Environ Sci Pollut Res DOI 10.1007/s11356-015-4673-6

DANIO RERIO AS A MODEL IN AQUATIC TOXICOLOGY AND SEDIMENT RESEARCH

Time-dependent expression and activity of cytochrome P450 1s in early life-stages of the zebrafish (Danio rerio) Jennifer Bräunig 1,3 & Sabrina Schiwy 1 & Oliver Broedel 2 & Yvonne Müller 1 & Marcus Frohme 2 & Henner Hollert 1 & Steffen H. Keiter 1,4

Received: 11 March 2015 / Accepted: 6 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Zebrafish embryos are being increasingly used as model organisms for the assessment of single substances and complex environmental samples for regulatory purposes. Thus, it is essential to fully understand the xenobiotic metabolism during the different life-stages of early development. The aim of the present study was to determine arylhydrocarbon receptor (AhR)-mediated activity during selected times of early development using qPCR, enzymatic activity through measurement of 7-ethoxyresorufin-Odeethylase (EROD) activity, and protein expression analysis. In the present study, gene expression of cyp1a, cyp1b1, cyp1c1, cyp1c2, and ahr2 as well as EROD activity were investigated up to 120 h postfertilization (hpf) after exposure

Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (doi:10.1007/s11356-015-4673-6) contains supplementary material, which is available to authorized users. * Jennifer Bräunig [email protected] * Steffen H. Keiter [email protected] 1

Institute for Environmental Research, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany

2

Molecular Biotechnology and Functional Genomics, Technical University of Applied Sciences Wildau, Hochschulring 1, 15745 Wildau, Germany

3

Present address: National Research Centre for Environmental Toxicology (Entox), The University of Queensland, 39 Kessels Road, 4108 Brisbane, Queensland, Australia

4

Present address: Man-Technology-Environment Research Centre, School of Science and Technology, Örebro University, 70182 Örebro, Sweden

to either β-naphthoflavone (BNF) or a polycyclic aromatic hydrocarbons (PAH)-contaminated sediment extract from Vering Kanal in Hamburg (VK). Protein expression was measured at 72 hpf after exposure to 20 μg/L BNF. Altered proteins were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting. Distinct patterns of basal messenger RNA (mRNA) expression were found for each of the cyp1 genes, suggesting specific roles during embryonic development. All transcripts were induced by BNF and VK. ahr2 mRNA expression was significantly upregulated after exposure to VK. All cyp1 genes investigated showed a temporal decline in expression at 72 hpf. The significant decline of Hsp 90β protein at 72 hpf after exposure to BNF may suggest an explanation for the decline of cyp1 genes at this time point as Hsp 90β is of major importance for the functioning of the Ah-receptor. EROD activity measured in embryos was significantly induced after 96 hpf of exposure to BNF or VK. Together, these results demonstrate distinct temporal patterns of cyp1 genes and protein activities in zebrafish embryos as well as show a need to investigate further the xenobiotic biotransformation system during early development of zebrafish. Keywords Arylhydrocarbon receptor . β-naphthoflavone . Cytochrome P450 . EROD . Hsp 90β . Zebrafish

Introduction Sediments have been recognized as major sink for persistent toxic substances in the aquatic environment, but also as a potential source (Bartzke et al. 2010, Hollert et al. 2003); thus, contaminated sediments may affect aquatic organisms. However, the majority of existing bioassays for sediment toxicity testing do not provide sufficient information concerning

