Hypoxia and recovery perturb free radical processes and antioxidant potential in common carp (Cyprinus carpio) tissues

Abstract

The effects of hypoxia exposure and subsequent normoxic recovery on the levels of lipid peroxides (LOOH), thiobarbituric acid reactive substances (TBARS), carbonylproteins, total glutathione levels, and the activities of six antioxidant enzymes were measured in brain, liver, kidney and skeletal muscle of the common carp Cyprinus carpio. Hypoxia exposure (25% of normal oxygen level) for 5 h generally decreased the levels of oxidative damage products, but in liver TBARS content were elevated. Hypoxia stimulated increases in the activities of catalase (by 1.7-fold) and glutathione peroxidase (GPx) (by 1.3-fold) in brain supporting the idea that anticipatory preparation takes place in order to deal with the oxidative stress that will occur during reoxygenation. In liver, only GPx activity was reduced under hypoxia and reoxygenation while other enzymes were unaffected. Kidney showed decreased activity of GPx under aerobic recovery but superoxide dismutase (SOD) and catalase responded with sharp increases in activities. Skeletal muscle showed minor changes with a reduction in GPx activity under hypoxia exposure and an increase in SOD activity under recovery. Responses by antioxidant defenses in carp organs appear to include preparatory increases during hypoxia by some antioxidant enzymes in brain but a more direct response to oxidative insult during recovery appears to trigger enzyme responses in kidney and skeletal muscle.

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.