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Seasonal Variation in the Antioxidant Defense System of the Brain of the Ground Squirrel (Citellus citellus) and Response to Low Temperature Compared with Rat
Comp. Biochem. Physiol. Vol. 117C, No. 2, pp. 141–149, 1997 Copyright 1997 Elsevier Science Inc.
ISSN 0742-8413/97/$17.00 PII S0742-8413(97)00061-3
Seasonal Variation in the Antioxidant Defense System of the Brain of the Ground Squirrel (Citellus citellus) and Response to Low Temperature Compared with Rat B. Buzadzˇic´, D. Blagojevic´, B. Korac´, Z. S. Saicˇic´, M. B. Spasic´ and V. M. Petrovic´ Department of Physiology, Institute for Biological Research ‘‘Sinisˇa Stankovic´,’’ 11060 Belgrade, Yugoslavia ABSTRACT. Seasonal variation in the activity of antioxidant enzymes (superoxide dismutase (EC 1.15.1.1.; SOD), catalase (EC 1.11.1.6; CAT), glutathione peroxidase (EC 1.11.1.9; GSH-Px), glutathione reductase (EC 1.6.4.2; GR), glutathione-S-transferase (EC 2.5.1.18; GST) and low-molecular-weight antioxidants: ascorbic acid (AsA), vitamin E (VIT E) and glutathione (GSH1GSSG) were examined in the brain of the ground squirrels (Citellus citellus) maintained at 30°C during the whole year. The highest activity (per mg protein) of antioxidant defense (AD) enzymes was found in the spring and was much lower in the summer. A further decrease in activity of CAT, GSH-Px and GST was observed in the winter. The highest levels of AsA and glutathione were recorded in winter in comparison with spring and summer. AD system in the brain of the ground squirrel and rats (maintained at thermoneutrality) exposed to low temperature (4°C) for 3, 6 or 24 hr during the summer was studied as well. Summer was chosen as a period of stable euthermia for ground squirrels and in thermoregulation similar to rats. Consumption of free fatty acid and glucose during the acute exposure to low temperature was found to be species specific. In the ground squirrel, an increase in the specific activities of SOD, after 3, 6 and 24 hr, CAT after 3 and 6 hr and GR after 6 hr of exposure to low temperature was detected. When activities were expressed in U/g wet mass, an increase of SOD after 3, 6 and 24 hr (P , 0.02, P , 0.02, P, 0.005) and CAT and GSH-Px 3 hr (P , 0.01) upon exposure to low temperature was observed. In the rats, no changes in the specific activities of these enzymes after exposure to low temperature were recorded and only an increase in GST activity (U/g wet mass) after 6 hr exposure was registered. Low-molecular-weight AD components in both animal species were unchanged upon short-term exposure to low temperature. The species-specific differences in brain AD between the rats and the ground squirrels after short exposure to low temperature may be ascribed to seasonal changes of the brain activity in the latter. comp biochem physiol 117C; 2:141–149, 1997. 1997 Elsevier Science Inc. KEY WORDS. Antioxidant defense, antioxidant enzymes, brain, Citellus citellus, ground squirrel, low-molecular antioxidants, low temperature, rat
INTRODUCTION Free radicals, molecules with an unpaired electron in the outer shell, can be produced in various ways, during normal metabolic processes (3,20). Because of their high reactivity and possibility for consequent disturbances of biological membranes, as well as of important biomolecules in aerobic Address reprint requests to: B. Buzadzˇ ic´, Senior Research Associate, Institute for Biological Research ‘‘Sinisˇa Stankovic´,’’ Department of Physiology, 29 Novembra 142, 11060 Belgrade, Yugoslavia. Tel. 381-11-764-422, ext.125; Fax 381-11-761-433; E-mail: [email protected]. Abbreviations–AD: antioxidant defense; AsA: ascorbic acid; CAT: catalase; FFA: free fatty acids; GSH: glutathione; GSH-Px: glutathione peroxidase; GST: glutathione-S-transferase; GR: glutathione reductase; MAO: monoamine oxidase; SOD: superoxide dismutase; VIT E: vitamin E, tocopherol. Received 1 February 1996; accepted 26 November 1996
organisms, an antioxidant defense (AD) system has been developed during the evolution. Under normal conditions, this system preserves a stable equilibrium between production and scavenging of free radical species. It consists of numerous enzymes and low-molecular-weight components that scavenge produced radicals and other reactive oxygen species and prevent production of more reactive radical species, such as the hydroxyl radical (⋅OH). It also removes lipid peroxides, preventing further propagation and conjugations of potentially harmful electrophillic compounds (41). In the brain as a central regulatory tissue, balance between free radical production and AD is of utmost importance for several reasons. The brain requires about one-fifth of total oxygen demand of the body (32). Additionally, this tissue is characterized by a high rate of oxidative metabolic activity, numerous membranes and a high concentration of
