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Analysis of Lethality and Malformations During Zebrafish (Danio rerio) Development

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Teratogenicity Testing

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1797))

Abstract

The versatility offered by zebrafish (Danio rerio) makes it a powerful and an attractive vertebrate model in developmental toxicity and teratogenicity assays. Apart from the newly introduced chemicals as drugs, xenobiotics also induce abnormal developmental abnormalities and congenital malformations in living organisms. Over the recent decades, zebrafish embryo/larva has emerged as a potential tool to test teratogenicity potential of these chemicals. Zebrafish responds to compounds as mammals do as they share similarities in their development, metabolism, physiology, and signaling pathways with that of mammals. The methodology used by the different scientists varies enormously in the zebrafish embryotoxicity test. In this chapter, we present methods to assess lethality and malformations during zebrafish development. We propose two major malformations scoring systems: binomial and relative morphological scoring systems to assess the malformations in zebrafish embryos/larvae. Based on the scoring of the malformations, the test compound can be classified as a teratogen or a nonteratogen and its teratogenic potential is evaluated.

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References

  1. Kimmel CB, Ballard WW, Kimmel SR et al (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310

    Article  CAS  Google Scholar 

  2. Zon LI, Peterson RT (2005) In vivo drug discovery in the zebrafish. Nat Rev Drug Discov 4:35–44

    Article  PubMed  CAS  Google Scholar 

  3. Teraoka H, Dong W, Hiraga T (2003) Zebrafish as a novel experimental model for developmental toxicology. Congenit Anom (Kyoto) 43:123–132

    Article  CAS  Google Scholar 

  4. Brannen KC, Panzica-Kelly JM, Danberry TL et al (2010) Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. Birth Defects Res B Dev Reprod Toxicol 89:66–77

    Article  PubMed  CAS  Google Scholar 

  5. Cortemeglia C, Beitinger TL (2005) Temperature tolerances of wildtype and red transgenic zebra danios. Trans Am Fish Soc 134:1431–1437

    Article  Google Scholar 

  6. Matthews M, Trevarrow B, Matthews J (2002) A virtual tour of the guide for zebrafish users. Lab Anim 31:34–40

    Google Scholar 

  7. Braunbeck T, Boettcher M, Hollert H et al (2005) Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species - an update. ALTEX 22:87–102

    PubMed  Google Scholar 

  8. Esmail MY, Astrofsky KM, Lawrence C et al (2015) Chapter 20: The biology and management of the zebrafish. In: Anderson LC, Otto G, Pritchett-Corning KR, Whary MT, Fox JG (eds) Laboratory animal medicine, 3rd edn. Academic Press, Amesterdam

    Google Scholar 

  9. Takahashi H (1977) Juvenile hermaphroditism in the zebrafish, Brachydanio rerio. Bull Fac Fish Hokkaido Univ 28:57–65

    Google Scholar 

  10. Goolish EM, Evans R, Max R (1998) Chamber volume requirements for reproduction of the zebrafish Danio rerio. Prog Fish Cult 60:127–132

    Article  Google Scholar 

  11. Ellis T, North B, Scott AP et al (2002) The relationships between stocking density and welfare in farmed rainbow trout. J Fish Biol 61:493–531

    Article  Google Scholar 

  12. Ramsay JM, Feist GW, Matthews JL et al (2006) Whole-body cortisol is an indicator of crowding stress in adult zebrafish, Danio rerio. Aquaculture 258:565–574

    Article  CAS  Google Scholar 

  13. Lawrence C (2007) The husbandry of zebrafish (Danio rerio): a review. Aquaculture 269:1–20

    Article  Google Scholar 

  14. Lawrence C, Adatto I, Best J et al (2012a) Generation time of zebrafish (Danio rerio) and medakas (Oryzias latipes) housed in the same aquaculture facility. Lab Anim (NY) 41:158–165

    Article  Google Scholar 

  15. Niimi AJ, LaHam QN (1974) Influence of breeding time interval on egg number, mortality, and hatching of the zebra fish Brachydanio rerio. Can J Zool 52:515–517

