldwide. The main objective of the study was a first evaluation of the effects of DCF and MTP on xenobiotic metabolism and elimination in Nile tilapia primary hepatocytes. Therefore, established biomarkers (CYP1A, GST, MDRP; Ziková et al., 2010) were used. The second objective was the analysis of the endocrine-disruption potential of the test substances concerning estrogenicity, and thus VTG gene expression in primary hepatocytes of male fish was analyzed.

Section snippets

Fish

Male Nile tilapia (O. niloticus), weighing between 100 and 200 g, were obtained from the stock of Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany. Prior to cell isolation, fish were kept in an aerated 800 L tank with water flow-through (26 ± 1 °C, natural photoperiod) for several weeks and fed daily ad libitum with a commercial fish food (Aller Aqua Primo, Golßen, Germany).

Chemicals

All chemicals were obtained from Sigma Aldrich (Steinheim, Germany) or as indicated otherwise.

Results

During the experiment, O. niloticus primary hepatocytes could be kept alive. The integrity of these cells was verified using a microscope as described by Segner et al. (1993). According to a positive control, the expression level of a reference gene (EF1-α) was nearly constant in all samples (data not shown).

Discussion

Pharmaceuticals are frequently found in the aquatic environment, but knowledge about their effects on the physiology of non-target organisms is still limited. Here, we investigated the effects of two pharmaceuticals with high environmental relevance, namely DCF and MTP, on primary hepatocytes of Nile tilapia (O. niloticus). Biomarkers of biotransformation phase I (CYP1A), II (GST) and III (MDRP) as well as of endocrine disruption potential, namely estrogenicity, (VTG) were studied.

Conclusion

In conclusion, our findings suggest the occurrence of physiological changes in O. niloticus primary hepatocytes due to exposure to DCF and MTP. Interestingly, remarkable differences occurred concerning gene expression patterns and sensitivities. DCF seems to be metabolized through a CYP1A-GST-MDRP route while no such evidence was found for MTP. Additionally, DCF caused significant changes of selected biomarkers already at environmentally relevant concentrations.

Overall, it could be shown that

Acknowledgments

The authors would like to thank Wibke Kleiner for technical assistance with the molecular work. This study was funded by the Federal Ministry of Education and Research (BMBF) within the project “INNOVATE” (01LL0904C).

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