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

  • Danio rerio as a Model in Aquatic Toxicology and Sediment Research
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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-O-deethylase (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 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.

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References

  • Aguiar M, Masse R, Gibbs BF (2005) Regulation of cytochrome P450 by posttranslational modification. Drug Metab Rev 37:379–404

    Article  CAS  Google Scholar 

  • Andreasen EA, Spitsbergen JM, Tanguay RL, Stegeman JJ, Heideman W, Peterson RE (2002) Tissue-specific expression of AHR2, ARNT2, and CYP1A in zebrafish embryos and larvae: effects of developmental stage and 2,3,7,8-tetrachlorodibenzo-p-dioxin exposure. Toxicol Sci 68:403–419

    Article  CAS  Google Scholar 

  • Bartzke M, Delov V, Stahlschmidt-Allner P, Allner B, Oehlmann J (2010) Integrating the fish embryo toxicity test as triad element for sediment toxicity assessment based on the Water Framework Directive approach. J Soils Sediments 10:389–399

    Article  CAS  Google Scholar 

  • Bernhardt R (1996) Cytochrome P450: Structure, function, and generation of reactive oxygen species, Reviews of Physiology Biochemistry and Pharmacology, 33rd edn. Springer-Verlag Berlin, Berlin, pp 137–221

    Google Scholar 

  • Brack W, Schirmer K, Erdinger L, Hollert H (2005) Effect-directed analysis of mutagens and ethoxyresorufin-o-deethylase inducers in aquatic sediments. Environ Toxicol Chem 24:2445–2458

    Article  CAS  Google Scholar 

  • Braunbeck T, Kais B, Lammer E, Otte J, Schneider K, Stengel D, Strecker R (2014) The fish embryo test (FET): origin, applications, and future. Environ Sci Pollut Res. doi:10.1007/s11356-014-3814-7

    Google Scholar 

  • Carver LA, Jackiw V, Bradfield CA (1994) The 90-kDa heat-shock protein is essential for Ah receptor signalling in a yeast expression system. J Biol Chem 269:30109–30112

    CAS  Google Scholar 

  • Chang TK, Waxman DJ (2006) Enzymatic analysis of cDNA-expressed human CYP1A1, CYP1A2, and CYP1B1 with 7-ethoxyresorufin as substrate. Methods Mol Biol 320:85–90

    CAS  Google Scholar 

  • Deane EE, Woo NYS (2011) Advances and perspectives on the regulation and expression of piscine heat shock proteins. Rev Fish Biol Fish 21:153–185

    Article  Google Scholar 

  • Denison MS, Nagy SR (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Ann Rev Pharmacol Toxicol 43:309–334

    Article  CAS  Google Scholar 

  • Doering JA, Wiseman S, Beitel SC, Tendler BJ, Giesy JP, Hecker M (2012) Tissue specificity of aryl hydrocarbon receptor (AhR) mediated responses and relative sensitivity of white sturgeon (Acipenser transmontanus) to an AhR agonist. Aquat Toxicol 114:125–133

    Article  Google Scholar 

  • Eravci M, Fuxius S, Broedel O, Weist S, Eravci S, Mansmann U, Schluter H, Tiemann J, Baumgartner A (2007) Improved comparative proteome analysis based on two-dimensional gel electrophoresis. Proteomics 7:513–523

    Article  CAS  Google Scholar 

  • Goldstone JV, Jonsson ME, Behrendt L, Woodin BR, Jenny MJ, Nelson DR, Stegeman JJ (2009) Cytochrome P450 1D1: A novel CYP1A-related gene that is not transcriptionally activated by PCB126 or TCDD. Arch Biochem Biophys 482:7–16

    Article  CAS  Google Scholar 

  • Goldstone JV, McArthur AG, Kubota A, Zanette J, Parente T, Jonsson ME, Nelson DR, Stegeman JJ (2010) Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish. BMC Genomics 11:643

    Article  Google Scholar 

  • Hafner C, Gartiser S, Garcia-Kaeufer M, Schiwy S, Hercher C, Wiebke M, Achten C, Keiter S, Hollert H (2015) Investigations on sediment toxicity of German rivers applying a standardized bioassay battery. Environ Sci Poll Res. doi:10.1007/s11356-015-4482-y

  • Hinger G, Brinkmann M, Bluhm K, Sagner A, Takner H, Eisenträger A, Braunbeck T, Engwall M, Tiehm A, Hollert H (2011) Some heterocyclic aromatic compounds are Ah receptor agonists in the DR-CALUX assay and the EROD assay with RTL-W1 cells. Environ Sci Pollut Res 18:1297–1304

