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UDC 57.044:577.3:639.3:612.062
CHANGES IN OXIDATIVE STRESS BIOMARKERS IN THE MUSCLE TISSUE OF RAINBOW TROUT (ONCORHYNCHUS MYKISS WALBAUM) DURING THERMAL ACCLIMATION
HALYNA TKACHENKO1, JOANNA GRUDNIEWSKA2
department of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University in Slupsk, Poland
2Department of Salmonid Research, Stanislaw Sakowicz Inland Fisheries Institute, 83-330 Zukowo, Poland
(Поступила в редакцию 29.01.2016)
Резюме. Компенсационное повышение аэробного метаболизма у рыб, которые остаются активными в холодной среде, и у рыб, которые становятся бездействующими при низких температурах, часто наблюдается в ответ на холодовую акклиматизацию. Мы использовали биомаркеры, окислительного стресса, чтобы исследовать, как изменение температуры влияет на функционирование мышечной ткани у радужной форели. Мы исследовали маркеры перекисного окисления липидов (ПОЛ) и окислительного повреждения белков в мышечной ткани радужной форели (Oncorhynchus mykiss Walbaum) при температурах 11—12 °С и 5,8 °С Наши результаты показали, что переохлаждение рыб сопровождается активацией процессов ПОЛ с существенным увеличением содержания альдегидных и кетоновых производных окислительной модификации белков. Поскольку полиненасыщенные жирные кислоты и фосфатидилэтаноламин уязвимы именно при окислительном стрессе. Вполне вероятно, что более высокое содержание этих липидов при низкой температуре тела неотъемлемо увеличивает восприимчивость мембранн к действию активных форм кислорода (АФК). Хотя мембраны клеток животных, живущих при низких температурах, могут быть более склонны к окислению, АФК и ПОЛ являются чувствительными к температуре (Crockett, 2008). Это подтверждает повышенный уровень маркеров окислительного стресса в мышечной ткани форели в условиях гипотермии.
Ключевые слова: гипотермия, радужная форель (OncorhynchusmykissWalbaum), мышечная ткань, перекисное окисление липидов, окислительная модификация белков
Summary. Compensatory increases of the aerobic capacity both in fish that remain active in the cold and in fish that become dormant at cold temperatures are frequently observed in response to cold acclimation. We used oxidative stress biomarkers to investigate how temperature change affects muscle tissue function in rainbow trout. We examined the lipid peroxida-tion (LPO) and protein damage biomarkers in the muscle tissue of rainbow trout (Oncorhyn-chus mykiss Walbaum) at the temperatures of 11—12 °C and 5,8 °C. Our results showed that hypothermia increase LPO with significant magnification of aldehydic and ketonic derivatives of protein damage.Since polyunsaturated fatty acids andphosphatidylethanolamine are particularly vulnerable to oxidation, it is likely that higher contents of these lipids at low body temperature elevate the inherent susceptibility of membranes to LPO. Although membranes from animals living at low body temperatures may be more prone to oxidation, ROS and LPO are sensitive to temperature (Crockett, 2008). This confirmed the increased level of LPO in muscle tissue of hypothermia-exposed trout.
Key words: hypothermia, rainbow trout (Oncorhynchus mykiss Walbaum), muscle tissue, lipid peroxidation, oxidatively modified proteins.
Introduction. For most fish, body temperature is very close to that of the habitat. The diversity of thermal habitats exploited by fish as well as their capacity to adapt to thermal change makes them excellent organisms in which to examine the responses to temperature (Guderley, 2004). Compensatory increases of the aerobic capacity of fish swimming muscle are frequently observed in response to cold acclimation. Such thermal compensation occurs both in fish that remain active in the cold and in fish that become dormant at cold temperatures. For cold-active fish, positive thermal compensation is best explained by conservation of the capacity for aerobic metabolic flux at low temperatures. The compensatory responses of cold-active species can be used to suggest the temperature range over which the activities of glycolytic and tricarboxylic acid cycle enzymes in a muscle, i.e., the muscle's «metabolic profile», can suffice (Guderley, 1990).
As body temperature decreases, changes in the physical chemistry of the cell produce a reduction in metabolic activity (Johnston and Dunn, 1987). In temperate fish, cold water temperatures either lead to dormancy or else trigger a range of homeostatic responses which serve to offset the passive effects of reduced temperature. Compensatory adjustments to temperature occur with time courses ranging from less than a second to more than a month. Although swimming performance may increase with cold-acclimation, active metabolic rate remains significantly below that for warm-acclimated fish. Compensatory and dormancy responses are not mutually exclusive and sometimes occur in the same species depending on the temperature (Johnston and Dunn, 1987).
