Journal of Comparative Physiology B

, Volume 189, Issue 1, pp 97–108 | Cite as

Acute hypoxia/reoxygenation affects muscle mitochondrial respiration and redox state as well as swimming endurance in zebrafish

  • G. Napolitano
  • Paola Venditti
  • G. Fasciolo
  • D. Esposito
  • E. Uliano
  • C. Agnisola
Original Paper


Rapid fluctuations of the oxygen content of both natural and anthropogenic origin are relatively common in freshwater environments. Fish adaptation to these conditions implies tolerance of both low levels of oxygen availability and reoxygenation. Hypoxia tolerance in fish has been widely studied, but the involvement of mitochondria in the response of fish to rapid hypoxia/reoxygenation stress is less known. Zebrafish, a floodplain species, is likely facing significant changes in dissolved oxygen in its natural environment and displays a moderate ability to tolerate hypoxia. In the present study, we report the effects of an acute hypoxia/reoxygenation stress (H/R) protocol on mitochondrial functionality (respiration, complex activities, rate of H2O2 release) and redox state (level of HPs and protein oxidation) of muscle tissue. In parallel, the animal metabolic performance (routine metabolism, nitrogen excretion and swimming performance) was measured. Additionally, the recovery from H/R was tested 20 h after treatment. A significant stimulation by H/R of muscle mitochondrial respiration and H2O2 release was observed, which was only in part counteracted by stimulation of the antioxidant system, resulting in an increased level of lipid peroxides and protein carbonyls. In parallel, H/R increased the animal oxygen consumption and urea excretion rate and reduced routine activity. A significant strong reduction of endurance at 80% Ucrit was also observed. Most of the altered parameter did not recover 20 h after reoxygenation. These data indicate a significant alteration of zebrafish muscle mitochondrial state after acute H/R, associated with changes in tissue redox state and locomotor performance.


Zebrafish Hypoxia/reoxygenation Oxidative stress Endurance Routine oxygen consumption Ammonia excretion Mitochondrial functionality. 



AA acknowledges support from the Basic Research Funding of the Biology Department of the Naples University Federico II.

Author contributions

PV conceived and coordinated the study together with CA and helped draft the manuscript. GN and DB attended to the experiment concerning the evaluation of mitochondrial bioenergetic state, oxidative damage and antioxidant status. DE and EU attended to the experimental set up of in vivo experiments, collected the related experimental data and participated in the data analysis. CA proposed the underlying hypothesis of the study, conceived and coordinated the study together with PV, designed the study, and helped draft the manuscript. All authors gave final approval for publication.


