Effect of food deprivation on oxidative stress biomarkers in fish (Sparus aurata)

Abstract

Oxidative stress in fish (Sparus aurata) as a consequence of food restriction and fasting, has been studied. Four groups of fish were maintained for 46 days under different conditions of food supplementation: a control group with no food restriction (ratio of food/fish of 2% w/w), two groups of animals with restricted food supplement (1 and 0.5%) and a fasting group (no meal addition). Finally, all the fish were provided with food at the same ratio as the control group for the last 7 days. Sampling and weighing of fish were carried out every week and their livers were used for the analysis of known biomarkers of oxidative stress. Malondialdehyde and oxidized glutathione levels increased at the third week in fish with partial or total food deprivation, but these levels returned to normal values when the fish readapted to the control conditions. Antioxidant enzymes were also analyzed and significant increases in superoxide dismutase (SOD), glutathione reductase and glutathione peroxidase activities were found in parallel with food restriction; however catalase activity decreased in fasting fish. New SOD isoforms were detected by isoelectrofocusing in fish under food restriction at the second week, which disappeared when starved fish returned to the control conditions. These new SOD isoforms were detected before the appearance of other usual oxidative stress biomarkers.

Introduction

Food deprivation enhances aging [1], ischemia-reperfusion injury [2], toxicity to chemicals [3] and morbidity under certain pathological situations. These effects could be mainly attributed to the participation of reactive oxygen species generated by starvation, leading to oxidative stress [4]. Oxidative stress results when the antioxidant defenses are overcome by pro-oxidant forces and reactive oxygen species are not adequately removed [5]. Living organisms are protected from reactive oxygen species by several defense mechanisms, including antioxidant enzymes such as superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase, etc. Low molecular weight antioxidants, such as reduced glutathione and vitamins E and C, act in conjunction with these enzymatic defenses.

Studies regarding generation of oxidative stress by food restriction have mainly focused in mammals [2], [4], [6], [7], [8], [9]. Decreased feeding leads to depletion of organ antioxidant stores and increases the generation of oxygen free radicals, particularly in liver [4]. Rats with a 72-h fasting suffered changes in free radical-scavenging mechanisms including antioxidant enzymes in different organs [6]. Regarding GSH, a significant diminution was observed in rat liver, muscle and heart after an 18-h fasting [8]. Remarkably, GSH decreases by fasting did not alter the hepatic levels of glutathione-related antioxidant enzymes [6]. Compared with mammals, biochemical and physiological changes under food restriction have not been so extensively studied in fish [10], [11]. Oxidative stress conditions have been observed in rainbow trout as a consequence of deficient Zn supplement [12]. Starvation in this fish results in protein repression and, particularly, glucose-6-phosphate dehydrogenase shifted to fully oxidized forms, making it more sensitive to proteolysis [13].

In addition, free radicals have been implicated in the deleterious effects of many environmental contaminants [14], [15], [16] and, therefore, some antioxidant enzymes have been proposed as biological indicators of pollutant exposure in aquatic organisms [17]. In previous studies, we have shown that some xenobiotics induce oxidative stress in fish and we have analyzed the molecular changes [15]. We observed that SOD suffers oxidative modifications and new acidic isoforms are generated by reaction with oxygen species or with aldehydes derived from lipid peroxidation, such as malondialdehyde (MDA) or 4-hydroxy-2-nonenal [18]. We proposed that these new isoforms of SOD might be used as bioindicators of oxidative stress provoked by pollutants [15]. However, injection and handling of fish in these experiments could have induced an additional stress in the animals, i.e., lack of appetite was noticed [15]. This fact prompted us to evaluate the effect of a deficient diet in fish.

Changes on the levels of certain metabolites, such as glutathione and MDA, and on the activity of some antioxidant enzymes have been described as biomarkers of oxidative stress provoked either by different nutritional conditions [2], [4], [6], [7], [8], [9], [12] or by exposition to xenobiotics [14], [15]. The aim of this work was to study the effects of food restriction or deprivation on the physiological levels of several antioxidant defenses, as well as the levels of some metabolites (glutathione status and MDA), in gilthead seabream liver. In addition, we have evaluated the generation of new SOD isoforms as a consequence of oxidative stress caused by starvation.