Marine Biotechnology

, Volume 19, Issue 5, pp 430–440 | Cite as

Carbonic Anhydrase Inhibitors Induce Developmental Toxicity During Zebrafish Embryogenesis, Especially in the Inner Ear

  • Hiroko Matsumoto
  • Shoko Fujiwara
  • Hisako Miyagi
  • Nobuhiro Nakamura
  • Yasuhiro Shiga
  • Toshihiro Ohta
  • Mikio Tsuzuki
Original Article

Abstract

In vertebrates, carbonic anhydrases (CAs) play important roles in ion transport and pH regulation in many organs, including the eyes, kidneys, central nervous system, and inner ear. In aquatic organisms, the enzyme is inhibited by various chemicals present in the environment, such as heavy metals, pesticides, and pharmaceuticals. In this study, the effects of CA inhibitors, i.e., sulfonamides [ethoxyzolamide (EZA), acetazolamide (AZA), and dorzolamide (DZA)], on zebrafish embryogenesis were investigated. In embryos treated with the sulfonamides, abnormal development, such as smaller otoliths, an enlarged heart, an irregular pectoral fin, and aberrant swimming behavior, was observed. Especially, the development of otoliths and locomotor activity was severely affected by all the sulfonamides, and EZA was a consistently stronger inhibitor than AZA or DZA. In the embryos treated with EZA, inner ear hair cells containing several CA isoforms, which provide HCO3 to the endolymph for otolith calcification and maintain an appropriate pH there, were affected. Acridine orange/ethidium bromide staining indicated that the hair cell damage in the inner ear and pectral fin is due to apoptosis. Moreover, RNA measurement demonstrated that altered gene expression of cell cycle arrest- and apoptosis-related proteins p53, p21, p27, and Bcl-2 occurred even at 0.08 ppm with which normal development was observed. This finding suggests that a low concentration of EZA may affect embryogenesis via the apoptosis pathway. Thus, our findings demonstrated the importance of potential risk assessment of CA inhibition, especially regarding the formation of otoliths as a one of the most sensitive organs in embryogenesis.

Keywords

Carbonic anhydrase Ethoxyzolamide Zebrafish Embryogenesis Inner ear Otoliths 

Notes

Acknowledgements

We are grateful to Mr. N. J. Halewood for correcting the English version of this paper. This work was supported by a Grant-in-Aid from the Ministry of Education, Science, Sports and Culture, Japan (16K07427), and the Promotion and Mutual Aid Corporation for Private Schools.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interest.

Supplementary material

10126_2017_9763_MOESM1_ESM.docx (379 kb)
Tables S1 (DOCX 378 kb)
10126_2017_9763_MOESM2_ESM.docx (150 kb)
Tables S2 (DOCX 149 kb)
10126_2017_9763_MOESM3_ESM.docx (175 kb)
Tables S3 (DOCX 174 kb)
10126_2017_9763_Fig11_ESM.jpg (41 kb)
Figure S1

(JPEG 41 kb)

