Combined effects of temperature and metal exposure on the fatty acid composition of cell membranes, antioxidant enzyme activities and lipid peroxidation in yellow perch (Perca flavescens)
Introduction
Biological membranes are semipermeable barriers surrounding cells and organelles. They are composed of a lipid bilayer and a variety of proteins. Membranes are highly sensitive to temperature fluctuations. This sensitivity is due to the effects of temperature on membrane lipids and consequently on the proteins embedded in the membranes (Hochachka and Somero, 2002). The internal temperature of fish and other poikilothermic organisms largely reflects ambient environmental temperature. These organisms counteract the effects of fluctuations in environmental temperature on the properties and function of their cell membranes by remodelling membrane lipids, a process known as homeoviscous adaptation (Hazel, 1995) involving changes in phospholipid head groups, acyl-chain composition and cholesterol content (Hazel and Williams, 1990). Shifts in phospholipid classes surrounding proteins modulate their activity (Frick et al., 2010, Robinson, 1993). This homeoviscous adaptation ensures the maintenance of membrane functions (Hazel, 1995, Kraffe et al., 2007, Pernet et al., 2007). Desaturases and elongases are the key enzymes involved in fatty acid synthesis and remodelling pathways. It was demonstrated that cold-challenged ectothermic fish display an upregulation of these enzymes to restore the fluidity of cold-rigidified membranes (Tiku et al., 1996, Tocher et al., 2004, Trueman et al., 2000).
Aerobic organisms depend on oxygen for energy production through oxidative phosphorylation. Reactive oxygen species (ROS) are constantly generated during normal cell metabolism. These species include superoxide anion (O2−), hydrogen peroxide (H2O2), hydroxyl radical (OH) and others (Halliwell and Gutteridge, 1999). Organisms cope with increasing ROS production by up-regulating their antioxidant defense system through non-enzymatic (glutathione, ascorbic and uric acid, tocopherols, etc.) and enzymatic components (Livingstone, 2003). General cellular antioxidant enzymes are superoxide dismutase (SOD, converts O2− to H2O2), catalase (CAT, reduces H2O2 to H2O), glutathione peroxidase (GPx, detoxifies H2O2 or organic hydroperoxides produced, for example, by lipid peroxidation (Halliwell and Gutteridge, 1999)) and glutathione S-transferase (GST, catalyzes the conjugation of glutathione (GSH) with various electrophilic substances). If antioxidant systems fail to eliminate excessive ROS production, significant damage can occur including DNA damage, protein degradation, enzyme inactivation and lipid peroxidation (Halliwell and Gutteridge, 1999). Indeed, ROS that possess sufficient energy to remove a hydrogen atom within lipid chains from methylene groups (-CH2-), can initiate lipid peroxidation (Girotti, 1985). During initiation, a lipid radical (L) produced after the abstraction of a hydrogen atom reacts with dioxygen to generate a lipid peroxyl radical (LOO). This peroxyl radical then reacts with another lipid to generate another radical (L) and a lipid hydroperoxide (LOOH). It has been demonstrated that more polyunsaturated fatty acids, and hence membranes with a higher degree of unsaturation, are more prone to LPO (Lin and Huang, 2007).
The intensity of LPO is assessed by measuring the concentrations of primary products, lipid peroxides or end products of LPO such as MDA and other aldehydes (Halliwell and Gutteridge, 1999). Common products of LPO released within the non-polar interior of biological membranes affect membrane stability by disruption of the non covalent bonds (e.g., van der Waals interactions). Furthermore, LOOH damages membrane integrity by affecting lipid-lipid and lipid-protein interactions (Kuhn and Borchert, 2002). In addition to damaging membrane physical properties, LPO can be deleterious by affecting membrane protein functions. For example, the activity of Na+/k+-ATPase was reduced by approximately 90% after LPO in brain synaptosomes (Chakraborty et al., 2003).
Thermal stress induces oxidative stress in ectotherms (Bagnyukova et al., 2003, Bocchetti et al., 2008, Verlecar et al., 2007). An increase of temperature stimulates metabolic processes, enhances oxygen consumption and consequently may increase ROS production (Lushchak, 2011).