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readily oxidizable substrates (e.g., membrane lipid polyunsaturated fatty acids); high amounts of iron, especially in some regions (52); and endogenous generation of reactive oxygen metabolites by specific neurochemical reactions (monoamine oxidase [MAO; EC 1.4.3.4] catalyzed oxidation of catecholamines), prostaglandin metabolism (13), activation of macrophage-type microglial cells (19) and nitric oxide generation by endothelial cells and neurones (44). In addition, low levels of antioxidant enzymes have been detected in the rat brain in comparison with other tissues (10,22,26). Numerous studies were devoted to hibernators, including the ground squirrels (1,24,31,37). In our previous papers the results on AD system in the ground squirrels of different physiological conditions were presented (6–8). In the present work the first evidence on the ground squirrel (Citellus citellus) AD system in the brain, including superoxide dismutase (EC 1.15.1.1; SOD), catalase (EC 1.11.1.6; CAT), glutathione peroxidase (EC 1.11.1.9; GSH-Px), glutathione reductase (EC 1.6.4.2; GR), glutathione-S-transferase (EC 2.5.1.18; GST) and low-molecular-weight antioxidants ascorbic acid (AsA), vitamin E (VIT E) and glutathione (GSH 1 GSSG), is provided. Initial studies were related to seasonal changes (May, August, February) of AD system activity in the brain of the animals maintained at 30°C during the entire year. Body weight measurements were performed to check for the preservation of annual changes of this parameter and to compare it with the corresponding changes recorded in the ground squirrels that were transferred to low temperatures during the winter and hibernated. Rectal temperature measurements served to demonstrate that the animals were euthermic throughout a year. The activity of AD system in the brain of the two mammalian species differing with regard to thermoregulation, the rat a homeotherm and the ground squirrel (Citellus citellus) a hibernator-heterotherm, was studied upon short-term exposure to low temperature. Both species were kept at the temperature of thermoneutrality to avoid body temperature variations in the ground squirrels, and under such conditions, all changes of oxidative metabolism can be ascribed to endogenously regulated seasonal variability. For experiments on seasonal hibernators, it is very important to choose and determine well the stage of the annual cycle, because they are the subjected to many phases during the year, especially when cold stress is applied such as in the present work. Morrison and Galster (30) categorized thermoregulatory behavior of hibernators and divided the annual cycle into a homeothermal (active) and heterothermal (hibernation) season. The same authors subdivided a homothermal season during which our examinations were performed into a reproductive, a recovery and growth, a maintenance and a preparative stage. For the study on the activity of AD in the brain of the ground squirrel, we chose a summer season (maintenance phase) as a period of high
metabolic activity, stable homeothermy and as a stage of ‘‘fighting the cold’’ (31). MATERIALS AND METHODS Male ground squirrels (C. citellus) caught during April (233 6 20 g body weight) were kept at 30 6 2°C and 12–12 hr light–dark cycle. They were fed a standard laboratory chow and had free access to water. Male Mill Hill hybrid hooded rats (Rattus norvegicus, Berkenhout 1769) were maintained at 28 6 2°C (mean 6 SD) under the identical light–dark cycle and feeding conditions as the ground squirrels. They were killed at 3 months of age (230 6 20 g body weight). The effects of circannual rhythm on the AD status in the brain of the ground squirrel were determined in the middle of May (spring), at the beginning of August (summer) and in February (winter). One group of animals was kept in the cold room (7°C) in the dark from the beginning of December until the beginning of March to mimic natural conditions and enable the hibernation during the period when it occurs in the field. After that, the animals were transferred to 22 6 2°C (means 6 SD) for 7 days and then to 30°C. In this group of individuals, the changes in body weight were recorded and compared with those observed in the ground squirrels kept at the temperature of thermoneutrality during the entire year. Body weight represents an important factor in the estimation of cyclic variations in seasonal hibernators. Ground squirrels and rats were exposed to low environmental temperature of 4°C for 3, 6 and 24 hr at the beginning of August. The ground squirrels and the rats were killed by decapitation, between 8 and 10 a.m. to avoid any possible cyclic daily variation in antioxidant levels. Rectal temperature of the animals was measured before decapitation. Five milliliters of fresh blood was collected in the tube containing 0.2 mL heparin. Whole brains were dissected within 3 min and homogenized with a Janke and Kunkel KaWerk UltraTurrax homogenizer at 0–4°C in 0.25 M sucrose, 1 mM EDTA and 0.05 M Tris–HCl solution, pH 7.4. The homogenates were sonicated as described by Takada et al. (49) and used to determine the content of AsA, VIT E and GSH 1 GSSG. The remaining sonicates were centrifuged (90 min, 85,000 g, 4°C), and total protein, and enzyme activities were determined in the supernates. Blood glucose concentration, plasma free fatty acid (FFA) level, GSH content in the tissues and GST activity in the blood plasma were determined immediately after the animals were killed. For all other analyses performed successively during a month period upon death, the sonicates and supernates prepared by ultracentrifugation and stored at 220°C were used. Once thawed, samples were not refrozen. Dextrostrix reagent strips were obtained from Ames Divi-
Antioxidant Defense of Ground Squirrel and Rat
sion, Miles Laboratories Ltd. All other chemicals were Sigma (St Louis, MO, U.S.A.) products. SOD activity was determined by the epinephrine method (29), which is a ‘‘negative’’ type method based on the measurement of a degree of epinephrine autoxidation inhibition by SOD contained in the examined samples in 50 mM sodium carbonate buffer, pH 10.2, within a linear range of autoxidation curve. A unit of SOD was defined as the amount of enzyme inhibiting oxidation of epinephrine by 50% under the appropriate reaction conditions (39). CAT activity was assayed as recommended in the Sigma catalogue and activity expressed as µmol H2O2 min21 mg 21 protein. The method is based on the rate of H2O2 degradation by the action of CAT contained in the examined samples followed spectrophotometrically at 230 nm in 5 mM EDTA, Tris–HCl solution, pH 8.0. GSH-Px activity was evaluated by a spectrophotometric method of Paglia and Valentine (36) based on the measurement of NADPH consumption (i.e., NADPH oxidation by GR [106 U/mg protein; a Sigma, type III product] at 340nm). This reaction is preceded by the action of GSH-Px contained in the samples examined on t-butyl hydroperoxide (3 mM) as a substrate in 0.5 phosphate buffer, pH 7.0, at 37°C. The activity was expressed as nmol NADPH oxidized min21 mg 21 protein. GR activity was assayed by the method of Glatzle et al. (16) and expressed as nmol NADPH min21 mg21 protein. This procedure is based on GR catalyzed reduction of oxidized glutathione with NADPH and oxidation of the latter was determined spectrophotometrically at 340nm, using 2 mM GSSG and 0.1 mM NADPH, in phosphate buffer, pH 7.4, at 37°C. GST activity was determined as suggested by Habig et al. (18). The method is based on reaction of 1-chloro2,4-dinitrobenzene with the -SH group of glutathione catalyzed by GST contained in the samples. The reaction proceeded in the presence of 1 mM GSH in phosphate buffer, pH 6.5, at 37°C. The activity was expressed as nmol GSH consumed min21 mg 21 protein. All enzyme assays were performed with two or three dilutions of each homogenate to check that the amount of activity measured was proportional to the amount of homogenate assayed. Total GSH 1 GSSG was measured by an enzyme recycling assay (17). Here, glutathione oxidation by 5,5′-dithio-bis-(2-nitrobenzoic acid) and GR-catalyzed reduction by NADPH occur alternately, and the rate of 2-nitro-5-thiobenzoic acid was measured spectrophotometrically at 412 nm. AsA content was measured by the method of Okamura (34) based on reduction of Fe31 to Fe21 by ascorbic acid in an acid medium and coupling of Fe21 with 2,2′-dipyridyl. The absorbance of the complex formed was spectrophotometrically recorded at 525 nm. VIT E was measured by the procedure of Desai (11) based on the reduction of Fe31 in Fe21 in the presence of tocopherol and production of a colored complex with βphenanthroline. Glucose content in the blood was measured using Dextrostrix reagent strips prepared on the basis
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of the method suggested by and Barnett and Cash (4). Here, the glucose gets oxidized by oxygen in the reaction catalyzed by glucosooxidase, producing gluconic acid and H2O2. The latter oxidizes chromogenes in the reagent strips, and the intensity of change in the reagent strip color corresponds to glucose concentration in the samples examined. Plasma FFA were determined by the method of Ducombe (12) based on the reaction of copper ions present in copper salts of fatty acids extracted with chloroform, with diethyldithiocarbamate affording a colored complex measured spectrophotometrically at 436 nm. Protein content was measured by the method of Lowry et al. (28). Student’s t-test was used for data