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bioavailability and mechanism-specific toxicity of contaminants (Keiter et al. 2010). The present study is part of the DanTox project, which aims at developing a combined vertebrate-based sediment contact assay which will help to investigate mechanism-specific effects in embryos of the zebrafish (Keiter et al. 2010). Within this study, the focus was set on the investigation of the arylhydrocarbon receptor (AhR)-regulated phase I mechanism in zebrafish during selected stages of the early development. The zebrafish has achieved high popularity as a model organism due to specific properties: easy to maintain, small size, high fecundity, rapid development, optical transparency, amenability to genetic and chemical screens, and an extensive literature base (Braunbeck et al. 2014, Strähle et al. 2012). In ecotoxicology, the zebrafish embryo is used within the regulatory framework of chemical risk assessments (OECD 2013) and it is used to determine the acute toxicity of wastewater at an international level (ISO 2007). Strähle et al. (2012) have addressed the need to develop assays that will investigate specific mechanisms of action using the zebrafish embryo in order to further develop its use as a model organism. The cytochrome P450-dependent monooxygenase (CYP) is a diverse supergene family that can be found in most organisms (Bernhardt 1996). CYPs catalyze oxidative transformation, leading to activation or inactivation of many endogenous and exogenous chemicals which have consequences on normal physiology and disease processes (Bernhardt 1996, Goldstone et al. 2010). Specific P450 systems are induced by xenobiotics, and the inductive response is usually a process by which the rate of gene transcription is stimulated, resulting in increased levels of messenger RNA (mRNA) and synthesis of P450 protein (Whyte et al. 2000). In zebrafish, 94 CYP genes have been identified which fall into 51 CYP gene families (Goldstone et al. 2010). These can be divided into two major groups: those that are primarily involved in synthesis, activation, or inactivation of endogenous regulatory molecules (CYP 5–51), and those that are primarily involved in the oxidation of xenobiotics, drugs, and fatty acids (CYP families 1–3, and to a lesser extent family 4) (Bernhardt 1996, Goldstone et al. 2010, Whyte et al. 2000). In this study, main focus was laid on the CYP1 family. In zebrafish, five CYP1 genes in four subfamilies have been identified: cyp1a, cyp1b1, cyp1c1, cyp1c2, and cyp1d1 (Goldstone et al. 2010). Many CYP1s are regulated by the AhR, which is a ligandactivated transcription factor belonging to the basic helixloop-helix-Per-Arnt-Sim (bHLH-PAS) family of proteins, located in the cytosol (Whyte et al. 2000). AhR is activated by planar congeners of polychlorinated dibenzodioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), several polycyclic aromatic hydrocarbons (PAHs), and NSO heterocyclic compounds (Denison and Nagy 2003, Hinger et al. 2011). These chemicals are ubiquitous environmental contaminants, and

several toxic responses have been observed in mammals, birds, and fish (Jönsson et al. 2007a, Peddinghaus et al. 2012, van den Berg et al. 1994). Knockdown of ahr2 in zebrafish has shown that cyp1a, cyp1b1, and the cyp1cs are primarily regulated by the ahr2 and induced by agonists for this receptor (Jönsson et al. 2007a, Jönsson et al. 2007b, Yin et al. 2008). However, unlike the other cyps, cyp1d1 is not induced by AhR agonists (e.g., 2,3,7, 8-Tetrachlorodibenzo-p-dioxin (TCDD) and PCB 126) and is not regulated by the AhR (Goldstone et al. 2009). Andreasen et al. (2002) detected Cyp1a protein in zebrafish larvae by whole mount immunolocalization as early as 36 h postfertilization (hpf) after exposure to TCDD. Mattingly and Toscano (2001), however, were neither able to detect Cyp1a protein nor its activity by Western blot and catalytic assay until 3 days postfertilization after exposure to TCDD and therefore suggested posttranscriptional silencing of the protein during embryogenesis. Otte et al. (2010) on the other hand were able to detect Cyp1 activity as early as 8 hpf in distinct tissues through in vivo localisation of 7-ethoxyresorufin-Odeethylase (EROD) activity imaged by confocal laser scanning microscopy. Between the three studies, there seems to be only little consensus about Cyp1 activity in early lifestages of zebrafish embryos. Additionally, the catalytic activity of Cyp1 protein in fish was exclusively attributed to Cyp1a (Whyte et al. 2000). Yet, the discoveries of other CYP1 isoforms leads to the assumption that measured Cyp1 activity can not solely be attributed to Cyp1a, but rather also to other Cyp1 isoform (Goldstone et al. 2009), which may explain diverging results in the studies. As a part of the DanTox project, the aim of this study was to investigate (a) the activation of the cytochrome P450 1 biotransformation system in whole embryos of the zebrafish on a genetic as well as protein-activity level, (b) to determine fluctuating basal levels of the investigated genes and protein activity during embryonic development, and (c) to select appropriate exposure times for measurement of EROD activity in zebrafish embryos. To achieve these goals, zebrafish embryos were exposed to the known AhR agonist β-naphthoflavone and an environmental sediment sample highly contaminated with PAHs.