Toxic trace metals induce uncontrolled reactive species production and oxidative stress. Cadmium (Cd) and nickel (Ni) are widespread pollutants in aquatics systems. Previous investigations have demonstrated that Cd does not generate ROS directly. Instead, Cd-induced oxidative stress results from the displacement of endogenous Fe leading to ROS generation (Schlenk and Benson, 2003, Valko et al., 2006). In turn, this affects GSH and thiol concentrations and antioxidant enzyme activities and can lead to lipid peroxidation (Sevcikova et al., 2011, Valko et al., 2005, Wang and Wang, 2009). Nickel is also highly toxic to living organisms. It can mediate directly or indirectly the oxidation of macromolecules (DNA, lipids and proteins) (Palermo et al., 2015). It can induce oxidative stress through ROS formation via Haber-Weiss/Fenton reactions (Torreilles and Guerin, 1990), depletion of intracellular free radical scavengers such as GSH (Krezel et al., 2003) or inhibition of the activity of antioxidant enzymes (Attig et al., 2014, Kubrak et al., 2012).
In a natural environment, organisms are typically exposed to multiple stressors, including natural factors, such as changes in temperature, oxygen concentrations or food availability, and anthropogenic stressors, such as contaminants. Several studies have investigated the effects of these stressors separately on different fish species and an increasing number of studies have examined their combined effects (Cai and Curtis, 1990, Cailleaud et al., 2007, Grasset et al., 2016, Kefaloyianni et al., 2005, Tocher et al., 2004). Yet, to our knowledge, this is the first study to examine the responses of cell membrane phospholipid fatty acid composition, lipid peroxidation and oxidative stress to variations in acclimation temperature and metal exposure. We investigated this question in the muscle of yellow perch (Perca flavescens), a freshwater fish species commonly found in areas affected by metal contamination. To this end, fish were acclimated to a cold or a warm temperature under clean conditions or combined with environmentally-relevant aqueous concentrations of Cd or Ni. Membrane phospholipid fatty acid composition was measured, along with indicators of oxidative stress (MDA) and oxidizability (PI) of membrane phospholipids and cellular antioxidant capacities (SOD, CAT, GST, GPx, GSH).
Section snippets
Experimental design: thermal acclimation and metal exposure
Yellow perch (Perca flavescens) were obtained from Trevor Thomas, Abbey Road Fish Farm, (Wainfleet, ON) and transported to the Laboratoire de Recherche en Sciences Aquatiques (LARSA) at Université Laval (Québec, QC) for thermal acclimation. Fish were maintained in a 1 m3 circular tank for one month to be acclimated to laboratory conditions at a temperature of 20 °C. During this period, fish were fed with Hikari® frozen brine shrimp (Artemia salina) with a daily ration of 3% of their biomass.
Fish condition and metal contamination
Fish exposed to Ni at 9 °C accumulated this metal significantly in their kidney compared to controls and Ni accumulation was enhanced 4-fold at 28 °C compared to 9 °C (Table 1). In control fish, kidney Ni concentrations were low and not affected by temperature. Exposure to Cd also led to a significant kidney accumulation of this metal at both temperatures. Like for Ni, fish exposed to the higher temperature accumulated higher concentrations of Cd in their kidney. However, in contrast to Ni, kidney
Effects of temperature on membrane composition
Cell membranes of poikilotherms subjected to variations in temperature restructure their phospholipids to maintain cellular integrity. In our study, the proportion of UFA, including PUFA and MUFA, was higher in muscle phospholipids of fish acclimated to the colder temperature, in agreement with the general theory of homeoviscous adaptation (Hazel, 1972, Hazel et al., 1991). The major decrease of DHA in the muscle of warm-acclimated fish supports the role of this major fatty acid in the thermal
Conclusion
Data from this study provide novel information about combined temperature and metal effects on fatty acid membrane composition, antioxidant defense system and lipid peroxidation. The higher PUFA content in the muscle cell membranes of cold-acclimated yellow perch allowing maintenance of membrane fluidity and function is consistent with the theory of homeoviscous adaptation. Our study also supports that cold acclimation of membrane composition results from modifications in the activity of key
Acknowledgements
We wish to thank Julie Grasset, who carried out fish exposures and provided us with samples for this study. We are also grateful to Mohamed Ali Ben Alaya for his help in the statistical part related to PCA. This study was funded by a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada to P. Couture.
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