comparison between different groups (23). RESULTS Circannual Examinations of the Ground Squirrels Changes in body mass of the ground squirrels kept at 30°C during the whole year and those kept at temperature of thermic neutrality and transferred to 7°C from December to March so that they could hibernate are depicted in Fig. 1, A and B, respectively. As seen, both groups of animals expressed the same seasonal rhythmicity with a maximum in the period September–October and a minimum in February. Rectal temperature of the ground squirrels kept at 30°C was constant throughout a year (Fig. 2); in this way, euthermic (normothermic) (33) animals were obtained for a whole year. Annual Changes in Activity of AD System in the Brain of Ground Squirrels Maintained at the Temperature of Thermoneutrality Figure 3 shows the activity of AD enzymes expressed in units per mg protein (A) and units per gram wet brain tissue (B). Content of low-molecular weight AD components is depicted in Fig. 3, C. As shown, the highest specific activities (per mg protein) of all AD enzymes examined were recorded in spring to be decreasing during the summer, and CAT, GSH-Px and GST activities were the lowest during the winter. When expressed per gram of wet tissue, the activities were about the same in the spring and summer with the exception of SOD and GSH-Px, which were somewhat reduced during the summer. In the winter, although we were dealing with euthermic ground squirrels, the lowest activities of CAT, GSH-Px and GST were recorded, accompanied by the highest levels of AsA and GSH 1 GSSG. Exposure of Ground Squirrels and Rats to Low Environmental Temperature Ground squirrels and rats maintained at temperature of thermoneutrality were exposed to low environmental tem-
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FIG. 1. Body mass of ground squirrels (Citellus citellus) kept at 30°C throughout the entire year (A) or at 30°C and 4°C (B).
FIG. 2. Rectal temperature of ground squirrels (Citellus citel-
lus) kept at 30°C throughout the entire year.
perature of 4°C for 3, 6 or 24 hr. As seen from Table 1 (A and B, respectively), rectal temperature remained unchanged in both animal species. The first differences in response to the exposure to cold between these two mammalian species were observed at the level of blood glucose (Fig. 4, A) and plasma FFA concentration (Fig. 4, B). An increased blood glucose concentration in ground squirrels exposed to low temperature during the summer was recorded only after 6 hr exposure to be returned to control level after 24 hr. In the rats exposed to cold, blood glucose content was increased already after 3 hr exposure to remain at a high level in the two later experimental points (6 and 24 hr). Plasma FFA concentration was remarkably higher in ground squirrels during the summer in comparison with rats. The curve of FFA changes upon various periods of exposure to low temperature was quite different in comparison with the corresponding curve obtained for rats. The level of FFA was
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FIG. 4. Blood glucose (A) and plasma FFA concentrations (B) in ground squirrels (Citellus citellus) and rats exposed to low ambient temperature (4°C). Results are means 6 SEM.
FIG. 3. The activity of antioxidative defense enzymes ex-
pressed in the units per mg protein (A) and per g wet mass (B) and the amount of the low-molecular-weight antioxidative components (C) in the brain of ground squirrels (Citellus citellus) in different seasons. Results are means 6 SEM, n 5 6.
decreased in ground squirrels 6 hr upon exposure to low temperature, whereas in the rats, an increased FFA concentration was recorded after 3 hr exposure to cold. Plasma content of FFA returned to control level in both species of animals 24 hr upon exposure to low temperature. This suggests a species-specific consumption of these substrates (glucose and FFA) during the acute exposure to low temperature. Specific activities of the antioxidative enzymes in the brain of ground squirrels and rats exposed to low environ-
TABLE 1. Rectal temperature of ground squirrels (Citellus citellus) (n 5 6) (A) and rats (n 5 10) (B) exposed to low ambient
temperature (4°C) during the summer
A. Ground squirrels Rectal temp. (°C) before cold exposure Rectal temp. (°C) after cold exposure B. Rats Rectal temp. (°C) before cold exposure Rectal temp. (°C) after cold exposure
Control*
3 hr at 4°C
6 hr at 4°C
24 hr at 4°C
36.7 6 0.3 (0.6)
36.3 6 0.5 (1) 35.6 6 0.6 (1)
36.4 6 0.6 (1.1) 36.4 6 0.4 (0.7)
36.4 6 0.8 (1.3) 35.6 6 0.5 (0.8)
36.7 6 0.2 (0.5)
36.9 6 0.4 (0.7) 36.6 6 0.3 (0.6)
36.6 6 0.5 (0.8) 36.7 6 0.1 (0.3)
36.7 6 0.2 (0.3) 36.3 6 0.2 (0.4)
Results are means 6 SEM; SD is given in parentheses. *Control temperature for ground squirrels was 30 6 2°C and for rats, 28 6 2°C.
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FIG. 6. The activity of antioxidant enzymes expressed in
FIG. 5. Specific activity of AD enzymes in the brain of
ground squirrels (Citellus citellus) ( n 5 6) (A) and rats ( n 5 10) (B) exposed to low temperature (4°C). Results are means 6 SEM, *P , 0.05, **P , 0.02, ***P , 0.005.
mental temperature during the summer are depicted in Fig. 5 (A and B, respectively). In the brain of ground squirrels, SOD activity was increased after 3, 6 and 24 hr of exposure, that of CAT after 3 and 6 hr and that of GR after 6 hr. The same treatment did not result in changes in specific activity of either of the examined enzymes in the rat brain. When enzymatic activities of ground squirrel were expressed in units per gram wet tissue (Fig. 6, A), SOD activity was enhanced in all experimental points, whereas CAT and GSH-Px activities were increased only after 3 hr exposure to low temperature. In the rat brain, only GST activity expressed per gram wet tissue was augmented after 6 hr of exposure to low temperature. The data on the content of total protein and lowmolecular-weight components of AD in the brain of ground squirrels and rats exposed to low temperature and of the corresponding controls are listed in Table 2 (A and B, respectively). The changes in protein content were registered after 6 hr of exposure but they were of opposite direction (i.e., in the rats, an increase was observed, whereas in ground squirrels, a decrease was found in relation to the corresponding controls), although the change in the latter
units per g wet brain mass of ground squirrels (Citellus citellus) ( n 5 6) (A) and rats (n 5 10) (B) exposed to cold. Results are mean 6 SEM; *P , 0.01, **P , 0.02, ***P , 0.005.
was at the limit of statistical significance. Content of lowmolecular antioxidants was unchanged in both animal species after the treatment described above. DISCUSSION Under conditions of thermoneutrality, energetic requirements of homeotherm organisms are reduced to the maintenance of basal metabolic level (15,31,48). As seen from the results presented in this work, a seasonal hibernator, the ground squirrel, although possessing the ability for an inherent endogenous metabolic regulation of body temperature changes (31), remains euthermic during the winter if kept at thermoneutrality throughout a year period, and under these conditions, seasonal rhythmicity of body mass changes remains preserved. It has been reported earlier that alterations of other environmental factors do not affect to a great extent normal rhythmicity (synchronizing behavioral physiology) of the ground squirrel. Based on these findings, it has been believed that the seasonal cycle changes (active and hibernating physiological state, body temperature, body weight, daily food consumption, etc.) are endogenously controlled (37). However, changes in the activity of AD enzymes occurred in the liver and interscapular brown adipose tissue of the ground squirrels kept at 30°C in autumn
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TABLE 2. The amount of protein and low-molecular-weight antioxidants in the brain of ground squirrels (Citellus citellus)
(A) and rats (B) exposed to low ambient temperature (4°C) Control (30°C)
3 hr at 4°C
6 hr at 4°C
24 hr at 4°C
A. Ground squirrels Protein (mg/g tissue) GSH (µM /g tissue) AsA (µM /g tissue) VIT E (µg/g tissue)
44.3 6 1.3 (2.9) n 5 6 0.64 6 0.06 (0.14) n 5 6 0.7 6 0.1 (0.28) n 5 6 861 (3.94) n 5 6
43.4 6 2 (3.3) n 5 6 0.5 6 0.1 (0.181) n 5 4 0.71 6 0.04 (0.07) n 5 4 7.2 6 1.3 (3.5) n 5 4
39 6 1* (1.3) n 5 6 0.68 6 0.04 (0.067) n 5 4 0.69 6 0.05 (0.09) n 5 4 7.6 6 1.4 (3.69) n 5 4
45 6 2 (2.7) n 5 6 0.58 6 0.05 (0.086) n 5 4 0.87 6 0.07 (0.1) n 5 4 8 6 1.5 (3.69) n 5 4
B. Rats Protein (mg/g tissue) GSH (µM /g tissue) AsA (µM /g tissue)
26 6 1 (2) n 5 10 0.39 6 0.01 (0.021) n 5 4 0.7 6 0.1 (0.237) n 5 6
25 6 1 (2.7) n 5 10 0.39 6 0.04 (0.039) n 5 2 0.6 6 0.1 (0.247) n 5 4
31 6 1*** (2.7) n 5 10 0.41 6 0.06 (0.059) n 5 2 0.82 6 0.04 (0.08) n 5 4
28 6 2 (4.9) n 5 10 0.4 6 0.01 (0.013) n 5 2 0.66 6 0.17 (0.24) n 5 3
Results are means 6 SEM; SD are given in parentheses. n represents number of examined animals. ***P , 0.005, *P , 0.05.