Materials and methods Animal husbandry Zebrafish were obtained from Fraunhofer IME laboratories and maintained according to Hollert et al. (2003). Fish were kept in glass aquaria at a water temperature of 26±1 °C. Water was constantly circulated by a pump, and a constant day to night rhythm (14/10 h) was maintained. Fish were fed 5 days a week, in the mornings with dry flake food (Tetra Min®; Tetra

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GmbH; Melle, Germany) and in the evenings with live Artemia nauplii (Silver Star Artemia, Inter Ryba GmbH; Zeven, Germany). Fertilized fish embryos were obtained from breeding groups of 2:1 male to female. Chemicals and sample preparation β-naphthoflavone (BNF), a synthetic derivate of a naturally occurring flavonoid, and a PAH containing fraction of a sediment extract from the outer Vering Kanal in HamburgWilhelmsburg were used for exposure (later on referred to as VK). Chemical analysis reported high levels of PAH in the extract, Σ 16 EPA-PAHs=1324 mg/kg (Hafner et al. 2015). Sediment preparation and extraction was conducted as described previously (Keiter et al. 2008). Sulfur was removed from the extract through the addition of acid-activated copper to the solvent during extraction. Alumina-fractionation according to Brack et al. (2005) resulted in a fraction containing non-polar, aromatic compounds, which was used for subsequent experiments. Exposure of zebrafish embryos Fertilized fish eggs were selected visually using a binocular microscope (SMZ 1500, Nikon GmbH, Düsseldorf, Germany). Only fish eggs without deficiencies at least in the 8-cell stage were chosen for further testing. Eggs were transferred into artificial water, which was prepared according to ISO 15088 (294 mg/l CaCl2°2 H2O, 123.3 mg/l MgSO4°7 H2O, 63 mg/l NaHCO3, 5.5 mg/l KCl) (2007) in order to guarantee equal water quality and ion concentration in each test series. Since focus of this study was set on the time dependence rather than on concentration dependence of gene expression and related protein activity eggs were exposed to either 20 μg/L BNF (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) or 0.2 mg sediment equivalent per milliliter solvent (SEQ/ml) of the sediment extract from VK, concentrations were selected based on results of Schiwy and coworkers (2014) and below the LC10 determined in the fish embryo toxicity test. Per time point and representing one replicate, a pool of either 30 or 45 embryos were exposed under static conditions for qPCR analysis and measurement of EROD induction, respectively. For each exposure scenario, at least three independent replicates were conducted on different days. Eggs were placed in glass dishes (Labomedic GmbH, Bonn, Germany) in the appropriate testing solution, covered with Parafilm® (Parafilm, Menasha, WI, USA) and incubated at 26±1 °C. Dimethyl sulfoxide (DMSO, Sigma-Aldrich) was used as a solvent control and ISO water (ISO 2007) as a negative control. DMSO concentrations were kept below 0.2 % for all tests. Only tests with a survival rate ≥90 % were used.

FE-EROD assay The fish embryo EROD assay (FE-EROD) was conducted according to the method recently introduced by Schiwy et al. (2014). Exposure of embryos was terminated after 24, 48, 72, 96, or 120 hpf.

