The International Journal of Biochemistry & Cell Biology
Hypoxia and recovery perturb free radical processes and antioxidant potential in common carp (Cyprinus carpio) tissues
Introduction
The common carp, Cyprinus carpio, is one of several fish species that are capable of surviving hypoxia and even anoxia for periods ranging from several hours to several days (van den Thillart & van Waarde, 1985). Species-specific biochemical mechanisms support long-term survival at low oxygen (Hochachka & Somero, 1984). One of the most important is metabolic rate depression (Hochachka, Buck, Doll, & Land, 1996; Storey, 1987; Storey and Storey, 1990, Storey and Storey, 2004; van Wavesveld, Addink, & van den Thillart, 1989). By strongly reducing the energy demands of their tissues, animals can greatly extend the time that they can survive using internal carbohydrate reserves and ATP generated primarily or exclusively from anaerobic glycolysis. Carp tissues contain large reserves of glycogen and some additional metabolic adaptations are also well developed (Hochachka & Somero, 1984). However, metabolic damage associated with hypoxia/anoxia or ischemia arises not only from the effects of low oxygen, but can also occur during tissue reoxygenation. The reintroduction of oxygen into hypoxic/anoxic tissues results in a rapid transient increase in the levels of reactive oxygen species (ROS) causing oxidative stress (Chang, Yoo, & Rho, 2002; Di Guilio, Washburn, Wenning, Winston, & Jewell, 1989; Garnier et al., 2001; Halliwell & Gutteridge, 1989; Hermes-Lima, Storey, & Storey, 1998; Hermes-Lima & Zenteno-Savin, 2002; Lievre et al., 2000; Lushchak, 2001; Lushchak, Lushchak, Mota, & Hermes-Lima, 2001; Storey, 1996). Hence, to successfully survive hypoxia exposures an organism must not only maintain its viability under low oxygen conditions, but also have effective mechanisms to minimize or prevent oxidative stress during the transition back from hypoxic to aerobic conditions.
Cells possess multiple systems that protect them from the damaging effects of ROS (Hermes-Lima, 2004). They are equipped with both low and high molecular weight antioxidants. The first group includes glutathione, ascorbic acid, tocopherol, etc., and the second group is represented by antioxidant enzymes. Hypoxia/anoxia tolerant animals may display two modes of antioxidant defense. Some display constitutively high defenses whereas others enhance antioxidant defenses during the period of hypoxia exposure, in an anticipatory process that has been called “preparation for oxidative stress” (Hermes-Lima et al., 1998, Lushchak et al., 2001). The last mechanism occurs in stress-tolerant species that regularly experience wide variation of oxygen availability in nature, for example, during transitions between anoxia/hypoxia and normoxia, freeze-thaw, or estivation-arousal. Species where these mechanisms have been documented include goldfish Carassius auratus (Lushchak et al., 2001), carp C. carpio (Vig & Nemcsok, 1989), garter snakes Thamnophis sirtalis (Hermes-Lima & Storey, 1993) and the frog Rana pipiens (Hermes-Lima & Storey, 1996). Enhancement of antioxidant defenses during physiological states where oxygen free radical production should be reduced (anoxia/hypoxia, freezing, estivation) is a preparatory mechanism that minimizes potential injury due to oxidative stress during reoxygenation (or thawing, arousal) when oxygen consumption increases.
Previous work on the role of antioxidant defenses in hypoxia/anoxia survival by C. carpio focused only on the responses of two antioxidant enzymes to hypoxia and showed that the activity of superoxide dismutase in liver was increased under hypoxic conditions. Other antioxidant enzymes were not investigated nor were markers of oxidative stress (Vig & Nemcsok, 1989). The present study aimed to analyze the tissue-specific responses of common carp to environmentally relevant hypoxia exposure and subsequent aerobic recovery based on a hypothesis of preparation for oxidative stress.
Section snippets
Reagents
Phenylmethylsulfonyl fluoride (PMSF), 5,5′-dithiobis-2-nitrobenzoic acid (DTNB), thiobarbituric acid (TBA), butylated hydroxytoluene (BHT), 2,4-dinitrophenylhydrazine (DNPH), guanidine–HCl, xylenol orange, cumene hydroperoxide, yeast glutathione reductase (GR), 1-chloro-2,4-dinitrobenzene (CDNB), reduced glutathione (GSH), oxidized glutathione (GSSG), NADP+, NADPH, glucose-6-phosphate (G6P) and ethylenediamine-tetraacetic acid (EDTA) were purchased from Sigma Chemical Co. (USA), Tris–HCl was
Protein carbonyls, thiobarbituric reactive substances and lipid peroxides
The level of carbonylproteins (CP) was evaluated either per milligram of protein solubilized by guanidine chloride or per gram wet weight of tissue. In both cases we found similar ratios of CP levels between the different tissues and virtually the same percentage changes in CP levels between the different experimental groups in each tissue. Therefore, CP results are shown only as nmol per gram wet weight (gww) of tissue. In the tissues studied, CP levels in control fish were highest in kidney
Discussion
Several carp fish species are highly tolerant of hypoxia, and even anoxia, and this adaptation is critically important for allowing them to occupy specific ecological niches where water oxygen content can drop to low levels. According to present knowledge the most tolerant species are the crucian carp (Carassius carassius) and the goldfish (Carassius auratus), followed by the common carp (C. carpio) (van den Thillart & van Waarde, 1985). The latter species can survive for 5 h at 15 °C under
Acknowledgements
We are grateful to L. Luzhna for technical assistance. The research received partial support from NSERC grant #6793 to KBS.
References (33)
- et al.
Induction of oxidative stress in Rana ridibunda during recovery from winter hibernation
Journal of Thermal Biology
(2003) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding
Analytical Biochemistry
(1976)- et al.
Stress response to hypoxia in gerbil brain: HO-1 and Mn-SOD expression and glial activation
Brain Research
(2001) - et al.
Antioxidant defenses and metabolic depression. The hypothesis of preparation for oxidative stress in land snails
Comparative Biochemistry and Physiology B
(1998) - et al.
Quantification of lipid peroxidation in tissue extracts based on Fe(III)xylenol orange complex formation
Free Radical Biology and Medicine
(1995) - et al.
Animal response to drastic changes in oxygen availability and physiological oxidative stress
Comparative Biochemistry and Physiology C
(2002) - et al.
Determination of carbonyl groups in oxidatively modified of proteins by reduction with tritiated sodium borohydride
Analytical Biochemistry
(1989) - et al.
Free radical production and changes in superoxide dismutases associated with hypoxia/reoxygenation-induced apoptosis of embryonic rat forebrain neurons in culture
Free Radical Biology and Medicine
(2000) - et al.
Free Radical Biology and Medicine
(1994) - et al.
Iron-catalyzed oxidative modification of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides
Journal of Biological Chemistry
(1992)
Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: Applications to mammalian blood and other tissues
Analytical Biochemistry
A simple computer program with statistical tests for the analysis of enzyme kinetics
BioTechniques
Transcriptional regulation and environmental induction of genes encoding copper- and zinc-containing superoxide dismutase
Methods in Enzymology
Biochemical responses in aquatic animals: A review of determinants of oxidative stress
Environmental Toxicology and Chemistry
Free Radicals in Biology and Medicine
Oxygen in biology and biochemistry: Role of free radicals
Cited by (284)
Effects of environmental hypoxia on the goldfish skeletal muscle: Focus on oxidative status and mitochondrial dynamics
2024, Journal of Contaminant HydrologyOleic acid as modulator of oxidative stress in European sea bass (Dicentrarchus labrax) juveniles fed high dietary lipid levels
2024, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology