Tissue repair: Antioxidant influences processes in rainbow trout

Tissue damage

The economics and profitability of fish farming operations depend on the size and quality of the fish. If they become injured in some way, during handling and transport, or succumb to bacterial infections, then the muscle texture can be affected and the quality of the fish suffers. One unlikely source of protection appears to come from an antioxidant which is added to the diet of the fish.

Astaxanthin is a carotenoid compound which imparts the pink colour to salmonid fish like rainbow trout. Without this antioxidant, the fish do not develop the colour that consumers have come to expect but it also confers some unexpected properties. Several research studies have shown that astaxanthin also inhibits inflammation, which is the first stage of tissue damage, as well as limiting damage to muscles and improving the resistance to disease.

Despite this knowledge, there have been few studies looking at tissue damage at the cellular level on lower vertebrates such as fish. Those that have been carried out looked at the zebrafish, which is often used as a model species. Now, European scientists have taken a look at the effects of tissue injury in the rainbow trout, using proteomics techniques to dig deep into the protein patterns.

Tune Wulff and Michael Engelbrecht Nielsen from the National Food Institute, Technical University of Denmark, Lyngby, and Tomé Silva from the Centre of Marine Sciences of Algarve, University of Algarve, Faro, Portugal, also looked at the effects of astaxanthin on the proteome to see if it played a role in the processes that followed injury.

Fish feeding

One-year-old rainbow trout were fed a conventional commercial organic trout feed. For some of the fish, the feed was supplemented by astaxanthin at a level of 25 ppm, which is in line with legislation for organic fish farming. After two months, some of the fish from the supplemented and non-supplemented groups were injured by puncturing the skin with a hollow needle to produce two rows of five holes, while other fish were left undamaged.

Seven days later, damaged and undamaged fish from both groups were killed and the muscle tissue was removed. The proteins were extracted from the sarcoplasmic fraction of the tissue which consists of 25-30% of the total muscle protein. Although this has the potential to miss certain proteins, it has been shown to increase the sensitivity 3-5-fold during gel electrophoresis for those proteins that are extracted.

The proteins were separated by 2D gel electrophoresis and stained so that the abundances from the injured and uninjured fish could be compared, along with the effects of the antioxidant. Those proteins of interest were then collected from preparative gels and identified by a tandem mass spectrometric method.

Antioxidant effects

A total of 874 protein spots were identified from the gels and the researchers applied a statistical procedure to identify those with abundances that showed a significant change between gels. This identified a subset of 96 proteins that were influenced by tissue damage.

The proteins were involved in a range of cellular processes. For instance, the altered abundances of α- and β-actin and the β-tubulin chain reflect the long-term changes in the cell structure. Other pathways that are impacted by damage include apoptosis, glycolysis, iron homeostasis, muscle contraction and cellular respiration.

The presence of astaxanthin had a marked affect on one group of proteins. Annexin IV and V were up-regulated and annexin VI was down-regulated in damaged tissue compared with undamaged tissue but this effect was only observed in the fish that were fed astaxanthin. Annexins have been reported to play an important role in response to cellular damage in cultures but this is the first report of its importance during the later stages of tissue repair.

The joint persistence of annexin IV and V at increased abundances suggests that these proteins continue to be involved in cell repair. The researchers suggested that astaxanthin interacts directly with the annexins, since the proteins and the antioxidant all enter the cell membrane.

The effects of astaxanthin and the protein pathways uncovered in this study will help to understand the processes which occur following tissue damage in lower vertebrates like fish. In the long term, they may lead to new food supplements which will accelerate tissue repair and maintain the integrity of the flesh of those species involved in food production, and might also help to develop new treatments for damaged tissue.