  1. Amérand A, Vettier A, Sébert P, Moisan C (2006) A comparative study of reactive oxygen species in red muscle: pressure effects. Undersea Hyperb Med 33:161–167Google Scholar
  2. Barré H, Bailly L, Rouanet JL (1987) Increased oxidative capacity in skeletal muscles from cold-acclimated ducklings: a comparison with rats. Comp Biochem Physiol Part B Comp Biochem 88:519–522. CrossRefGoogle Scholar
  3. Barrionuevo WR, Fernandes MN, Rocha O (2010) Aerobic and anaerobic metabolism for the zebrafish, Danio rerio, reared under normoxic and hypoxic conditions and exposed to acute hypoxia during development. Braz J Biol 70:425–434CrossRefGoogle Scholar
  4. Bayley PB (1995) Understanding large river: floodplain ecosystems. Bioscience 45:153–158. CrossRefGoogle Scholar
  5. Bickler PE, Buck LT (2007) Hypoxia tolerance in reptiles, amphibians, and fishes: life with variable oxygen availability. Annu Rev Physiol 69:145–170. CrossRefGoogle Scholar
  6. Bosworth CA, Chou C-W, Cole RB, Rees BB (2005) Protein expression patterns in zebrafish skeletal muscle: initial characterization and the effects of hypoxic exposure. Proteomics 5:1362–1371. CrossRefGoogle Scholar
  7. Bourdineaud J-P, Rossignol R, Brèthes D (2013) Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics. Int J Biochem Cell Biol 45:16–22. CrossRefGoogle Scholar
  8. Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 134:707–716CrossRefGoogle Scholar
  9. Burgetz IJ, Rojas-Vargas A, Hinch SG, Randall DJ (1998) Initial recruitment of anaerobic metabolism during sub-maximal swimming in rainbow trout (Oncorhynchus mykiss). J Exp Biol 201:2711–2721Google Scholar
  10. Cao C, Leng Y, Huang W, Liu X, Kufe D (2003) Glutathione peroxidase 1 Is regulated by the c-Abl and Arg tyrosine kinases. J Biol Chem 278:39609–39614. CrossRefGoogle Scholar
  11. Carlberg I, Mannervik B (1975) Purification and characterization of the flavoenzyme glutathione reductase from rat liver. J Biol Chem 250:5475–5480Google Scholar
  12. Chakravarty S, Reddy BR, Sudhakar SR, Saxena S, Das T, Meghah V, Swamy B, Kumar CV, Idris A, M.M (2013) Chronic unpredictable stress (CUS)-induced anxiety and related mood disorders in a zebrafish model: altered brain proteome profile implicates mitochondrial dysfunction. PLoS One 8:e63302. CrossRefGoogle Scholar
  13. Chapman LJ, Mckenzie DJ (2009) Chapter 2 behavioral responses and ecological consequences. In: Richards JG, Farrell AP, Brauner CJ (eds) Fish physiology, hypoxia. Academic Press, Cambridge, pp 25–77. CrossRefGoogle Scholar
  14. Cheek A, Landry C, Steele S, Manning S (2009) Diel hypoxia in marsh creeks impairs the reproductive capacity of estuarine fish populations. Mar Ecol Prog Ser 392:211–221. CrossRefGoogle Scholar
  15. Cresci A, De Rosa R, Putman NF, Agnisola C (2017) Earth-strength magnetic field affects the rheotactic threshold of zebrafish swimming in shoals. Comp Biochem Physiol A Mol Integr Physiol 204:169–176. CrossRefGoogle Scholar
  16. Diaz RJ, Breitburg DL (2009) The hypoxic environment. In: Richards JG, Farrell AP, Brauner CJ (eds) Hypoxia, fish physiology. Academic Press, Amsterdam, pp 1–23. Google Scholar
  17. DiMichele L, Powers DA (1982) Physiological basis for swimming endurance differences between LDH-B genotypes of Fundulus heteroclitus. Science 216:1014–1016. CrossRefGoogle Scholar
  18. Du SNN, Mahalingam S, Borowiec BG, Scott GR (2016) Mitochondrial physiology and reactive oxygen species production are altered by hypoxia acclimation in killifish (Fundulus heteroclitus). J Exp Biol 219:1130–1138. CrossRefGoogle Scholar
  19. Farrell AP, Richards JG (2009) Defining hypoxia: an integrative synthesis of the responses of fish to hypoxia. In: Richards JG, Farrell AP, Brauner CJ (eds) Hypoxia, fish physiology. Academic Press, Amsterdam, pp 487–503. CrossRefGoogle Scholar
  20. Filho DW (2007) Reactive oxygen species, antioxidants and fish mitochondria. Front Biosci 12:1229. CrossRefGoogle Scholar