and winter (8), thus indicating that during the examination periods, the animals were in different metabolic states and consequently characterized by different levels and rates of free radicals production. Armitage and Shulenberger (2) reported that due to circannual cycle of body weight change, a weight-specific oxygen consumption occurs even when there is no metabolic cycle. However, it has been shown that oxygen consumption levels can be only partially evaluated based on the body mass (i.e., the cycle of oxygen consumption is probably endogenously regulated). The results presented here on the ground squirrels kept at 30°C, in the phase of an increasing metabolic rate during the spring, the phase of high metabolic level before the hibernation period (summer) and during the phase of low metabolic rate in the winter (2), revealed the differences in the brain AD system activity. It is possible that the changes of the enzymatic components of the brain AD system coincide with the variations in the central nervous system activity during different seasons. It has been proposed that, in general, thermoregulation of euthermic hibernators does not differ from that of nonhibernators and that a hibernating mammal functions physiologically and behaviorally during the euthermia in the same manner as any non-hibernating mammal such as the rat (43,46). However, we observed the differences at the level of antioxidant defense in the brain between euthermic ground squirrels and the rats exposed to low environmental temperature. Both an increased heat preservation and an enhanced heat production during exposure to cold are thought to be induced by some central nervous system mechanism (48). The intensity of these activities is influenced by thermal input from centrally and peripherally
localized cold receptors (48). Preoptic area in the hypothalamus of the mammals represents a principal thermoregulatory center both in euthermic and hibernating animals (21). Glucose and FFA represent the most important substrates for oxidative processes in the muscles that participate in thermogenesis by shivering as a response to low temperature action. Differences in the level of these substrates in the blood of ground squirrels and rats exposed to cold very probably result from higher FFA and lipid levels in the plasma (14) and tissues (50) of the ground squirrels during the summer in comparison with the rats, so that increased requirements after 3 hr of exposure to cold can be met by using energetic tissue pools. Acute stress evoked by exposure to low temperature results in firing of neural response and activation of hypothalamus and other brain regions. All that together leads to an increase of both metabolic activity and oxygen supply (45,47). Differences in the response of AD system in the brain of both the ground squirrels and the rats after a shortterm exposure to low temperature could be the consequence of a complex variability of brain processes and seasonal variability of their central regulation in the ground squirrel. During the summer, MAO activity was found to be increased, and thus the amount of generated H2O2 should also be increased in the ground squirrels in relation to other seasons (38). These changes may account for the enhanced CAT and GSH-Px activities recorded during the present work. In addition, the possibility for lipid peroxidation is increased during the summer, when higher content of unsaturated fatty acids in the cell membranes of the ground squirrels has been detected (1). The variations at the level of antioxidant defense in the brain of the rat after short-
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term exposure to low environmental temperature were found to be of low intensity, suggesting that this mammalian species is better adapted to rapid changes of metabolic rate during the summer in comparison with the ground squirrel. Some changes registered in the brain of the ground squirrels exposed for a short time to cold were also observed in the brain of the rats maintained at low ambient temperature for 35 days (42) (e.g., GR activity was found to be enhanced). These results support the hypothesis that thermoregulation of euthermic hibernators does not differ from that of nonhibernators (43,46), and the only difference between the ground squirrels and the rats during the summer was higher sensitivity of the former to changes of temperature in comparison with the latter. Differences between the ground squirrel and the rat brain were recorded at the level of somatostatin binding sites. Namely, in the ground squirrel, a single population of somatostatin binding sites was recorded during both hibernation and non-hibernation phases, whereas in the rat brain cortex, two types of somatostatin binding sites occur (27). In relation to the rat, ground squirrels possess a capability for seasonal modification of thermoregulation and regression of brain activity accompanied by seasonal fluctuations in the content of individual transmitters (24), as well as sensitivity of some brain regions toward these transmitters (51). Also, a total regulation of sensitivity and reactivity level of brain regions to certain stimuli was found to change (9). Variation in brain SOD activity of the ground squirrels exposed to low temperature during the summer could lead to alterations in free radical equilibrium. Bearing in mind interactions of superoxide anion radicals with nitric oxide (5,25,40), as well as the fact that a neurotransmitter role of nitric oxide has been confirmed (35,44), these changes could be connected to both alterations in oxygen metabolism and regulatory changes at the level of central nervous system. Seasonal changes of AD in the brain of ground squirrel may be the cause of the species-specific differences in variation in brain AD between rat and ground squirrel after short exposure to low temperature. This work was supported by Ministry for Science and Technology of Serbia, Grant No.03E18.
