Nutritional status as the key modulator of antioxidant responses induced by high environmental ammonia and salinity stress in European sea bass (Dicentrarchus labrax)

Salinity fluctuation is one of the main factors affecting the overall fitness of marine fish. In addition, water borne ammonia may occur simultaneously with salinity stress. Additionally, under such stressful circumstances, fish may encounter food deprivation. The physiological and ion-osmo regulatory adaptive capacities to cope with all these stressors alone or in combination are extensively addressed in fish. To date, studies revealing the modulation of antioxidant potential as compensatory response to multiple stressors are rather lacking. Therefore, the present work evaluated the individual and combined effects of salinity challenge, ammonia toxicity and nutritional status on oxidative stress and antioxidant status in a marine teleost, European sea bass (Dicentrarchus labrax). Fish were acclimated to normal seawater (32 ppt), to brackish water (20 ppt and 10 ppt) and to hypo-saline water (2.5 ppt). Following acclimation to different salinities for two weeks, fish were exposed to high environmental ammonia (HEA, 20 mg/L representing 50% of 96h LC50 value for ammonia) for 12 h, 48 h, 84 h and 180 h, and were either fed (2% body weight) or fasted (unfed for 7 days prior to HEA exposure). Results show that in response to decreasing salinities, oxidative stress indices such as xanthine oxidase activity, levels of hydrogen peroxide (H2O2) and lipid peroxidation (malondialdehyde, MDA) increased in the hepatic tissue of fasted fish but remained unaffected in fed fish. HEA exposure at normal salinity (32 ppt) and at reduced salinities (20 ppt and 10 ppt) increased ammonia accumulation significantly (84 h-180 h) in both feeding regimes which was associated with an increment of H2O2 and MDA contents. Unlike in fasted fish, H2O2 and MDA levels in fed fish were restored to control levels (84 h-180 h); with a concomitant increase in superoxide dismutase (SOD), catalase (CAT), components of the glutathione redox cycle (reduced glutathione, glutathione peroxidase and glutathione reductase), ascorbate peroxidase (APX) activity and reduced ascorbate (ASC) content. On the contrary, fasted fish could not activate many of these protective systems and rely mainly on CAT and ASC dependent pathways as antioxidative sentinels. The present findings exemplify that in fed fish single factors and a combination of HEA exposure and reduced seawater salinities (upto 10 ppt) were insufficient to cause oxidative damage due to the highly competent antioxidant system compared to fasted fish. However, the impact of HEA exposure at a hypo-saline environment (2.5 ppt) also defied antioxidant defence system in fed fish, suggesting this combined factor is beyond the tolerance range for both feeding groups. Overall, our results indicate that the oxidative stress mediated by the experimental conditions were exacerbated during starvation, and also suggest that feed deprivation particularly at reduced seawater salinities can instigate fish more susceptible to ammonia toxicity

Ammonia accumulation.

<p>Ammonia accumulation (μmol/g) in liver of fed and fasted fish during acclimation to different salinities and exposure to HEA. Values are mean ± S.E. Asterisk (*) indicates a significant difference between the ammonia exposed fish and its respective control at each salinity (*<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.001), bullet (•) indicates a significant difference between experimental salinities (20 ppt -2.5 ppt) and the 32 ppt-acclimated fish at the same sampling period (^•<i>P</i> < 0.05), dagger (†) denotes the significant difference between fed fish and its respective fasted fish counterpart (^†<i>P</i> < 0.05).</p

Principal Component Analysis.

<p>Principal Component Analysis (PCA) representing the contribution of biochemical parameters for fed (F) and starved (S) fish. The variable coordination is presented by the complementary cases analysis showing distribution of salinity acclimation groups (32, 20, 10, and 2.5 ppt) and HEA exposure (C, 12 h, 48 h, 84 h, 180 h) in the (PC 1 ×PC 2) coordination plane.</p

Malondialdehyde content.

<p>Malondialdehyde (MDA) content (nmol/g) in liver of fed and fasted fish during acclimation to different salinities and exposure to HEA. Values are mean ± S.E. Asterisk (*) indicates a significant difference between the ammonia exposed fish and its respective control at each salinity (*<i>P</i> < 0.05; **<i>P</i> < 0.01), bullet (•) indicates a significant difference between experimental salinities (20 ppt -2.5 ppt) and the 32 ppt-acclimated fish at the same sampling period (^•<i>P</i> < 0.05), dagger (†) denotes the significant difference between fed fish and its respective fasted fish counterpart (^†<i>P</i> < 0.05).</p

Ascorbate peroxidase acivity.

<p>Ascorbate peroxidase (APX) acivity (μmol ASC/min/mg protein) in liver of fed and fasted fish during acclimation to different salinities and exposure to HEA. Values are mean ± S.E. Asterisk (*) indicates a significant difference between the ammonia exposed fish and its respective control at each salinity (*<i>P</i> < 0.05; **<i>P</i> < 0.01).</p

Superoxide dismutase activity.

<p>Superoxide dismutase (SOD) activity (units SOD/mg protein) in liver of fed and fasted fish during acclimation to different salinities and exposure to HEA. Values are mean ± S.E. Asterisk (*) indicates a significant difference between the ammonia exposed fish and its respective control at each salinity (*<i>P</i> < 0.05; **<i>P</i> < 0.01), bullet (•) indicates a significant difference between experimental salinities (20 ppt -2.5 ppt) and the 32 ppt-acclimated fish at the same sampling period (^•<i>P</i> < 0.05), dagger (†) denotes the significant difference between fed fish and its respective fasted fish counterpart (^†<i>P</i> < 0.05).</p

Hydrogen peroxide content.

<p>Hydrogen peroxide (H₂O₂) content (nmol/g) in liver of fed and fasted fish during acclimation to different salinities and exposure to HEA. Values are mean ± S.E. Asterisk (*) indicates a significant difference between the ammonia exposed fish and its respective control at each salinity (*<i>P</i> < 0.05; **<i>P</i> < 0.01; ***<i>P</i> < 0.001), bullet (•) indicates a significant difference between experimental salinities (20 ppt -2.5 ppt) and the 32 ppt-acclimated fish at the same sampling period (^•<i>P</i> < 0.05), dagger (†) denotes the significant difference between fed fish and its respective fasted fish counterpart (^†<i>P</i> < 0.05).</p

Glutathione reductase activity.

<p>Glutathione reductase (GR) activity (nmol NADPH/min/mg protein) in liver of fed and fasted fish during acclimation to different salinities and exposure to HEA. Values are mean ± S.E. Asterisk (*) indicates a significant difference between the ammonia exposed fish and its respective control at each salinity (*<i>P</i> < 0.05; **<i>P</i> < 0.01), bullet (•) indicates a significant difference between experimental salinities (20 ppt -2.5 ppt) and the 32 ppt-acclimated fish at the same sampling period (^•<i>P</i> < 0.05), dagger (†) denotes the significant difference between fed fish and its respective fasted fish counterpart (^†<i>P</i> < 0.05).</p