  21. Flohé L, Günzler WA (1984) Assays of glutathione peroxidase. Methods Enzymol 105:114–120Google Scholar
  22. Galli GLJ, Richards JG (2014) Mitochondria from anoxia-tolerant animals reveal common strategies to survive without oxygen. J Comp Physiol B 184:285–302. CrossRefGoogle Scholar
  23. Gornall AG, Bardawill CJ, David MM (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177:751–766Google Scholar
  24. Granger DN, Kvietys PR (2015) Reperfusion injury and reactive oxygen species: the evolution of a concept. Redox Biol 6:524–551. CrossRefGoogle Scholar
  25. Griffith RW (1980) Chemistry of the body fluids of the coelacanth, latimeria chalumnae. Proc R Soc B Biol Sci 208:329–347. Google Scholar
  26. Griffiths E (2012) Mitochondria and heart disease. In: Scatena R, Bottoni P, Giardina B (eds) Advances in mitochondrial medicine, advances in experimental medicine and biology. Springer, Amsterdam, pp 249–267Google Scholar
  27. Hafner RP, Brown GC, Brand MD (1990) Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the ‘top-down’ approach of metabolic control theory. Eur J Biochem 188:313–319. CrossRefGoogle Scholar
  28. Hagenaars A, Vergauwen L, Benoot D, Laukens K, Knapen D (2013) Mechanistic toxicity study of perfluorooctanoic acid in zebrafish suggests mitochondrial dysfunction to play a key role in PFOA toxicity. Chemosphere 91:844–856. CrossRefGoogle Scholar
  29. Heath RL, Tappel AL (1976) A new sensitive assay for the measurement of hydroperoxides. Anal Biochem 76:184–191. CrossRefGoogle Scholar
  30. Hermes-Lima M, Zenteno-Savın T (2002) Animal response to drastic changes in oxygen availability and physiological oxidative stress. Comp Biochem Physiol Part C Toxicol Pharmacol 133:537–556CrossRefGoogle Scholar
  31. Hermes-Lima M, Storey JM, Storey KB (1998) Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails. Comp Biochem Physiol B Biochem Mol Biol 120:437–448CrossRefGoogle Scholar
  32. Hyslop PA, Sklar LA (1984) A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: Its use in the simultaneous fluorimetric assay of cellular activation processes. Anal Biochem 141:280–286. CrossRefGoogle Scholar
  33. Ivanov AS, Putvinskiĭ AV, Antonov VF, Vladimirov IA (1977) Magnitude of the protein permeability of liposomes following photoperoxidation of lipids. Biofizika 22:621–624Google Scholar
  34. Jain KE, Hamilton JC, Farrell AP (1997) Use of a ramp velocity test to measure critical swimming speed in rainbow trout (Onchorhynchus mykiss). Comp Biochem Physiol 117A:441–444CrossRefGoogle Scholar
  35. Keller ET, Murtha JM (2004) The use of mature zebrafish (Danio rerio) as a model for human aging and disease. Comp Biochem Physiol Part C Toxicol Pharmacol 138:335–341. CrossRefGoogle Scholar
  36. Landman MJ, Van Den Heuvel MR, Ling N (2005) Relative sensitivities of common freshwater fish and invertebrates to acute hypoxia. N Z J Mar Freshw Res 39:1061–1067. CrossRefGoogle Scholar
  37. Lenaz G (2012) Mitochondria and reactive oxygen species. Which role in physiology and pathology? In: Scatena R, Bottoni P, Giardina B (eds), Advances in mitochondrial medicine, advances in experimental medicine and biology. Springer, Amsterdam, pp 96–136Google Scholar
  38. Lerebours A, Gonzalez P, Adam C, Camilleri V, Bourdineaud J-P, Garnier-Laplace J (2009) Comparative analysis of gene expression in brain, liver, skeletal muscles, and gills of zebrafish (Danio rerio) exposed to environmentally relevant waterborne uranium concentrations. Environ Toxicol Chem 28:1271–1278CrossRefGoogle Scholar
  39. Leveelahti L, Rytkönen KT, Renshaw GMC, Nikinmaa M (2014) Revisiting redox-active antioxidant defenses in response to hypoxic challenge in both hypoxia-tolerant and hypoxia-sensitive fish species. Fish Physiol Biochem 40:183–191. CrossRefGoogle Scholar