During phenotypic cold acclimation, mitochondrial volume density increases in oxidative muscle of some species (striped bass Morone saxatilis, crucian carp Carassius carassius), but remains stable in others (rainbow trout Oncorhynchus mykiss). A role for the mitochondrial reticulum in distributing oxygen through the complex architecture of skeletal muscle fibres may explain mitochondrial proliferation. In rainbow trout, compensatory increases in the protein-specific rates of mitochondrial substrate oxidation maintain constant capacities except at winter extremes (Guderley, 2004). Changes in mitochondrial properties (membrane phospholipids, enzymatic complement and cristae densities) can enhance the oxidative capacity of muscle in the absence of changes in mitochondrial volume density. Changes in the unsaturation of membrane phospholipids are a direct response to temperature and occur in isolated cells. This fundamental response maintains the dynamic phase behaviour of the membrane and adjusts the rates of membrane processes. However, these adjustments may have deleterious consequences. For fish living at low temperatures, the increased polyunsatu-ration of mitochondrial membranes should raise rates of mitochondrial respiration which would in turn enhance the formation of reactive oxygen spe-
cies (ROS), increase proton leak and favour peroxidation of these membranes. Minimization of mitochondrial oxidative capacities in organisms living at low temperatures would reduce such damage (Guderley, 2004).
In this study, we used oxidative stress biomarkers to investigate how temperature change affects muscle tissue function in rainbow trout. We examined the lipid peroxidation (LPO) and protein damage biomarkers in the muscle tissue of rainbow trout (Oncorhynchus mykiss Walbaum) at the temperatures of 11-12 °C and 5,8 °C.
Materials and methods. Fish. Thirty clinically healthy rainbow trout were used in the experiments. The study was carried out in a Department of Salmonid Research, Inland Fisheries Institute near the village of Zukowo, Poland. Experiments were performed at a water temperature of 16±2 °C and the pH was 7,5. The dissolved oxygen level was about 12 ppm with additional oxygen supply. All biochemical assays were carried out at De part-ment of Zoology and Animal Physiology, Institute of Biology and Environmental Protection, Pomeranian University (Slupsk, Poland).
The fish were divided into two groups and held in 250-L square tanks (70 fish per tank). Fish were divided into the following groups: 1) control group, the water temperature 11-12 oC; 2) Experimental group, the water temperature 5,8 oC (hypothermia). Hypothermic exposure time was 2 hours. Muscle tissue was removed from trout after decapitation. One trout was used for each homogenate preparation. Fish were not anesthetized before tissue sampling.
Muscle tissue isolation. Muscle tissue were excised, weighted and washed in ice-cold buffer. The minced tissue was rinsed clear of blood with cold isolation buffer and homogenized in a glass Potter-Elvehjem homogenising vessel with a motor-driven Teflon pestle on ice. The isolation buffer contained 100 mM tris-HCl; pH of 7.2 was adjusted with HCl.
Analytical methods. All enzymatic assays were carried out at 25±0,5 °C using a Specol 11 spectrophotometer (Carl Zeiss Jena, Germany). The enzymatic reactions were started by adding the homogenate suspension. The specific assay conditions are presented subsequently. Each sample was analyzed in triplicate. The protein concentration in each sample was determined according to Bradford (1976) using bovine serum albumin as a standard.
TBARS assay for lipid peroxidation. LPO levelwas determined by quantifying the concentration of 2-thiobarbituric acid reactive substances (TBARS) according to Kamyshnikov (2004). The TBARS level was expressed in nmol MDA per mg protein by using 1.56 105 mM-1 cm-1 as molar extinction coefficient.
Carbonyl derivatives of oxidatively modified protein (OMP) assay.The rate of protein oxidative destruction was estimated from the reaction of the resultant carbonyl derivatives of amino acid reaction with DNFH as de-
scribed by Levine et al. (1990) and as modified by Dubinina et al. (1995). The carbonyl content was calculated from the absorbance measurement at 370 nm and 430 nm and an absorption coefficient 22,000 M-1xm-1. Carbonyl groups were determined spectrophotometrically from the difference in absorbance at 370 nm (aldehydic derivatives) and 430 nm (ketonic derivatives) and expressed in nmol per mg of tissue protein.