References

  1. Amsterdam A, Nissen RM, Sun Z, Swindell EC, Farrington S, Hopkins N (2004) Identification of 315 genes essential for early zebrafish development. Proc Natl Acad Sci U S A 101:12792–12797CrossRefPubMedPubMedCentralGoogle Scholar
  2. Aspatwar A, Tolvanen MEE, Jokitalo E, Parikka M, Ortutay C, Harjula SKE, Rämet M, Vihinen M, Parkkila S (2013) Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish. Hum Mol Genet 22:417–432Google Scholar
  3. Beier M, Anken R (2006) On the role of carbonic anhydrase in the early phase of fish otolith mineralization. Adv Space Res 38:1119–1122CrossRefGoogle Scholar
  4. Beier M, Hilbig R, Anken R (2008) Histochemical localisation of carbonic anhydrase in the inner ear of developing cichlid fish, Oreochromis mossambicus. Adv Space Res 42:1986–1994CrossRefGoogle Scholar
  5. Brubaker KD, Mao F, Gay CV (1999) Localization of carbonic anhydrase in living osteoclasts with bodipy 558/568–modified acetazolamide, a thiadiazole carbonic anhydrase inhibitor. J Histochem Cytochem 47:545–550CrossRefPubMedGoogle Scholar
  6. Braunbeck T, Böttcher M, Hollert H, Kosmehl T, Lammer E, Leist E, Rudolf M, Seitz N (2005) Towards an alternative for the acute fish LC50 test in chemical assessment: the fish embryo toxicity test goes multi–species—an update. ALTEX 22:87–102PubMedGoogle Scholar
  7. Coffin AB, Williamson KL, Mamiya A, Raible DW, Rubel EW (2013) Profiling drug–induced cell death pathways in the zebrafish lateral line. Apoptosis 18:393–408CrossRefPubMedPubMedCentralGoogle Scholar
  8. Doğan S (2006) The in vitro effects of some pesticides on carbonic anhydrase activity of Oncorhynchus mykiss and Cyprinus carpio carpio fish. J Hazard Mater A132:171–176Google Scholar
  9. Gustafson AL, Stedman DB, Ball J, Hillegass JM, Flood A, Zhang CX, Panzica-Kelly J, Cao J, Coburn A, Enright BP, Tornesi MB, Hetheridge M, Augustine-Rauch KA (2012) Inter–laboratory assessment of a harmonized zebrafish developmental toxicology assay—progress report on phase I. Reprod Toxicol 33:155–164CrossRefPubMedGoogle Scholar
  10. He Y, Yu H, Sun S, Wang Y, Liu L, Chen Z, Li H (2013) Trans-2-phenylcyclopropylamine regulates zebrafish lateral line neuromast development mediated by depression of LSD1 activity. Int J Dev Biol 57:363–373CrossRefGoogle Scholar
  11. Hruska K, Franek M (2012) Sulfonamides in the environment: a review and a case report. Vet Med 57:1–35Google Scholar
  12. Hsu CJ (1991) Ultrastructural study of cytochemical localization of carbonic anhydrase in the inner ear. Acta Otolaryngol 111:75–84CrossRefPubMedGoogle Scholar
  13. Ito Y, Kobayashi S, Nakamura N, Miyagi H, Esaki M, Hoshijima K, Hirose S (2013) Close association of carbonic anhydrase (CA2a and CA15a), Na+/H+ exchanger (Nhe3b), and ammonia transporter Rhcg1 in zebrafish ionocytes responsible for Na+ uptake. Front Phys 4:59CrossRefGoogle Scholar
  14. Komori K, Suzuki Y (2009) Occurrence of pharmaceuticals and their environmental risk assessment of urban streams whose basins have different wastewater treatment conditions. J Jpn Soc Water Environ 32:133–138CrossRefGoogle Scholar
  15. Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T (2009) Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol C 149:196–209Google Scholar
  16. Levine AJ, Oren M (2009) The first 30 years of p53: growing ever more complex. Nat Rev Cancer 9:749–758CrossRefPubMedPubMedCentralGoogle Scholar
  17. Liao BK, Chen RD, Hwang PP (2009) Expression regulation of Na+–K+–ATPase alpha1-subunit subtypes in zebrafish gill ionocytes. Am J Physiol Regul Integr Comp Physiol 296:R1897–R1906Google Scholar
  18. Lin TY, Liao B, Horng J, Yan J, Hsiao C, Hwang P (2008) Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na+ uptake in zebrafish H+-ATPase-rich cells. Am J Physiol Cell Physiol 294:C1250-C1260Google Scholar
  19. Liu Z, Zuo J (2008) Cell cycle regulation in hair cell development and regeneration in the mouse cochlea. Cell Cycle 7:2129–2133CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lowenheim H, Furness DN, Kil J, Zinn C, Gultig K, Fero ML (1999) Gene disruption of p27 (Kip1) allows cell proliferation in the postnatal and adult organ of corti. Proc Natl Acad Sci U S A 96:4084–4088CrossRefPubMedPubMedCentralGoogle Scholar
  21. Mantela J, Jiang Z, Ylikoski J, Fritzsch B, Zacksenhaus E, Pirvola U (2005) The retinoblastoma gene pathway regulates the postmitotic state of hair cells of the mouse inner ear. Development 132:2377–2388CrossRefPubMedPubMedCentralGoogle Scholar
  22. Maren TH (1992) Direct measurements of the rate constants of sulfonamides with carbonic anhydrase. Mol Pharmacol 41:419–426PubMedGoogle Scholar
  23. Maren TH, Conroy CW (1993) A new class of carbonic anhydrase inhibitor. J Biol Chem 268:26233–26239PubMedGoogle Scholar
  24. Mayer-Gostan N, Kossmann H, Watrin A, Payan P, Boeuf G (1997) Distribution of ionocytes in the saccular epithelium of the inner ear of two teleosts (Oncorhynchus mykiss and Scophthalmus maximus). Cell Tissue Res 289:53–61CrossRefPubMedGoogle Scholar