  40. Li J, Xie X (2018) Inconsistent responses of liver mitochondria metabolism and standard metabolism in Silurus meridionalis when exposed to waterborne cadmium. Comp Biochem Physiol Part C Toxicol Pharmacol 214:17–22. CrossRefGoogle Scholar
  41. Lucas MC, Priede IG (1992) Utilization of metabolic scope in relation to feeding and activity by individual and grouped zebrafish, Brachydanio rerio (Hamilton-Buchanan). J Fish Biol 41:175–190. CrossRefGoogle Scholar
  42. Lushchak VI (2011) Environmentally induced oxidative stress in aquatic animals. Aquat Toxicol 101:13–30. CrossRefGoogle Scholar
  43. Lushchak VI (2012) Glutathione homeostasis and functions: potential targets for medical interventions. J Amino Acids. Google Scholar
  44. Lushchak VI, Bagnyukova TV (2006) Effects of different environmental oxygen levels on free radical processes in fish. Comp Biochem Physiol B Biochem Mol Biol 144:283–289. CrossRefGoogle Scholar
  45. Mandic M, Todgham AE, Richards JG (2009) Mechanisms and evolution of hypoxia tolerance in fish. Proc R Soc B Biol Sci 276:735–744. CrossRefGoogle Scholar
  46. Martos-Sitcha JA, Bermejo-Nogales A, Calduch-Giner JA, Pérez-Sánchez J (2017) Gene expression profiling of whole blood cells supports a more efficient mitochondrial respiration in hypoxia-challenged gilthead sea bream (Sparus aurata). Front Zool. Google Scholar
  47. Mayzaud P, Conover RJ (1988) O:N atomic ratio as a tool to describe zooplankton metabolism. Mar Ecol Prog Ser 45:289–302CrossRefGoogle Scholar
  48. McDonald MD, Wood CM (2004) The effect of chronic cortisol elevation on urea metabolism and excretion in the rainbow trout (Oncorhynchus mykiss). J Comp Physiol 174:71–81. CrossRefGoogle Scholar
  49. Onukwufor JO, MacDonald N, Kibenge F, Stevens D, Kamunde C (2014) Effects of hypoxia-cadmium interactions on rainbow trout (Oncorhynchus mykiss) mitochondrial bioenergetics: attenuation of hypoxia-induced proton leak by low doses of cadmium. J Exp Biol 217:831–840. CrossRefGoogle Scholar
  50. Onukwufor JO, Kibenge F, Stevens D, Kamunde C (2016) Hypoxia-reoxygenation differentially alters the thermal sensitivity of complex I basal and maximal mitochondrial oxidative capacity. Comp Biochem Physiol A Mol Integr Physiol 201:87–94. CrossRefGoogle Scholar
  51. Paital B (2013) Antioxidant and oxidative stress parameters in brain of Heteropneustes fossilis under air exposure condition; role of mitochondrial electron transport chain. Ecotoxicol Environ Saf 95:69–77. CrossRefGoogle Scholar
  52. Palmer G, Horgan DJ, Tisdale H, Singer TP, Beinert H (1968) Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase XIV. Location of the sites of inhibition of rotenone, barbiturates, and piericidin by means of electron paramagnetic resonance spectroscopy. J Biol Chem 243:844–847Google Scholar
  53. Putvinskiĭ AV (1977) Decrease in the electrical stability of lipid membranes following UV-irradiation. Biofizika 22:725–727Google Scholar
  54. Ragan CI, Wilson MT, Darley-Usmar VM, Lowe PN (1987) Sub-fractionation of mitochondria and isolation of the proteins of oxidative phosphorylation. In: Darley-Usmar VM, Rickwood D, Wilson MT (eds) Mitochondria: a practical approach. IRL Press, Oxford, pp 79–112Google Scholar
  55. Rees BB, Sudradjat F, Love JW (2001) Acclimation to hypoxia increases survival time of zebrafish, Danio rerio, during lethal hypoxia. J Exp Zool 247Google Scholar
  56. Reznick AZ, Packer L (1994) [38] Oxidative damage to proteins: spectrophotometric method for carbonyl assay. In: methods in enzymology, oxygen radicals in biological systems part C. Academic Press, Cambridge, pp 357–363. CrossRefGoogle Scholar
  57. Richards JG (2009) Metabolic and molecular responses of fish to hypoxia. In: Richards JG, Farrell AP, Brauner CJ (eds) Hypoxia, fish physiology. Academic Press, Amsterdam, pp 443–485. CrossRefGoogle Scholar
  58. Robertson CE, Wright PA, Koblitz L, Bernier NJ (2014) Hypoxia-inducible factor-1 mediates adaptive developmental plasticity of hypoxia tolerance in zebrafish, Danio rerio. Proc R Soc B Biol Sci 281:20140637–20140637. CrossRefGoogle Scholar
  59. Simon LM, Robin ED (1971) Relationship of cytochrome oxidase activity to vertebrate total and organ oxygen consumption—ScienceDirect. Int J Biochem 2:569–573CrossRefGoogle Scholar
  60. Solórzano L (1969) Determination of ammonia in natural waters by the phenolhypochlorite method. Limnol Oceanogr 14:799–801. CrossRefGoogle Scholar
  61. Speers-Roesch B, Sandblom E, Lau GY, Farrell AP, Richards JG (2010) Effects of environmental hypoxia on cardiac energy metabolism and performance in tilapia. Am J Physiol Regul Integr Comp Physiol 298:R104–R119. CrossRefGoogle Scholar
  62. Spence R, Gerlach G, Lawrence C, Smith C (2008) The behaviour and ecology of the zebrafish, Danio rerio. Biol Rev 83:13–34. CrossRefGoogle Scholar
  63. Thengchaisri N, Hein TW, Wang W, Xu X, Li Z, Fossum TW, Kuo L (2006) Upregulation of arginase by H 2 O 2 impairs endothelium-dependent nitric oxide-mediated dilation of coronary arterioles. Arterioscler Thromb Vasc Biol 26:2035–2042. CrossRefGoogle Scholar
  64. Thorarensen H (2011) The effect of exercise on respiration. In: Farrell AP, Cech JJ, Richards JG, Stevens ED (eds) Encyclopedia of fish: from genome to environment. Academic Press, Cambridge, pp 812–819Google Scholar
  65. Tierney KB (2011) Swimming performance assessment in fishes. J Vis Exp 1:e2572. Google Scholar
  66. Ton C, Stamatiou D, Liew C-C (2003) Gene expression profile of zebrafish exposed to hypoxia during development. Physiol Genom 13:97–106. CrossRefGoogle Scholar
  67. Turrens JF, Alexandre A, Lehninger AL (1985) Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. Arch Biochem Biophys 237:408–414. CrossRefGoogle Scholar
  68. Uliano E, Cataldi M, Carella F, Migliaccio O, Iaccarino D, Agnisola C (2010) Effects of acute changes in salinity and temperature on routine metabolism and nitrogen excretion in gambusia (Gambusia affinis) and zebrafish (Danio rerio). Comp Biochem Physiol Mol Integr Physiol 157:283–290. CrossRefGoogle Scholar
  69. van der Meer DLM, van den Thillart GEEJM, Witte F, de Bakker MAG, Besser J, Richardson MK, Spaink HP, Leito JTD, Bagowski CP (2005) Gene expression profiling of the long-term adaptive response to hypoxia in the gills of adult zebrafish. Am J Physiol Regul Integr Comp Physiol 289:R1512–R1519. CrossRefGoogle Scholar
  70. Venditti P, Masullo P, Meo SD (2001) Effects of myocardial ischemia and reperfusion on mitochondrial function and susceptibility to oxidative stress. Cell Mol Life Sci CMLS 58:1528–1537. CrossRefGoogle Scholar
  71. Venditti P, De Rosa R, Di Meo S (2003) Effect of thyroid state on H2O2 production by rat liver mitochondria. Mol Cell Endocrinol 205:185–192CrossRefGoogle Scholar
  72. Venditti P, De Rosa R, Cigliano L, Agnisola C, Di Meo S (2004) Role of nitric oxide in the functional response to ischemia–reperfusion of heart mitochondria from hyperthyroid rats. Cell Mol Life Sci CMLS 61:2244–2252. CrossRefGoogle Scholar
  73. Verkerk AO, Remme CA (2012) Zebrafish: a novel research tool for cardiac (patho)electrophysiology and ion channel disorders. Front Physiol. Google Scholar
  74. Ward AC, Lieschke GJ (2002) The zebrafish as a model system for human disease. Front Biosci 7:d827–d833CrossRefGoogle Scholar
  75. Welker AF, Campos ÉG, Cardoso LA, Hermes-Lima M (2012) Role of catalase on the hypoxia/reoxygenation stress in the hypoxia-tolerant Nile tilapia. Am J Physiol Regul Integr Comp Physiol 302:R1111–R1118. CrossRefGoogle Scholar
  76. Wu G, Morris SM (1998) Arginine metabolism: nitric oxide and beyond. Biochem J 336:1–17. CrossRefGoogle Scholar

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© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of BiologyUniversity of Naples Federico II, Complesso Universitario Monte Sant’AngeloNaplesItaly

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