    Article  CAS  Google Scholar 

  • Hollert H, Keiter S, Koenig N, Rudolf M, Ulrich M, Braunbeck T (2003) A new sediment contact assay to assess particle-bound pollutants using zebrafish (Danio rerio) embryos. J Soils Sediments 3:197–207

    Article  Google Scholar 

  • Hughes D, Guttenplan JB, Marcus CB, Subbaramaiah K, Dannenberg AJ (2008) Heat shock protein 90 inhibitors suppress aryl hydrocarbon receptor-mediated activation of CYP1A1 and CYP1B1 transcription and DNA adduct formation. Cancer Prev Res 1:485–493

    Article  CAS  Google Scholar 

  • ISO (2007) Water quality—determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio). ISO 15088:2007

    Google Scholar 

  • Jönsson ME, Jenny MJ, Woodin BR, Hahn ME, Stegeman JJ (2007a) Role of AHR2 in the expression of novel cytochrome p450 1 family genes, cell cycle genes, and morphological defects in developing zebra fish exposed to 3,3',4,4',5-pentachlorobiphenyl or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Sci 100:180–193

    Article  Google Scholar 

  • Jönsson ME, Orrego R, Woodin BR, Goldstone JV, Stegeman JJ (2007b) Basal and 3,3',4,4',5-pentachlorobiphenyl-induced expression of cytochrome P450 1A, 1B and 1C genes in zebrafish. Toxicol Appl Pharmacol 221:29–41

    Article  Google Scholar 

  • Jönsson ME, Franks DG, Woodin BR, Jenny MJ, Garrick RA, Behrendt L, Hahn ME, Stegeman JJ (2009) The tryptophan photoproduct 6-formylindolo 3,2-b carbazole (FICZ) binds multiple AHRs and induces multiple CYP1 genes via AHR2 in zebrafish. Chem Biol Interact 181:447–454

    Article  Google Scholar 

  • Keiter S, Grund S, van Bavel B, Engwall M, Hagberg J, Kammann U, Klempt M, Manz W, Olsman H, Braunbeck T, Hollert H (2008) Activities and identification of Ah receptor agonists in sediments from the Danube River. Anal Bioanal Chem 390:2009–2020

    Article  CAS  Google Scholar 

  • Keiter S, Peddinghaus S, Feiler U, von der Goltz B, Hafner C, Ho NY, Rastegar S, Otte JC, Ottermanns R, Reifferscheid G, Strähle U, Braunbeck T, Hammers-Wirtz M, Hollert H (2010) DanTox—a novel joint research project using zebrafish (Danio rerio) to identify specific toxicity and molecular modes of action of sediment-bound pollutants. J Soils Sediments 10:714–717

    Article  CAS  Google Scholar 

  • Krone PH, Sass JB (1994) Hsp 90-alpha and Hsp 90-beta genes are present in the zebrafish and are differentially regulated in developing embryos. Biochem Biophys Res Commun 204:746–752

    Article  CAS  Google Scholar 

  • Mattingly CJ, Toscano WA (2001) Posttranscriptional silencing of cytochrome P4501A1 (CYP1A1) during zebrafish (Danio rerio) development. Dev Dyn 222:645–654

    Article  CAS  Google Scholar 

  • McCurley AT, Callard GV (2008) Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment. BMC Mol Biol 9:102

    Article  Google Scholar 

  • Nebert DW, Dalton TP (2006) The role of cytochrome P450 enzymes in endogenous signalling pathways and environmental carcinogenesis. Nat Rev Cancer 6:947–960

    Article  CAS  Google Scholar 

  • OECD (2013) Test No. 236: Fish Embryo Acute Toxicity (FET) Test. OECD Publishing

  • Omiecinski CJ, Remmel RP, Hosagrahara VP (1999) Concise review of the cytochrome P450s and their roles in toxicology. Toxicol Sci 48:151–156

    Article  CAS  Google Scholar 

  • Otte JC, Schmidt AD, Hollert H, Braunbeck T (2010) Spatio-temporal development of CYP1 activity in early life-stages of zebrafish (Danio rerio). Aquat Toxicol 100:38–50

    Article  CAS  Google Scholar 

  • Peddinghaus S, Brinkmann M, Bluhm K, Sagner A, Hinger G, Braunbeck T, Eisenträger A, Tiehm A, Hollert H, Keiter SH (2012) Quantitative assessment of the embryotoxic potential of NSO-heterocyclic compounds using zebrafish (Danio rerio). Reprod Toxicol 33:224–232