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ISSN 0742-8413/97/$17.00 PII S0742-8413(97)00061-3
Seasonal Variation in the Antioxidant Defense System of the Brain of the Ground Squirrel (Citellus citellus) and Response to Low Temperature Compared with Rat B. Buzadzˇic´, D. Blagojevic´, B. Korac´, Z. S. Saicˇic´, M. B. Spasic´ and V. M. Petrovic´ Department of Physiology, Institute for Biological Research ‘‘Sinisˇa Stankovic´,’’ 11060 Belgrade, Yugoslavia ABSTRACT. Seasonal variation in the activity of antioxidant enzymes (superoxide dismutase (EC 1.15.1.1.; SOD), catalase (EC 1.11.1.6; CAT), glutathione peroxidase (EC 1.11.1.9; GSH-Px), glutathione reductase (EC 1.6.4.2; GR), glutathione-S-transferase (EC 2.5.1.18; GST) and low-molecular-weight antioxidants: ascorbic acid (AsA), vitamin E (VIT E) and glutathione (GSH1GSSG) were examined in the brain of the ground squirrels (Citellus citellus) maintained at 30°C during the whole year. The highest activity (per mg protein) of antioxidant defense (AD) enzymes was found in the spring and was much lower in the summer. A further decrease in activity of CAT, GSH-Px and GST was observed in the winter. The highest levels of AsA and glutathione were recorded in winter in comparison with spring and summer. AD system in the brain of the ground squirrel and rats (maintained at thermoneutrality) exposed to low temperature (4°C) for 3, 6 or 24 hr during the summer was studied as well. Summer was chosen as a period of stable euthermia for ground squirrels and in thermoregulation similar to rats. Consumption of free fatty acid and glucose during the acute exposure to low temperature was found to be species specific. In the ground squirrel, an increase in the specific activities of SOD, after 3, 6 and 24 hr, CAT after 3 and 6 hr and GR after 6 hr of exposure to low temperature was detected. When activities were expressed in U/g wet mass, an increase of SOD after 3, 6 and 24 hr (P , 0.02, P , 0.02, P, 0.005) and CAT and GSH-Px 3 hr (P , 0.01) upon exposure to low temperature was observed. In the rats, no changes in the specific activities of these enzymes after exposure to low temperature were recorded and only an increase in GST activity (U/g wet mass) after 6 hr exposure was registered. Low-molecular-weight AD components in both animal species were unchanged upon short-term exposure to low temperature. The species-specific differences in brain AD between the rats and the ground squirrels after short exposure to low temperature may be ascribed to seasonal changes of the brain activity in the latter. comp biochem physiol 117C; 2:141–149, 1997. 1997 Elsevier Science Inc. KEY WORDS. Antioxidant defense, antioxidant enzymes, brain, Citellus citellus, ground squirrel, low-molecular antioxidants, low temperature, rat
INTRODUCTION Free radicals, molecules with an unpaired electron in the outer shell, can be produced in various ways, during normal metabolic processes (3,20). Because of their high reactivity and possibility for consequent disturbances of biological membranes, as well as of important biomolecules in aerobic Address reprint requests to: B. Buzadzˇ ic´, Senior Research Associate, Institute for Biological Research ‘‘Sinisˇa Stankovic´,’’ Department of Physiology, 29 Novembra 142, 11060 Belgrade, Yugoslavia. Tel. 381-11-764-422, ext.125; Fax 381-11-761-433; E-mail: [email protected]. Abbreviations–AD: antioxidant defense; AsA: ascorbic acid; CAT: catalase; FFA: free fatty acids; GSH: glutathione; GSH-Px: glutathione peroxidase; GST: glutathione-S-transferase; GR: glutathione reductase; MAO: monoamine oxidase; SOD: superoxide dismutase; VIT E: vitamin E, tocopherol. Received 1 February 1996; accepted 26 November 1996
organisms, an antioxidant defense (AD) system has been developed during the evolution. Under normal conditions, this system preserves a stable equilibrium between production and scavenging of free radical species. It consists of numerous enzymes and low-molecular-weight components that scavenge produced radicals and other reactive oxygen species and prevent production of more reactive radical species, such as the hydroxyl radical (⋅OH). It also removes lipid peroxides, preventing further propagation and conjugations of potentially harmful electrophillic compounds (41). In the brain as a central regulatory tissue, balance between free radical production and AD is of utmost importance for several reasons. The brain requires about one-fifth of total oxygen demand of the body (32). Additionally, this tissue is characterized by a high rate of oxidative metabolic activity, numerous membranes and a high concentration of
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readily oxidizable substrates (e.g., membrane lipid polyunsaturated fatty acids); high amounts of iron, especially in some regions (52); and endogenous generation of reactive oxygen metabolites by specific neurochemical reactions (monoamine oxidase [MAO; EC 1.4.3.4] catalyzed oxidation of catecholamines), prostaglandin metabolism (13), activation of macrophage-type microglial cells (19) and nitric oxide generation by endothelial cells and neurones (44). In addition, low levels of antioxidant enzymes have been detected in the rat brain in comparison with other tissues (10,22,26). Numerous studies were devoted to hibernators, including the ground squirrels (1,24,31,37). In our previous papers the results on AD system in the ground squirrels of different physiological conditions were presented (6–8). In the present work the first evidence on the ground squirrel (Citellus citellus) AD system in the brain, including superoxide dismutase (EC 1.15.1.1; SOD), catalase (EC 1.11.1.6; CAT), glutathione peroxidase (EC 1.11.1.9; GSH-Px), glutathione reductase (EC 1.6.4.2; GR), glutathione-S-transferase (EC 2.5.1.18; GST) and low-molecular-weight antioxidants ascorbic acid (AsA), vitamin E (VIT E) and glutathione (GSH 1 GSSG), is provided. Initial studies were related to seasonal changes (May, August, February) of AD system activity in the brain of the animals maintained at 30°C during the entire year. Body weight measurements were performed to check for the preservation of annual changes of this parameter and to compare it with the corresponding changes recorded in the ground squirrels that were transferred to low temperatures during the winter and hibernated. Rectal temperature measurements served to demonstrate that the animals were euthermic throughout a year. The activity of AD system in the brain of the two mammalian species differing with regard to thermoregulation, the rat a homeotherm and the ground squirrel (Citellus citellus) a hibernator-heterotherm, was studied upon short-term exposure to low temperature. Both species were kept at the temperature of thermoneutrality to avoid body temperature variations in the ground squirrels, and under such conditions, all changes of oxidative metabolism can be ascribed to endogenously regulated seasonal variability. For experiments on seasonal hibernators, it is very important to choose and determine well the stage of the annual cycle, because they are the subjected to many phases during the year, especially when cold stress is applied such as in the present work. Morrison and Galster (30) categorized thermoregulatory behavior of hibernators and divided the annual cycle into a homeothermal (active) and heterothermal (hibernation) season. The same authors subdivided a homothermal season during which our examinations were performed into a reproductive, a recovery and growth, a maintenance and a preparative stage. For the study on the activity of AD in the brain of the ground squirrel, we chose a summer season (maintenance phase) as a period of high
metabolic activity, stable homeothermy and as a stage of ‘‘fighting the cold’’ (31). MATERIALS AND METHODS Male ground squirrels (C. citellus) caught during April (233 6 20 g body weight) were kept at 30 6 2°C and 12–12 hr light–dark cycle. They were fed a standard laboratory chow and had free access to water. Male Mill Hill hybrid hooded rats (Rattus norvegicus, Berkenhout 1769) were maintained at 28 6 2°C (mean 6 SD) under the identical light–dark cycle and feeding conditions as the ground squirrels. They were killed at 3 months of age (230 6 20 g body weight). The effects of circannual rhythm on the AD status in the brain of the ground squirrel were determined in the middle of May (spring), at the beginning of August (summer) and in February (winter). One group of animals was kept in the cold room (7°C) in the dark from the beginning of December until the beginning of March to mimic natural conditions and enable the hibernation during the period when it occurs in the field. After that, the animals were transferred to 22 6 2°C (means 6 SD) for 7 days and then to 30°C. In this group of individuals, the changes in body weight were recorded and compared with those observed in the ground squirrels kept at the temperature of thermoneutrality during the entire year. Body weight represents an important factor in the estimation of cyclic variations in seasonal hibernators. Ground squirrels and rats were exposed to low environmental temperature of 4°C for 3, 6 and 24 hr at the beginning of August. The ground squirrels and the rats were killed by decapitation, between 8 and 10 a.m. to avoid any possible cyclic daily variation in antioxidant levels. