Journal of Comparative Physiology B

, Volume 177, Issue 7, pp 765–777 | Cite as

Effects of seasonal and latitudinal cold on oxidative stress parameters and activation of hypoxia inducible factor (HIF-1) in zoarcid fish

  • K. Heise
  • M. S. Estevez
  • S. Puntarulo
  • M. Galleano
  • M. Nikinmaa
  • H. O. Pörtner
  • D. AbeleEmail author
Original Paper


Acute, short term cooling of North Sea eelpout Zoarces viviparus is associated with a reduction of tissue redox state and activation of hypoxia inducible factor (HIF-1) in the liver. The present study explores the response of HIF-1 to seasonal cold in Zoarces viviparus, and to latitudinal cold by comparing the eurythermal North Sea fish to stenothermal Antarctic eelpout (Pachycara brachycephalum). Hypoxic signalling (HIF-1 DNA binding activity) was studied in liver of summer and winter North Sea eelpout as well as of Antarctic eelpout at habitat temperature of 0°C and after long-term warming to 5°C. Biochemical parameters like tissue iron content, glutathione redox ratio, and oxidative stress indicators were analyzed to see whether the cellular redox state or reactive oxygen species formation and HIF activation in the fish correlate. HIF-1 DNA binding activity was significantly higher at cold temperature, both in the interspecific comparison, polar vs. temperate species, and when comparing winter and summer North Sea eelpout. Compared at the low acclimation temperatures (0°C for the polar and 6°C for the temperate eelpout) the polar fish showed lower levels of lipid peroxidation although the liver microsomal fraction turned out to be more susceptible to lipid radical formation. The level of radical scavenger, glutathione, was twofold higher in polar than in North Sea eelpout and also oxidised to over 50%. Under both conditions of cold exposure, latitudinal cold in the Antarctic and seasonal cold in the North Sea eelpout, the glutathione redox ratio was more oxidised when compared to the warmer condition. However, oxidative damage parameters (protein carbonyls and thiobarbituric acid reactive substances (TBARS) were elevated only during seasonal cold exposure in Z. viviparus. Obviously, Antarctic eelpout are keeping oxidative defence mechanisms high enough to avoid accumulation of oxidative damage products at low habitat temperature. The paper discusses how HIF could be instrumental in cold adaptation in fish.


North Sea eelpout Polar eelpout HIF-1 Oxidative stress Glutathione 



The authors would like to thank Maike Schmidt for her help in preparing the fish samples, as well as Tamara Zaobornyj and Laura Valdez for adjusting the chemiluminescence assay protocol to fish liver tissue. We further thank Hanna Tranberg, Eeva Rissanen and Kristiina Vuori for their contribution to adjust EMSA and Western blot methodology to eelpout samples. This study was supported by grants from Deutscher Akademischer Austauschdienst (DAAD) to KH.