Quantitative real-time PCR qPCR was carried out according to Schiwy et al. (2014), with minor modifications. For measurement of transcript abundance in zebrafish embryos, the exposure was terminated after 48, 72, 96, or 120 hpf. Measurement of transcript abundance at 24 hpf was not possible, due to the little amount of extracted RNA from the embryos. Overlaying testing solution was removed; the embryos were anesthetized with saturated benzocaine solution, transferred to 2 ml tubes, washed twice with phosphate-buffered saline, and stored in RNAlater® (SigmaAldrich). The samples were shock-frozen in liquid nitrogen to stop metabolism and stored at −80 °C until further use. Genespecific primers for eef1a1, ahr2, cyp1a, cyp1b1, cyp1c1, and cyp1c2 were either designed using the Primer 3 software or previously published sequences were used (eef1a1, cyp1c2). Primers were synthesized by Life Technologies (Life Technologies Corporation, Darmstadt, Germany; Table 1). Expression of target genes was quantified by use of the comparative quantification cycle method with adjusted PCR efficiency according to the methods reported by Simon (2003). The expression level of the target gene was normalized to the reference gene eef1a1 to calculate the normalized expression of the target genes. Levels of gene expression were expressed relative to the average value of the unexposed controls as a fold change.

Protein expression analysis Two-dimensional gel electrophoresis was performed according to Eravci et al. (2007), with minor modifications. In brief, embryos were anesthetized with saturated benzocaine solution 72 hpf after exposure to 20 μg/L BNF or artificial water, dechorionated, deyolked, and stored at −80 °C. For protein extraction, 45 embryos were pooled and homogenized in triplicates in a volume of 250 μl; 350 μg of protein were applied to the 2-D gel electrophoresis in the pH range of 4–7. After visualization of the proteins by ruthenium staining, gels were analyzed with the Decodon Delta2D software (Decodon, Greifswald, Germany). Differential protein spots (t test for independent samples, two-tailed, p ≤ 0.05) were identified by matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) peptide mass fingerprinting (Thiede et al. 2005).

Environ Sci Pollut Res Table 1 Primer sequences, amplicon sizes, and accession numbers investigated in quantitative real-time PCR

Gene

Primer sequence (5′-3′)

Amplicon size (bp)

Accession no.

eef1a1a

F R F R F R F R F R F R

358

AY 422992.1

ahr2 cyp1a cyp1b1 cyp1c1 cyp1c2b

CTTCTCAGGCTGACTGTGC CCGCTAGCATTACCCTCC GAAGAAGCCCGTTCAGAAAA GGGTTGGATTTCACACCATC AAAGACACCTGCGTGTTTGTAA GAGGGATCCTTCCACAGTTCT TGGATCATCCTGCTACTTGTCA TCCACTACCCTGTCCACGTC GACTGAGTGCTGATGGACGA CACAGAGCGCAGATGACATT GTGGTGGAGCACAGACTAAG TTCAGTATGAGCCTCAGTCAAAC

a

McCurley and Callard (2008)

b

Jönsson et al. (2007b)

Data analysis All spreadsheet calculations were performed using Microsoft ExcelTM 2010. Graphs were plotted using the software Sigma Plot 10.0 (Systat Software, Erkrath, Germany). Statistical analyses were conducted using the software Sigma Stat 3.5 (Systat Software). Outliers were excluded based on the Grubbs test. All datasets that did not pass the KolmogorovSmirnov test on Gaussian distribution (p < 0.05) or the Levene’s test for equal variance (p96>120 hpf. As observed after BNF exposure, cyp1a and cyp1b1 were induced higher than the cyp1cs between 72 and 120 hpf, only at 48 hpf cyp1c1 showed higher induction than cyp1b1. An overall lower induction of gene expression at time point 72 hpf was found for all genes investigated compared to earlier and later times after exposure to both BNF and the sediment fraction.

Transcript Abundance (Fold Change)

500

*

cyp1a cap1b1 cyp1c1 cyp1c2

400

300

* *

*

200

*

* 100

* 0

Fig. 2 Basal cyp1 expression in Danio rerio embryos at 48, 72, 96, and 120 hpf. Normalized expressions (to eef1a1) are given as median with 25th and 75th percentile (n = 5). Significant differences in basal expression between genes at selected time points were determined by Friedman’s repeated measures ANOVA on ranks and Tukey’s multiple comparison test (n=5). Bars at the same time point not sharing common letters are significantly different

cyp1a cyp1b1 cyp1c1 cyp1c2

*

48

* * * * * 72

*

*

96

* *

120

Hours post ferilization [h]

Fig. 4 VK-induced gene expression in Danio rerio embryos at 48, 72, 96, and 120 hpf. Expression is shown as the multiple of the control (fold change), normalized to eef1a. Bars represent median values with 25th and 75th percentile. Asterisks indicate significant differences compared to control at given time points (Mann-Whitney rank sum test, p≤0.05)

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Basal and induced gene expression of ahr2 Basal expression of ahr2 receptor measured in unexposed embryos was consistent over time, no significant difference could be found between the investigated points in development (Fig. 5; repeated measures oneway ANOVA; p ≤0.05). BNF-induced ahr2 expression showed 1.5- to 2-fold higher inductions compared to basal levels (Fig. 6); however, this was not a significant upregulation. Significant upregulation of transcript abundance (2.9- to 4.5-fold) was observed in embryos exposed to VK at each observed time in development (Fig. 6; Mann-Whitney rank sum test, p≤0.05) and significant upregulation of ahr2 transcript was found in VK-exposed embryos over the time investigated (repeated measures ANOVA on ranks, p≤0.05).

Regulation of proteins related to the AhR pathway Two-DE gels derived from 72 hpf old embryos are presented in the supporting information (Figures S1, S2, and S3). A characteristic pattern could be recognized in all three states shown, negative control (NC), solvent control (SC), and BNF. Analysis with the software Delta2D revealed 34 significantly altered protein spots. Of these spots, 24 were significantly altered in at least two combinations (BNF vs. NC, BNF vs. SC, and BNF vs. SC and NC) and were chosen for MALDI-TOF peptide mass fingerprinting. Twenty-two spots were successfully identified (Table S1). Four of the identified proteins were significantly upregulated, while the rest was significantly downregulated. Among the significantly regulated protein, only Hsp 90β is directly involved in the AhR pathway.

Fig. 5 Basal ahr2 expression in Danio rerio embryos at 48, 72, 96, and 120 hpf. Normalized expression (to eef1a) is shown as median with 25th and 75th percentile

Fig. 6 BNF- and VK-induced ahr2 expression in Danio rerio embryos at 48, 72, 96, and 120 hpf. Expression is shown as the multiple of the control (fold change), normalized to eef1a. Bars represent median with 25th and 75th percentiles (BNF: n=4; VK: n=3). Asterisks indicate significant differences compared with the control at given time points (MannWhitney rank sum test, p≤0.05)

Discussion This study investigated the time-dependent activation of the cytochrome P450 biotransformation system in embryonic stages of the zebrafish. Therefore, the induction of related cytochrome P450 genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) was investigated in basal as well as induced states and activity of the AhR-regulated detoxification system was measured using the newly developed FE-EROD assay (Schiwy et al. 2014).

EROD activity in zebrafish embryos Basal and induced activity of CYP1s was monitored through the FE-EROD assay in embryos of the zebrafish at five times during early development. It was shown that basal EROD activity increases during normal embryonic development. Endogenous physiological functions of CYP1s are known for mammals (Nebert and Dalton 2006) and assumed also for zebrafish (Jönsson et al. 2007b), explaining basal activity of the enzyme. Exposure to BNF led to induction of EROD activity starting at 72 hpf, but first significant induction was only found 4 days postfertilization. These results are in accordance with findings by Mattingly and Toscano (2001) and Otte et al. (2010). Specifically, Otte and coworkers showed a similar pattern of EROD induction during the early development of zebrafish with a clear increase up to 104 hpf and with a subsequent decline at 128 hpf. However, exposure to the heavily contaminated extract from VK showed a significant induction of EROD activity already at 24 hpf, but apparent

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increase in EROD activity was not supported by statistical analysis at 48 and 72 hpf. Mattingly and Toscano (2001) proposed posttranscriptional silencing of EROD translation as a possible reason for undetectable EROD activity before hatch. Posttranslational modification such as phosphorylation leading to modulation of enzyme activity has been reported for several CYP P450 enzymes in mammals (Aguiar et al. 2005). Posttranslational silencing during embryogenesis might reduce disturbance of endogenous processes, such as inappropriate metabolism of endogenous signaling molecules (Omiecinski et al. 1999). Silencing of the enzyme is therefore a feasible possibility. However, considering the early significant induction of EROD activity after exposure to extract, it must be considered that concentration-dependent exposure to strong inducers or a complex mixture of inducers at once may lead the organism to neglect endogenous processes and rather produce active enzymes for detoxification. Basal expression of cyp1 genes and the ahr2 in zebrafish embryos Each of the four investigated cyp1 genes showed a distinctive pattern of basal gene expression, with maximum expressions at different times during development of the unexposed embryos. Cyp1c1 and cyp1c2 were expressed to a higher extent than cyp1a and cyp1b1 at all developmental times investigated. These findings are in agreement with previously published results for zebrafish (Jönsson et al. 2007b) as well as for killifish embryos (Wang et al. 2006). However, fold-inductions between the cyp1cs and cyp1a and cyp1b1 were higher than the ones reported elsewhere (Jönsson et al. 2007b, Wang et al. 2006). The high basal levels of the cyp1c genes suggest that they play a specific role during early development, isolated from xenobiotic metabolizing abilities. Low constitutive levels of cyp1a, as found in this study, were also reported by Andreasen et al. (2002) and Jönsson et al. (2007b). Constant basal levels of ahr2 were detected at all points investigated, this is, however, not in accordance with findings reported by Tanguay et al. (1999), who via Northern blot analyses reported an increased ahr2 gene expression with further development of the embryo. Expression of cyp1 genes and the ahr2 in zebrafish embryos Transcript levels of cyp1 genes were significantly induced in zebrafish embryos after exposure to BNF. A comparable high induction (150-fold) was observed for cyp1a at 48 hpf. Other cyp genes investigated also showed significant induction at this time point; however, induction was between 15- and 20fold higher than in unexposed embryos. A similar pattern was found after exposure to VK sediment extract; however,

transcript abundance was overall higher. Cyp1a induction after VK exposure was upregulated 380-fold (48 hpf) compared to basal levels. This very high induction can be explained by high levels of potential AhR agonists found in the sediment extract. Chemical analysis had revealed 1.3 g/kg of Σ 16 EPAPAHs in the sediment (Hafner et al. 2015) and other AhR inducing compounds such as heterocyclic aromatic compounds with incorporated nitrogen, sulfur, or oxygen (NSOHet) (Hinger et al. 2011, Peddinghaus et al. 2012), or nonpriority PAHs could be present in the extract as well. After fractionation of the extract, many of these are assumed to be in the fraction containing non-polar, aromatic compounds (Brack et al. 2005), which was used for exposure in this study. Thus, the embryos were exposed to a high concentration and a diverse mixture of AhR agonists, which might also lead to additive or synergistic effects (Timme-Laragy et al. 2007). Each gene observed followed a very distinct path of expression at the four time points investigated. In accordance with previously published results (Jönsson et al. 2007b), induction of cyp1a and cyp1b1 was higher than that of the cyp1cs. All genes investigated had a temporal decline in gene expression at 72 hpf in common. A similar decline was found for cyp1a induction after exposure to 6-formylindolo[3,2b]carbazole for 6 h prior to sampling (Jönsson et al. 2009). Zebrafish embryos hatch sporadically during the third day (between 48 and 72 hpf) of development. Therefore, the decline in gene expression in BNF- and sediment extractinduced cyp1 gene expression coincides with the time of hatch. It is possible that the exposure scenario might have an influence on such a decline in gene expression. Several studies exposed the embryos statically for a short time during the first day, usually

Time-dependent expression and activity of cytochrome P450 1s in early life-stages of the zebrafish (Danio rerio).

Zebrafish embryos are being increasingly used as model organisms for the assessment of single substances and complex environmental samples for regulat...
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