  25. Miyachi S, Tsuzuki M, Avramova ST (1983) Utilization modes of inorganic carbon for photosynthesis in various species of Chlorella. Plant Cell Physiol 24:441–451Google Scholar
  26. Mohammed A (2013) Why are early life stages of aquatic organisms more sensitive to toxicants than adults? New insights into toxicology and drug testing. Chaper 3:39–62Google Scholar
  27. Mugiya Y (1977) Effect of acetazolamide on the otolith growth of goldfish. Bull Jpn Soc Sci Fish 43:1053–1058CrossRefGoogle Scholar
  28. Mugiya Y, Takahashi K (1985) Chemical properties of the saccular endolymph in the rainbow trout, Salmo gairdneri. Bull Fac Fish Hokkaido Univ 36:57–60Google Scholar
  29. Nagel R (2002) DarT: The embryo test with the zebrafish Danio rerio—a general model in ecotoxicology and toxicology. ALTEX 19:38–48Google Scholar
  30. Pietsch EC, Sykes SM, McMahon SB, Murphy ME (2008) The p53 family and programmed cell death. Oncogene 27:6507–6521CrossRefPubMedPubMedCentralGoogle Scholar
  31. Ralston S, Miyamoto GT (1983) Analyzing the width of daily otolith increments to age the Hawaiian snapper, Pristipomoides filamentosus. Fish Bull 81:523–535Google Scholar
  32. Russell RW, Gobas FAPC, Haffner GD (1999) Maternal transfer and in ovo exposure of organochlorines in oviparous organisms: a model and field verification. Environ Sci Technol 33:416–420CrossRefGoogle Scholar
  33. Seiler C, Nicolson T (1999) Defective calmodulin-dependent rapid apical endocytosis in zebrafish sensory hair cell mutants. J Neurobiol 41:424–434Google Scholar
  34. Seiler TB, Best N, Fernqvist MM, Hercht H, Smith KE, Braunbeck T, Mayer P, Hollert H (2014) PAH toxicity at aqueous solubility in the fish embryo test with Danio rerio using passive dosing. Chemosphere 112:77–84Google Scholar
  35. Shiao JC, Lin LY, Horng JL, Hwang PP, Kaneko T (2005) How can teleostean inner ear hair cells maintain the proper association with the accreting otolith? J Comp Neurol 488:331–341CrossRefPubMedGoogle Scholar
  36. Shinohara C, Yamashita K, Matsuo T, Kitamura S, Kawano F (2007) Effects of carbonic anhydrase inhibitor acetazolamide (AZ) on osteoclasts and bone structure. J Hard Tissue Biol 16:115–123CrossRefGoogle Scholar
  37. Stawicki TM, Owens KN, Linbo T, Reinhart KE, Rubel EW, Raible DW (2014) The zebrafish merovingian mutant reveals a role for pH regulation in hair cell toxicity and function. Dis Model Mech 7:847–856CrossRefPubMedPubMedCentralGoogle Scholar
  38. Tohse H, Ando H, Mugiya Y (2004) Biochemical properties and immunohistochemical localization of carbonic anhydrase in the sacculus of the inner ear in the salmon Oncorhynchus masou. Comp Biochem Physiol A Mol Integr Physiol 137:87–94CrossRefPubMedGoogle Scholar
  39. Tohse H, Murayama E, Ohira T, Takagi Y, Nagasawa H (2006) Localization and diurnal variations of carbonic anhydrase mRNA expression in the inner ear of the rainbow trout Oncorhynchus mykiss. Comp Biochem Physiol B 145:257–264CrossRefPubMedGoogle Scholar
  40. Topal A, Atamanalp M, Oruc E, Demir Y, Beydemir S, Isık A (2014) In vivo changes in carbonic anhydrase activity and histopathology of gill and liver tissues after acute exposure to chlorpyrifos in rainbow trout. Arh Hig Rada Toksikol 65:377–385CrossRefPubMedGoogle Scholar
  41. Tsuzuki M, Miyachi S (1989) The function of carbonic anhydrase in aquatic photosynthesis. Aquat Bot 34:85–104CrossRefGoogle Scholar
  42. Tytler P, Bell MV (1989) A study of diffusional permeability of water, sodium and chloride in yolk-sac larvae of cod (Gadus morhua L). J Exp Biol 147:125–132Google Scholar
  43. van Gelder MM, van Rooij IA, Miller RK, Zielhuis GA, de Jong-van den Berg LT, Roeleveld N (2010) Teratogenic mechanisms of medical drugs. Hum Reprod Update 16:378–394Google Scholar
  44. von Westernhagen H (1988) Sublethal effects of pollutants on fish eggs and larvae. In: Hoar WS, Randall DJ (eds) Fish physiology, Volume 11, Part A. The physiology of developing fish eggs and larvae. Academic Press, San Diego, pp 253–346Google Scholar
  45. Wang T, Wang J, Qiu J (1996) Observation on activity of carbonic anhydrase in the vestibule of guinea pigs. Zhonghua Er Bi Yan Hou Ke Za Zhi 31:24–25PubMedGoogle Scholar
  46. Wu L, Sagong B, Choi JY, Kim UK, Bok J (2013) A systematic survey of carbonic anhydrase mRNA expression during mammalian inner ear development. Dev Dyn 242:269–280CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2017

Authors and Affiliations

  • Hiroko Matsumoto
    • 1
  • Shoko Fujiwara
    • 1
  • Hisako Miyagi
    • 2
  • Nobuhiro Nakamura
    • 2
  • Yasuhiro Shiga
    • 1
  • Toshihiro Ohta
    • 1
  • Mikio Tsuzuki
    • 1
  1. 1.School of Life SciencesTokyo University of Pharmacy and Life SciencesTokyoJapan
  2. 2.Department of Life Science and TechnologyTokyo Institute of TechnologyYokohamaJapan

Personalised recommendations