  1. Abele D, Puntarulo S (2004) Formation of reactive species and induction of antioxidant defence systems in polar and temperate marine invertebrates and fish. Comp Biochem Physiol 138A:405–415Google Scholar
  2. Abele D, Philipp E, Gonzalez P, Puntarulo S (2007) Marine invertebrate mitochondria and oxidative stress. Front Biosci 12:933–946PubMedCrossRefGoogle Scholar
  3. Acker T, Acker H (2004) Cellular oxygen sensing need in CNS function: physiological and pathological implications. J Exp Biol 207:3171–3188PubMedCrossRefGoogle Scholar
  4. Acker T, Fandrey J, Acker H (2006) The good, the bad and the ugly in oxygen sensing: ROS, cytochromes and prolyl-hydroxylases. Cardiovasc Res 71:195–207PubMedCrossRefGoogle Scholar
  5. Atunes F, Boveris A, Cadenas E (2004) On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide. PNAS 101:16774–16779CrossRefGoogle Scholar
  6. Buettner GR (1987) Spin trapping: ESP parameters of spin adducts. Free Rad Biol Med 3:259–303PubMedCrossRefGoogle Scholar
  7. Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 71:248–254CrossRefGoogle Scholar
  8. Brodte E, Graeve M, Jacob U, Knust R, Pörtner H-O (2006a) Temperature dependent lipid levels and components in polar and temperate eelpout (Zoarcidae). Fish Physiol Biochem (in press)Google Scholar
  9. Brodte E, Knust R, Pörtner H-O, Arntz WE (2006b) Biology of the Antarctic eelpout Pachycara brachycephalum. Deep-Sea Res 53:1131–1140CrossRefGoogle Scholar
  10. Brodte E, Knust R, Pörtner HO (2006c) Temperature dependent energy allocation to growth in Antarctic and boreal eelpout (Zoarcidae). Polar Biol doi:10.1007/s00300-006-0165-yGoogle Scholar
  11. Brumby PE, Massey V (1967) Determination of non-heme iron, total iron and copper. Methods Enzymol 10:464–472Google Scholar
  12. Cleeter MWJ, Cooper JM, Darley-Usmar VM, Moncada S, Schapira AHV (1994) Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett 345:50–54PubMedCrossRefGoogle Scholar
  13. Czubryt MP, Panagia V, Pierce GN (1996) The roles of free radicals, peroxides and oxidized lipoproteins in second messenger system dysfunction. EXS 76:57–69PubMedGoogle Scholar
  14. Desai I (1984) Vitamin E analysis methods for animal tissues. Methods Enzymol 105:138–146PubMedCrossRefGoogle Scholar
  15. Doege K, Heine S, Jensen I, Jelkmann W, Metzen E (2005) Inhibition of mitochondrial respiration elevates oxygen concentration but leaves regulation of hypoxia-inducible factor (HIF) intact. Blood 106:2311–2317PubMedCrossRefGoogle Scholar
  16. Dunlap WC, FujisawaA , Yamamoto Y, Moylan TJ, Sidell BD (2002) Notothenoid fish, krill and phytoplankton from Antarctica contain a vitamin E constituent (α-tocopherol) functionally associated with cold-water adaptation. Comp Biochem Physiol 133B:299–305Google Scholar
  17. Egginton S, Sidell BD (1989) Thermal acclimation induces adaptive changes in subcellular structure of fish skeletal muscle. Am J Physiol 256:R1–R9PubMedGoogle Scholar
  18. Fandrey J, Gorr TA, Gassmann M (2006) Regulating cellular oxygen sensing by hydroxylation. Cardiovasc Res 71:642–651PubMedCrossRefGoogle Scholar
  19. Fariss MW, Reed DJ (1987) High-performance liquid chromatography of thiols and disufides: diphenol derivatives. Methods Enzymol 143:101–109PubMedGoogle Scholar
  20. Gieseg SP, Cuddihy S, Hill JV, Davison W (2000) A comparison of plasma vitamin C and E levels in two Antarctic and two temperate water fish species. Comp Biochem Physiol 125B:371–378Google Scholar
  21. Gonzalez Flecha B, Llesuy S, Boveris A (1991) Hydroperoxide-initiated chemiluminescence: an assay for oxidative stress in biopsies of heart, liver, and muscle. Free Rad Biol Med 10:93–100PubMedCrossRefGoogle Scholar
  22. Gorr TA, Gassmann M, Wappner P (2006) Sensing and responding to hypoxia via HIF in model invertebrates. J Insect Physiol 52:349–364PubMedCrossRefGoogle Scholar
  23. Gracey AY, Fraser FJ, Li W, Fang Y (2004) Coping with cold: an integrative analysis of the transcriptome of a poikilothermic vertebrate. Proc Natl Acad Sci USA 101(48):16970–16975PubMedCrossRefGoogle Scholar
  24. Günzler WA, Flohe L (1985) Glutathione peroxidase. In: Greenwald RA (ed) CRC handbooks of methods for oxygen radical research. CRC, Boca Raton, pp 285–289Google Scholar
  25. Haddad JJE, Olvers RE, Land SC (2000) Antioxidant/pro-oxidant equilibrium regulates HIF-1α and NF-κB redox sensitivity. J Biol Chem 275:21130–21139PubMedCrossRefGoogle Scholar