    Article  PubMed  CAS  Google Scholar 

  16. Trevarrow B (2004) Zebrafish facilities for small and large laboratories. Methods Cell Biol 77:565–591

    Article  PubMed  Google Scholar 

  17. Strecker R, Seiler TB, Hollert H et al (2011) Oxygen requirements of zebrafish (Danio rerio) embryos in embryo toxicity tests with environmental samples. Comp Biochem Physiol C Toxicol Pharmacol 153:318–327

    Article  PubMed  CAS  Google Scholar 

  18. Lawrence C, Mason T (2012) Zebrafish housing systems: a review of basic operating principles and considerations for design and functionality. ILAR J 53:179–191

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  19. Stoskopf M (2010) Fish medicine volume I, 2nd edn. ART Sciences LLC, Apex, NC

    Google Scholar 

  20. Noga EJ (2010) Fish disease: diagnosis and treatment, 2nd edn. Wiley-Blackwell, Hoboken, NJ

    Book  Google Scholar 

  21. Camargo JA, Alonso A, Salamanca A (2005) Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates. Chemosphere 58:1255–1267

    Article  PubMed  CAS  Google Scholar 

  22. Westerfield M (2007) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 5th edn. University of Oregon Press, Eugene, OR

    Google Scholar 

  23. Matthews M, Varga ZM (2012) Anesthesia and euthanasia in zebrafish. ILAR J 53:192–204

    Article  PubMed  CAS  Google Scholar 

  24. Selman K, Wallace RA, Sarka A et al (1993) Stages of oocyte development in the zebrafish Brachydanio rerio. J Morphol 218:203–224

    Article  PubMed  Google Scholar 

  25. Spence R, Fatema MK, Reichard M et al (2006) The distribution and habitat preferences of the zebrafish in Bangladesh. J Fish Biol 69:1435–1448

    Article  Google Scholar 

  26. Yelick PC, Schilling TF (2002) Molecular dissection of craniofacial development using zebrafish. Crit Rev Oral Biol Med 13:308–322

    Article  PubMed  Google Scholar 

  27. Bakkers J (2011) Zebrafish as a model to study cardiac development and human cardiac disease. Cardiovasc Res 91:279–288

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Hu N, Sedmera D, Yost HJ (2000) Structure and function of the developing zebrafish heart. Anat Rec 260:148–157

    Article  PubMed  CAS  Google Scholar 

  29. Grimes AC, Stadt HA, Shepherd IT et al (2006) Solving an enigma: arterial pole development in the zebrafish heart. Dev Biol 290:256–276

    Article  CAS  Google Scholar 

  30. Orr RT (1982) Vertebrate biology, 5th edn. Saunders College Publishing, Philadelphia, PA

    Google Scholar 

  31. Hickman CP, Roberts LS, Hickman FM (1988) Integrated principles of zoology. Times Mirror/Mosby College Publishing, St. Louis, MO

    Google Scholar 

  32. Stemple DL (2005) Structure and function of the notochord: an essential organ for chordate development. Development 132:2503–2512

    Article  PubMed  CAS  Google Scholar 

  33. Stickney HL, Barresi MJ, Devoto SH (2000) Somite development in zebrafish. Dev Dyn 219:287–303

    Article  PubMed  CAS  Google Scholar 

  34. Panzica-Kelly JM, Zhang CX, Augustine-Rauch K (2012) Zebrafish embryo developmental toxicology assay. Methods Mol Biol 889:25–50

    Article  PubMed  CAS  Google Scholar 

  35. Parichy DM, Elizondo MR, Mills MG (2009) Normal table of post-embryonic zebrafish development: staging by externally visible anatomy of the living fish. Dev Dyn 238:2975–3015

    Article  PubMed  PubMed Central  Google Scholar 

  36. Panzica-Kelly JM, Zhang CX, Danberry TL et al (2010) Morphological score assignment guidelines for the dechorionated zebra fish teratogenicity assay. Birth Defects Res B Dev Reprod Toxicol 89:382–395

    Article  PubMed  CAS  Google Scholar 

  37. Selderslaghs IW, Van Rompay AR, De Coen W et al (2009) Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo. Reprod Toxicol 28:308–320

    Article  PubMed  CAS  Google Scholar 

  38. Weigt S, Huebler N, Strecker R et al (2011) Zebrafish (Danio rerio) embryos as a model for testing proteratogens. Toxicology 281:25–36

    Article  PubMed  CAS  Google Scholar 

  39. Hermsen SA, van den Brandhof EJ, van der Ven LT et al (2011) Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies. Toxicol In Vitro 25:745–753

    Article  PubMed  CAS  Google Scholar 

  40. Padilla S, Corum D, Padnos B et al (2012) Zebrafish developmental screening of the ToxCastTM phase I chemical library. Reprod Toxicol 33:174–187

    Article  PubMed  CAS  Google Scholar 

  41. Beekhuijzen M, de Koning C, Flores-Guillen ME et al (2015) From cutting edge to guideline: a first step in harmonization of the zebrafish embryotoxicity test (ZET) by describing the most optimal test conditions and morphology scoring system. Reprod Toxicol 56:64–76

    Article  PubMed  CAS  Google Scholar 

  42. Incardona JP, Collier TK, Scholz NL (2004) Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol Appl Pharmacol 196:191–205

    Article  PubMed  CAS  Google Scholar 

  43. Prasch AL, Teraoka H, Carney SA et al (2003) Aryl hydrocarbon receptor 2 mediates 2,3,7,8- tetrachlorodibenzo-p-dioxin developmental toxicity in zebrafish. Toxicol Sci 76:138–150

    Article  PubMed  CAS  Google Scholar 

  44. Antkiewicz DS, Burns CG, Carney SA (2005) Heart malformation is an early response to TCDD in embryonic zebrafish. Toxicol Sci 84:368–377

    Article  PubMed  CAS  Google Scholar 

  45. Walker MB, Kimmel CB (2007) A two-color acid-free cartilage and bone stain for zebrafish larvae. Biotech Histochem 82:23–28

    Article  PubMed  CAS  Google Scholar 

  46. Kague E, Gallagher M, Burke S et al (2012) Skeletogenic fate of zebrafish cranial and trunk neural crest. PLoS One 7:e47394

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  47. Strecker R, Weigt S, Braunbeck T (2013) Cartilage and bone malformations in the head of zebrafish (Danio rerio) embryos following exposure to disulfiram and acetic acid hydrazide. Toxicol Appl Pharmacol 268:221–231

    Article  PubMed  CAS  Google Scholar 

  48. Ganesan S, Anaimalai Thirumurthi N, Raghunath A et al (2016) Acute and sub-lethal exposure to copper oxide nanoparticles causes oxidative stress and teratogenicity in zebrafish embryos. J Appl Toxicol 36:554–567

    Article  PubMed  CAS  Google Scholar 

  49. Busquet F, Nagel R, von Landenberg F et al (2008) Development of a new screening assay to identify proteratogenic substances using zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT). Toxicol Sci 104:177–188

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

Azhwar Raghunath is the recipient of a University Grants Commission—Basic Scientific Research Senior Research Fellowship (UGC-BSR-SRF—No.F.7-25/2007) funded by UGC-BSR, New Delhi, India.

This work was supported by the University Grants Commission—Special Assistance Programme (UGC-SAP-II:F-3-20/2013) and Department of Science and Technology, Fund for Improvement of Science and Technology infrastructure in universities and higher educational institutions (DST-FIST:SR/FST/LSI-618/2014), New Delhi, India.

The photomicrographs provided in this chapter are the work of research scholars in the Dr. P. Ekambaram’s laboratory. The authors thank the research scholars A.T. Naveen Kumar, E. Sankavi, and P. Reshma for contributing their precious time in taking the photomicrographs.

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Authors and Affiliations

  1. Molecular Toxicology Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, India

    Azhwar Raghunath & Ekambaram Perumal

Authors
  1. Azhwar Raghunath
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  2. Ekambaram Perumal
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Correspondence to Ekambaram Perumal .

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Editors and Affiliations

  1. Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trás-os-Montes e Alto Douro (UTAD), Vila Real, Portugal

    Luís Félix

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Raghunath, A., Perumal, E. (2018). Analysis of Lethality and Malformations During Zebrafish (Danio rerio) Development. In: Félix, L. (eds) Teratogenicity Testing. Methods in Molecular Biology, vol 1797. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7883-0_18

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  • DOI: https://doi.org/10.1007/978-1-4939-7883-0_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7882-3

  • Online ISBN: 978-1-4939-7883-0

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