    Article  CAS  Google Scholar 

  • Perdew GH (1988) Association of the Ah receptor with the 90-kDa heat-shock-protein. J Biological Chem 263:13802–13805

    CAS  Google Scholar 

  • Rupik W, Jasik K, Bembenek J, Widlak W (2011) The expression patterns of heat shock genes and proteins and their role during vertebrate’s development. Comp Biochem Physiol A Mol Integr Physiol 159:349–366

    Article  Google Scholar 

  • Schiwy S, Bräunig J, Alert H, Hollert H, Keiter S (2014) A novel contact assay for testing aryl hydrocarbon receptor (AhR)-mediated toxicity of chemicals and whole sediments in zebrafish (Danio rerio) embryos. Environ Sci Pollut Res. doi:10.1007/s11356-014-3185-0, 1–14

    Google Scholar 

  • Shimada T, Gillam EMJ, Sutter TR, Strickland PT, Guengerich FP, Yamazaki H (1997) Oxidation of xenobiotics by recombinant human cytochrome P450 1B1. Drug Metab Dispos 25:617–622

    CAS  Google Scholar 

  • Shimada T, Fujii-Kuriyama Y (2004) Metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes P450 1A1 and 1B1. Canc Sci 95:1–6

    Article  CAS  Google Scholar 

  • Simon P (2003) Q-Gene: processing quantitative real-time RT–PCR data. Bioinformatics 19:1439–1440

    Article  CAS  Google Scholar 

  • Strähle U, Scholz S, Geisler R, Greiner P, Hollert H, Rastegar S, Schumacher A, Selderslaghs I, Weiss C, Witters H, Braunbeck T (2012) Zebrafish embryos as an alternative to animal experiments—a commentary on the definition of the onset of protected life stages in animal welfare regulations. Reprod Toxicol 33:128–132

    Article  Google Scholar 

  • Tanguay RL, Abnet CC, Heideman W, Peterson RE (1999) Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor. Biochim Biophys Acta Gene Struct Expr 1444:35–48

    Article  CAS  Google Scholar 

  • Thiede B, Hohenwarter W, Krah A, Mattow J, Schmid M, Schmidt F, Jungblut PR (2005) Peptide mass fingerprinting. Methods 35:237–247

    Article  CAS  Google Scholar 

  • Timme-Laragy AR, Cockman CJ, Matson CW, Di Giulio RT (2007) Synergistic induction of AHR regulated genes in developmental toxicity from co-exposure to two model PAHs in zebrafish. Aquat Toxicol 85:241–250

    Article  CAS  Google Scholar 

  • van den Berg M, De Jongh J, Poiger H, Olson JR (1994) The toxicokinetics and metabolism of polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and their relevance for toxicity. Crit Rev Toxicol 24:1–74

    Article  Google Scholar 

  • Wang L, Scheffler BE, Willett KL (2006) CYP1C1 messenger RNA expression is inducible by benzo[a]pyrene in Fundulus heteroclitus embryos and adults. Toxicol Sci 93:331–340

    Article  CAS  Google Scholar 

  • Whyte JJ, Jung RE, Schmitt CJ, Tillitt DE (2000) Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit Rev Toxicol 30:347–570

    Article  CAS  Google Scholar 

  • Yin HC, Tseng HP, Chung HY, Ko CY, Tzou WS, Buhler DR, Hu CH (2008) Influence of TCDD on zebrafish CYP1B1 transcription during development. Toxicol Sci 103:158–168

    Article  CAS  Google Scholar 

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Acknowledgments

The authors acknowledge the financial support by the German Federal Ministry of Education and Research (grant nos. 02WU1053). The authors also want to thank the German Federal Institute of Hydrology (BfG) and the Hamburg Port Authority for providing the sediments and its related parameters.

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Author notes

  1. Jennifer Bräunig

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

  2. Steffen H. Keiter

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

Authors and Affiliations

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

    Jennifer Bräunig, Sabrina Schiwy, Yvonne Müller, Henner Hollert & Steffen H. Keiter

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

    Oliver Broedel & Marcus Frohme

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  1. Jennifer Bräunig
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Correspondence to Jennifer Bräunig or Steffen H. Keiter.

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Bräunig, J., Schiwy, S., Broedel, O. et al. Time-dependent expression and activity of cytochrome P450 1s in early life-stages of the zebrafish (Danio rerio). Environ Sci Pollut Res 22, 16319–16328 (2015). https://doi.org/10.1007/s11356-015-4673-6

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