**4. A wide vision of redox balance in fish: looking for new markers**

Fish, like all other organisms, must have a balance between the production of oxidative substances (ROS and RNS) and antioxidant defences, and are of particular interest as they experience a multitude of above-mentioned stressors. To protect themselves against the potentially highly damaging oxidants, organisms have evolved a system to either prevent or repair the effects of oxidative stress. Prevention comes in the form of antioxidants, which can either be enzymatic (SOD, CAT, GPX, GR) or non-enzymatic molecules (mainly glutathione and vitamins C and E), carotenoids and other small molecules. These are the most commonly measured oxidative markers and antioxidants in fish biology (recently revised [54]). However, recent advances on redox studies in mammals suggest other markers to be considered widening to associated pathways of redox balance. Some of them should be also considered when studying redox balance in fish. **Figure 2** attempts to provide this broad range of markers also in fish: including markers of protein oxidation levels, metabolic enzymes related to glutathione synthesis, repair/ refolding of oxidized proteins or main protein degradation processes (via ubiquitinproteasome system, UPS, or lysosomal proteolytic fate).

**103**

*Redox Balance Affects Fish Welfare*

*DOI: http://dx.doi.org/10.5772/intechopen.89842*

peroxidation (own data unpublished).

fish studies related to redox balance.

Beyond the LPO measurements (via TBARS analyses), it was recently reported that advanced oxidation protein products (AOPPs) could act as markers of oxidative stress, resulted from the direct oxidation of the amino acid side chains of proteins as a consequence of metal toxicity in freshwater species [55, 56] or produced at low temperatures in marine species [21]. Another marker recently used in mammals is the accumulation of conjugated proteins with 4-HNE as the major biomarker relating lipoperoxidation and protein oxidation [57, 58]. 4-HNE is the most toxic product of lipid peroxidation reported in mammals damaging proteins by adding covalent adducts and accelerating protein aggregation [59, 60]. Although no data exists in fish on HNE products analyses, a preliminary study in gilthead sea bream would demonstrate the existence of a singular pattern of 4-HNE oxidized proteins in liver with the proteins ranging 40–50 and 75–100 kDa the main target of lipid

As proteins are the major constituents of cellular organization and metabolism, effects of oxidative attack on protein structure, stability and folding deserve careful consideration in fish species. Several cellular pathways exist which repair and eliminate damaged proteins and thus prevent their accumulation and aggregation. One of the first mechanisms to cope with protein damage is binding with chaperones or 'heat shock proteins' (HSPs). Thus, a few studies recently addressed the changes on HSPs levels/expression in fish when the redox balance is challenged. For instance, it was reported on several fish tissues the expression of HSP70 and HSP90 protein expression levels in response to temperature seasonal challenges [61, 62]. When a protein is irremediably damaged, its fate is to be recycled via UPS degradation pathways, or to be removed/autophaged via a lysosomal degradation process. Protein degradation via UPS involves two discrete and successive steps: tagging of the substrate protein by the covalent attachment of multiple ubiquitin molecules, and the subsequent degradation of the tagged protein by the 26S proteasome, composed of the catalytic 20S core and the 19S regulator [63]. The capacity to remove damaged proteins by the proteasome may prevent oxidative stress and it has been suggested that this is also part of the antioxidant defences in mammals [64]. In fish which inhabiting permanently temperature-fluctuant aquatic environments, this enzymatic complex could play a key role in antioxidant defence systems [65]. The analysis of UPS system markers is still scarce or null in fish and to obtain a profile of protein-ubiquitination labelling according the MW or the analyses of main proteasome subunits (catalytic 20S core or 19S regulatory) should be of further interest on

The aggregation of proteins as a result of the accumulation of cross-linked 4-HNE proteins cannot be degraded through the UPS even could block its correct functioning. Then, lysosome acts to eliminate protein aggregates. The lysosomal system has an elevated non-selective protein degradation capacity as a result of the combined random and limited action of various proteases, with the cathepsin family being one of the most important in mammals [59, 66] and also in fish [67]. Thus, as the fate of 4-HNE protein conjugates is preferably the lysosomal degradation pathway, the cathepsin activities should be another target on the study of redox balance. Finally, we encourage fish biology researcher to study other enzymes which their activities are also strongly related with antioxidant defence. Both TBARS and HNE are aldehydic products, relatively stable and capable of roaming freely and attacking molecules, e.g. DNA, proteins, lipids far from their origin [68]. Thus, aldehyde dehydrogenase (ADH) is an enzyme involved in the oxidation pathway, and is complementary to the GST pathway, which reduces the potential damage of these peroxide products by oxidizing them and removing them from inside the cells. As the glutathione plays a central role in lipid peroxide detoxification, reducing the peroxides to their corresponding alcohols, the study of enzymes involved in

**Figure 2.** *'Wide view' of redox balance markers.*

### *Redox Balance Affects Fish Welfare DOI: http://dx.doi.org/10.5772/intechopen.89842*

*Redox*

28°C. Moreover, it was evaluated the effect of rearing water temperature in turbot juveniles, *Scophthalmus maximus* (15 days at normal 15°C and higher 20°C), evidencing lower CAT and GPX enzyme activities [53]. In Senegalese sole, *Solea senegalensis,* cold exposure at 12°C increased GR activity and LPO while decreased SOD activity in liver [19]. Moreover, in this specie decreasing protein content in diet from 55–45% worsened redox balance. All these recent studies reinforce the idea that temperature

challenges strongly compromise redox balance in fish temperate species.

proteasome system, UPS, or lysosomal proteolytic fate).

**4. A wide vision of redox balance in fish: looking for new markers**

Fish, like all other organisms, must have a balance between the production of oxidative substances (ROS and RNS) and antioxidant defences, and are of particular interest as they experience a multitude of above-mentioned stressors. To protect themselves against the potentially highly damaging oxidants, organisms have evolved a system to either prevent or repair the effects of oxidative stress. Prevention comes in the form of antioxidants, which can either be enzymatic (SOD, CAT, GPX, GR) or non-enzymatic molecules (mainly glutathione and vitamins C and E), carotenoids and other small molecules. These are the most commonly measured oxidative markers and antioxidants in fish biology (recently revised [54]). However, recent advances on redox studies in mammals suggest other markers to be considered widening to associated pathways of redox balance. Some of them should be also considered when studying redox balance in fish. **Figure 2** attempts to provide this broad range of markers also in fish: including markers of protein oxidation levels, metabolic enzymes related to glutathione synthesis, repair/ refolding of oxidized proteins or main protein degradation processes (via ubiquitin-

**102**

**Figure 2.**

*'Wide view' of redox balance markers.*

Beyond the LPO measurements (via TBARS analyses), it was recently reported that advanced oxidation protein products (AOPPs) could act as markers of oxidative stress, resulted from the direct oxidation of the amino acid side chains of proteins as a consequence of metal toxicity in freshwater species [55, 56] or produced at low temperatures in marine species [21]. Another marker recently used in mammals is the accumulation of conjugated proteins with 4-HNE as the major biomarker relating lipoperoxidation and protein oxidation [57, 58]. 4-HNE is the most toxic product of lipid peroxidation reported in mammals damaging proteins by adding covalent adducts and accelerating protein aggregation [59, 60]. Although no data exists in fish on HNE products analyses, a preliminary study in gilthead sea bream would demonstrate the existence of a singular pattern of 4-HNE oxidized proteins in liver with the proteins ranging 40–50 and 75–100 kDa the main target of lipid peroxidation (own data unpublished).

As proteins are the major constituents of cellular organization and metabolism, effects of oxidative attack on protein structure, stability and folding deserve careful consideration in fish species. Several cellular pathways exist which repair and eliminate damaged proteins and thus prevent their accumulation and aggregation. One of the first mechanisms to cope with protein damage is binding with chaperones or 'heat shock proteins' (HSPs). Thus, a few studies recently addressed the changes on HSPs levels/expression in fish when the redox balance is challenged. For instance, it was reported on several fish tissues the expression of HSP70 and HSP90 protein expression levels in response to temperature seasonal challenges [61, 62]. When a protein is irremediably damaged, its fate is to be recycled via UPS degradation pathways, or to be removed/autophaged via a lysosomal degradation process. Protein degradation via UPS involves two discrete and successive steps: tagging of the substrate protein by the covalent attachment of multiple ubiquitin molecules, and the subsequent degradation of the tagged protein by the 26S proteasome, composed of the catalytic 20S core and the 19S regulator [63]. The capacity to remove damaged proteins by the proteasome may prevent oxidative stress and it has been suggested that this is also part of the antioxidant defences in mammals [64]. In fish which inhabiting permanently temperature-fluctuant aquatic environments, this enzymatic complex could play a key role in antioxidant defence systems [65]. The analysis of UPS system markers is still scarce or null in fish and to obtain a profile of protein-ubiquitination labelling according the MW or the analyses of main proteasome subunits (catalytic 20S core or 19S regulatory) should be of further interest on fish studies related to redox balance.

The aggregation of proteins as a result of the accumulation of cross-linked 4-HNE proteins cannot be degraded through the UPS even could block its correct functioning. Then, lysosome acts to eliminate protein aggregates. The lysosomal system has an elevated non-selective protein degradation capacity as a result of the combined random and limited action of various proteases, with the cathepsin family being one of the most important in mammals [59, 66] and also in fish [67]. Thus, as the fate of 4-HNE protein conjugates is preferably the lysosomal degradation pathway, the cathepsin activities should be another target on the study of redox balance.

Finally, we encourage fish biology researcher to study other enzymes which their activities are also strongly related with antioxidant defence. Both TBARS and HNE are aldehydic products, relatively stable and capable of roaming freely and attacking molecules, e.g. DNA, proteins, lipids far from their origin [68]. Thus, aldehyde dehydrogenase (ADH) is an enzyme involved in the oxidation pathway, and is complementary to the GST pathway, which reduces the potential damage of these peroxide products by oxidizing them and removing them from inside the cells. As the glutathione plays a central role in lipid peroxide detoxification, reducing the peroxides to their corresponding alcohols, the study of enzymes involved in glutathione synthesis seems adequate to evaluate cell redox potential and response. The BHMT and the adenosine-methionine synthetase (SAM) are enzymes involved in antioxidant mechanisms through the synthesis of S-adenosylmethionine and through maintain its steady-state levels which is a crucial component of methylation reactions and a biosynthetic precursor of glutathione. Under acute cold stress the expression of these enzymes of glutathione synthesis as well as ADH are affected in gilthead sea bream [50].

Overall, we hope that the proposed markers in **Figure 2** from that 'wide view' on redox balance and related processes in fish. Can contribute to expanding knowledge of the relevant oxidant products (LPO, AOPPs, HNE), the classic enzymes studied (SOD, CAT, GPX, GR, GST) and the associated processes affected such as protein repairing/protection machinery and protein turnover by the oxidant insult.