  26. Halliwell B, Gutteridge JMC (1985) Free radicals in biology and medicine, 2nd edn. Clarendon, Oxford, pp 1–543Google Scholar
  27. Han D, Hanawa N, Saberi B, Kaplowitz N (2006) Mechanisms of liver injury: role of glutathione redox status in liver injury. Am J Physiol Gastrointest Liver Physiol 291:G1–G7PubMedCrossRefGoogle Scholar
  28. Heise K, Puntarulo S, Nikinmaa M, Lucassen M, Pörtner HO, Abele D (2006a) Oxidative stress and HIF-1 DNA binding during stressful cold exposure and recovery in the North Sea eelpout (Zoarces viviparus). Comp Biochem Physiol 143A:494–503Google Scholar
  29. Heise K, Puntarulo S, Nikinmaa M, Abele D, Pörtner HO (2006b) Oxidative stress during stressful heat exposure and recovery in the North Sea eelpout (Zoarces viviparus). J Exp Biol 209:353–363PubMedCrossRefGoogle Scholar
  30. Huang LE, Arany Z, Livingston DM, Bunn FH (1996) Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its α-subunit. J Biol Chem 271(50):32253–32259PubMedCrossRefGoogle Scholar
  31. Huang LE, Bunn FH (2003) Hypoxia-inducible factor and its biomedical relevance. J Biol Chem 278(22):19575–19578PubMedCrossRefGoogle Scholar
  32. Hulbert AJ (2006) The link between membrane composition, metabolic rate and lifespan. Comp Biochem Physiol A (in press)Google Scholar
  33. Ivan M, Kondo K, Yang H, Kim W, Valiando J, Ohh M, Salic A, Asara JM, Lane WS, Kaelin WG Jr (2001) HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Sci 292:464–468Google Scholar
  34. Johnston IA (1982) Capillarisation, oxygen diffusion distances and mitochondrial content of carp muscles following acclimation to summer and winter temperatures. Cell Tissue Res 222:325–337PubMedCrossRefGoogle Scholar
  35. Katschinski DM, Le L, Heinrich D, Wagner KF, Hofer T, Schindler SG, Wenger RH (2002) Heat induction of the unphosphorylated form of hypoxia-inducible factor-1α is dependent on heat shock protein-90 activity. J Biol Chem 277:9262–9267PubMedCrossRefGoogle Scholar
  36. Kidd PM (1997) Glutathione: systemic protectant against oxidative and free radical damage. Alt Med Rev 1:155–176Google Scholar
  37. Klein SM, Cohen G, Lieber CS, Cederbaum AI (1983) Increased microsomal oxidation of hydroxy radical scavengers and ethanol after chronic consumption of ethanol. Arch Biochem Biophys 223:425–433PubMedCrossRefGoogle Scholar
  38. Kvietikova I, Wenger RH, Marti HH, Gassmann M (1995) The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site. Nucl Acids Res 23:4542–4550PubMedCrossRefGoogle Scholar
  39. Lannig G, Storch D, Pörtner H-O (2005) Aerobic mitochondrial capacities in Antarctic and temperate eelpout (Zoarcidae) subjected to warm versus cold acclimation. Polar Biol 28:575–584CrossRefGoogle Scholar
  40. Larsen B., Pörtner HO, Jensen FB (1997) Extra- and intracellular acid-base balance and ionic regulation in cod (Gadus morhua) during combined and isolated exposures to hypercapnia and copper. Marine Biol 128:337–346CrossRefGoogle Scholar
  41. Law S, Wu R, Ng P, Yu R, Kong R (2006) Cloning and expression analysis of two distinct HIF-alpha isoforms—gcHIF-1alpha and gcHIF-4alpha—from the hypoxia-tolerant grass carp, Ctenopharyngodon idellus. BMC Mol Biol 7:15 doi:10.1186/1471-2199-7-15Google Scholar
  42. Lawrie S, Tancock N, McGrowth N, Roger J (1991) Influence of complexation of the uptake by plants of iron, manganese, copper and zinc. I Effect of EDTA in a multimetal and computer simulation study. J Exp Biol 42:509–515Google Scholar
  43. Levine RL, Garland D, Oliver CN, Amici A, Climent I, Lenz A-G, Ahn B-W, Shaltiel S, Stadtman ER (1990) Determination of carbonal content in oxidatively modified proteins. Methods Enzymol 186:464–485PubMedGoogle Scholar
  44. Linares E, Nakao LS, Augusto O, Kadiiska MB (2003) EPR studies of in vivo radical production by lipopolysaccharide: potential role of iron mobilized from iron-nitrosyl complexes. Free Rad Biol Med 34:766–773PubMedCrossRefGoogle Scholar
  45. Livingstone DR, Lips F, Martinez PG, Pipe RK (1992) Antioxidant enzymes in the digestive gland of the common mussel Mytilus edulis. Marine Biol 112:265–276CrossRefGoogle Scholar
  46. Mark FC, Bock C, Pörtner HO (2002a) Oxygen-limited thermal tolerance in Antarctic fish investigated by MRI and 31P-MRS. Am J Physiol, Regul Integr Comp Physiol 283:R1254–R1262Google Scholar
  47. Mark FC, Lucassen M, Pörtner HO (2006b) Thermal sensitivity of uncoupling proteins in polar and temperate fish. Comp Biochem Physiol D1:365–374Google Scholar
  48. Nikinmaa M (2002) Oxygen-dependent cellular functions—why fishes and their aquatic environment are a prime choice of study. Comp Biochem Physiol 133A:1–16Google Scholar
  49. Nikinmaa M, Pursiheimo S, Soitamo AJ (2004) Redox state regulates HIF-1α and its DNA binding and phosphorylation in salmonid cells. J Cell Sci 117:3201–3206PubMedCrossRefGoogle Scholar
  50. Nikinmaa M, Rees BB (2005) Oxygen-dependent gene expression in fishes. Am J Physiol Regul Integr Comp Physiol 288R:1079–1090Google Scholar
  51. Minet E, Mottet D, Michel G, Roland I, Raes M, Remacle J, Michiels C (1999) Hypoxia-induced activation of HIF-1: role of HIF-1alpha-Hsp90 interaction. FEBS Lett 460(2):251–256PubMedCrossRefGoogle Scholar
  52. Morin PJ, McMullen DC, Storey KB (2005) HIF-1α involvement in low temperature and anoxia survival by a freeze tolerant insect. Mol Cell Biochem 280:99–106PubMedCrossRefGoogle Scholar
  53. Philipp E, Brey T, Pörtner H-O, Abele D (2005) Chronological and physiological ageing in a polar and a temperate mud clam. Mech Ageing Dev 126:598–609PubMedCrossRefGoogle Scholar
  54. Philipp E, Brey T, Heilmayer O, Abele D, Pörtner H-O (2006) Physiological ageing in a polar and a temperate swimming scallop. Mar Ecol Prog Ser 307:187–198CrossRefGoogle Scholar
  55. Reed DJ (1990) Glutathione: toxicological implications. Annu Rev Pharmacol Toxicol 30:603–631PubMedCrossRefGoogle Scholar
  56. Rissanen E, Tranberg HK, Sollid J, Nilsson GE, Nikinmaa M (2006) Temperature regulates hypoxia-inducible factor-1 (HIF-1) in a poikilothermic vertebrate, crucian carp (Carassius carassius). J Exp Biol 209:994–1003PubMedCrossRefGoogle Scholar
  57. Sartoris FJ, Bock C, Pörtner HO (2003) Temperature-dependent pH regulation in eurythermal and stenothermal marine fish: an interspecies comparison using 31P-NMR. J Therm Biol 28:363–371CrossRefGoogle Scholar
  58. Schafer FQ, Buettner GR (2001) Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Rad Biol Med 30:1191–1212PubMedCrossRefGoogle Scholar
  59. Shams I, Nevo E, Avivi A (2004) Ontogenetic expression of erythropoietin and hypoxia inducible factor-1 alpha genes in subterranean blind mole rats. FASEB: doi:10.1096/fj.04-2758fjeGoogle Scholar
  60. Soitamo AJ, Rabergh CMI, Gassmann M, Sistonen L, Nikinmaa M (2001) Characterization of a hypoxia-inducible factor (HIF-1α) from rainbow trout. J Biol Chem 276:19677–19705CrossRefGoogle Scholar
  61. Stroka DM, Burkhardt T, Desbaillets I, Wenger RH, Neil DAH, Gassmann M, Candinas D (2001) HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia. FASEB J 15:2445–2453PubMedGoogle Scholar
  62. Uchiyama M, Mihara M (1978) Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Anal Biochem 86:271–278PubMedCrossRefGoogle Scholar
  63. Van Dijk PLM, Tesch C, Hardewig I, Pörtner H-O (1999) Physiological disturbances at critically high temperatures: a comparison between stenothermal antarctic and eurythermal temperate eelpouts (Zoarcidae). J Exp Biol 202:3611–3621PubMedGoogle Scholar
  64. Vegh M, Marton A,Horvath I (1988) Reduction of Fe(III) ADP complex by liver microsomes. Biochim Biophys Act 964:146–150Google Scholar
  65. Vuori KAM, Soitamo A, Vuorinen PJ, Nikinmaa M (2004) Baltic salmon (Salmo salar) yolk-sac fry mortality is associated with disturbances in the function of hypoxia-inducible transcription factor (HIF-1α) and consecutive gene expression. Aquat Toxicol 68:301–313PubMedCrossRefGoogle Scholar
  66. Wenger RH (2000) Mammalian oxygen sensing, signalling and gene regulation. J Exp Biol 203:1253–1263PubMedGoogle Scholar
  67. Woodmansee AN, Imlay JA (2002) Meth Enzymol 349:3–9PubMedGoogle Scholar
  68. Yegorov DY, Kozlov AV, Azizova OA, Vladimorov YA (1993) Simultaneous determination of Fe(III) and Fe(II) in water solutions and tissue homogenates using desferal and 1,10-phenanthroline. Free Rad Biol Med 15:565–574PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • K. Heise
    • 1
  • M. S. Estevez
    • 2
  • S. Puntarulo
    • 2
  • M. Galleano
    • 2
  • M. Nikinmaa
    • 3
  • H. O. Pörtner
    • 1
  • D. Abele
    • 1
    Email author
  1. 1.Alfred-Wegener-Institute for Polar and Marine ResearchBremerhavenGermany
  2. 2.Physical Chemistry-PRALIBSchool of Pharmacy and Biochemistry University of Buenos AiresBuenos AiresArgentina
  3. 3.Animal Physiology, Department of BiologyUniversity of TurkuTurkuFinland

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