Statistical analysis. The mean ± S.E.M. values was calculated for each group to determine the significance of inter group difference. All variables were tested for normal distribution using the Kolmogorov-Smirnov and Lilliefors test (p>0,05). Significance of differences between the oxidative stress biomarkers level (significance level, p<0,05) was examined using Kruskal-Wallis one-way analysis of variance by ranks test (Zar, 1999). All statistical calculation was performed on separate data from each individual with STATISTICA 10.0.
Results. Influence of hypothermia on LPO biomarker, measured as TBARS in the muscle tissue of trout are presented in Fig. 1A. Non-significantly higher TBARS level (by 19 %, p>0,05) in rainbow trout exposed to hypothermia compared to control group was observed (Fig. 1 A).
Aldehydic and ketonic derivatives of oxidatively modified proteins in the muscle tissue of hypothermia-exposed trout were significantly higher by 189% (p=0.000) and 130% (p=0.000) respectively compared to controls (Fig. 1B).
Discussion. Linkages between cold acclimation and oxidative stress in fishes are unclear and contradictory results have been published (Kammer et al., 2011). Our results showed that hypothermia increase LPO with significant magnification of aldehydic and ketonic derivatives of protein damage (Figs 1A and 1B).
A
TBARS
water temperature 11-12 oC water temperature 5.8 oC
B
Oxidatively modified proteins
Alilcliydir derivative.* OMP Ketonir ilprivalivps OMP
Owntrr temperature 11-12 o(" Dwnlrr Irmprraliirr ill
F i g. 1. Effect of hypothermia on lipid peroxidation biomarker, measured as 2-thiobarbituric acid reactive substances (TBARS, A), as well as aldehydic and ketonic derivatives of oxidatively modified proteins (B) in the muscle tissue of rainbow trout
* the significant difference was shown as p<0,05 when compared control and hypothermia-
exposed groups
An extensive literature links cold temperatures with enhanced oxidative capacities in fish tissues, particularly skeletal muscle. Closer examination of inter-species comparisons (i.e. the evolutionary perspective) indicates that the proportion of muscle fibres occupied by mitochondria increases at low temperatures, most clearly in moderately active demersal species. Isolated muscle mitochondria show no compensation of protein-specific rates of substrate oxidation during evolutionary adaptation to cold temperatures (Guderley, 2004). Cold-acclimation results in significant increases in the density of mitochondria and capillaries in skeletal muscle. This serves to reduce diffusion distances and increase the capacity for aerobic ATP production relative to fish acutely exposed to low temperature. There is evidence that cold acclimation has differential effects on the synthesis and degradation rates of mitochondrial proteins leading to a net increase in their concentration. In contrast, the activities of enzymes associated with glycol-ysis and phosphocreatine hydrolysis show no consistent changes with thermal acclimation suggesting that flux through these pathways is modulated by factors other than enzyme concentration. Higher mitochondrial densities have also been reported for the liver, brain and gill tissue of cold compared with warm acclimated fish. In spite of their increased concentration, the activities of aerobic enzymes remain much lower at cold than warm temperatures (Johnston and Dunn, 1987).
Our results are in agreement with data of other researchers. Changes in oxidative capacities and phospholipid remodeling accompany temperature acclimation in ectothermic animals. Both responses may alter redox status and membrane susceptibility to LPO. Grim et al. (2015) tested the hypothesis that phospholipid remodeling is sufficient to offset temperature-driven rates of LPO and, thus, membrane susceptibility to LPO is conserved. They also predicted that the content of LPO products is maintained over a range of physiological temperatures. To assess LPO susceptibility, rates of LPO were quantified with the fluorescent probe C11-BODIPY in mitochondria and sarcoplasmic reticulum from oxidative and glycolytic muscle of striped bass (Morone saxatilis) acclimated to 7°C and 25 °C. They also measured phospholipid compositions, contents of LPO products [i.e., individual classes of phospholipid hydroperoxides], and two membrane antioxidants. Despite phospholipid headgroup and acyl chain remodeling, these alterations do not counter the effect of temperature on LPO rates (i. e., LPO rates are generally not different among acclimation groups when normalized to phospholipid content and compared at a common temperature). Although absolute levels of phospholipid hydroperoxides are higher in muscles from cold- than warm-acclimated fish, this difference is lost when phospholipid hydroperoxides levels are normalized to total phospholipid. Contents of vitamin E and two homologs of ubiquinone are more than four times higher in mitochondria prepared from oxidative muscle of warm- than cold-acclimated fish. Collectively, Grim et al. (2015) demonstrated that although phospholipid remodeling does not provide a means for offsetting thermal effects on rates of LPO, differences in phospholipid quantity ensure a constant proportion of LPO products with temperature variation (Grim et al., 2015).
Kammer et al. (2011) determine whether oxidative stress occurs during cold acclimation of three spine stickleback (Gasterosteus aculeatus), and, if so, when it occurs and whether it varies among tissues. Fish were warm (20°C) or cold (8°C) acclimated for 9 weeks, and harvested during acclimation. Oxidative stress was assessed in oxidative and glycolytic muscles and liver by measuring levels of protein carbonyls and glutathione, and the activity and transcript levels of superoxide dismutase (SOD). Protein carbonyl levels increased in liver after 1 week at 8°C and then decreased after week 4, and remained unchanged in glycolytic and oxidative muscle. Glutathione levels increased in liver on day 3 of cold acclimation and may minimize oxidative stress later during acclimation (Kammer et al., 2011).
For cold-inactive species that remain normoxic during winter dormancy, the compensatory metabolic modifications may facilitate lipid catabolism. Alternately, an increased aerobic capacity may be adaptive during the relatively cold periods that precede and follow winter dormancy. For goldfish
and carp that encounter hypoxia and anoxia during winter dormancy, increased mitochondrial abundance could facilitate ethanol production during anoxia and the diffusion of oxygen to mitochondria during hypoxia. Finally, metabolic modifications during natural acclimatization indicate both thermal compensation and direct thermal effects and suggest that thermal compensation may be masked by reproductive and feeding activities (Guderley, 1990).
Bouchard and Guderley (2003) examined The time course of changes in the properties of mitochondria from oxidative muscle of rainbow trout during warm (15oC) and cold (5oC) acclimation. Mitochondrial properties changed more quickly during warm than cold acclimation. Warm acclimation reduced the proportion of cytochrome c oxidase and citrate synthase needed during mitochondrial substrate oxidation. Phospholipid concentrations per mg mitochondrial protein changed little with thermal acclimation. While the biochemical modifications during thermal acclimation may eventually compensate for the thermal change, compensation did not occur at its onset. The initial changes of mitochondrial oxidative capacity in response to temperature change accentuated the functional impact of the thermal change, and prolonged exposure to the new temperature was required to attain a degree of thermal compensation (Bouchard and Guderley, 2003).
We showed that thermal acclimation caused an oxidative stress response in muscle tissue. Particularly during cold acclimation, aldehydic and ketonic derivatives of protein damage followed much the same time course as oxi-dative stress expressed as LPO. These responses are compatible with a major influence of the phospholipid and fatty acid composition of the membrane and of the concentration of proteins in the muscle tissue. In rainbow trout (Oncorhynchus mykiss), cold acclimation and acclimatization increase the capacity of skeletal muscle mitochondria to oxidise pyruvate and acyl carnitines and increase polyunsaturation of mitochondrial phospholipids (Guderley et al., 1997). Since polyunsaturated fatty acids and phosphatidyl-ethanolamine are particularly vulnerable to oxidation, it is likely that higher contents of these lipids at low body temperature elevate the inherent susceptibility of membranes to LPO. Although membranes from animals living at low body temperatures may be more prone to oxidation, ROS and LPO are sensitive to temperature (Crockett, 2008). This confirmed the increased level of LPO in muscle tissue of hypothermia-exposed trout (Fig. 1A).
Conclusion. Our results suggest that hypothermia-induced oxidative stress caused increase of aldehydic and ketonic derivatives of protein damage (Fig. 1B). Mitochondria are viewed as one of the major contributors of ROS production (Zuo et al., 2011 b). In vivo experimentation has confirmed that increased oxidative stress impairs mitochondrial function (Williams et al., 1998; Berneburg et al., 1999). Cold acclimation and acclimatisation also
increase the activity of some mitochondrial enzymes: ß-hydroxyacyl CoA dehydrogenase (Guderley and Gawlicka, 1992), cytochrome c oxidase, citrate synthase and carnitine palmitoyl transferase (St. Pierre et al., 1998). Furthermore, the cristae surface density of mitochondria (St. Pierre et al., 1998) and the total mitochondrial volume in oxidative muscle fibres increase at low acclimatisation temperature (Egginton et al., 2000). ROS can also target proteins in the mitochondrial membrane and lead to mitochondrial permeability transition (MPT) (Cosso et al., 2002). ROS can contribute to the opening of the mitochondrial permeability transition pore (mPTP) (Lemasters et al., 1998). One such mechanism of ROS-mediated mPTP opening is by the oxidation of dithiols in the protein pore located on the inner mitochondrial membrane (Lemasters et al., 1998).
This work was supported by grant of the Pomeranian University for Young Scientists.
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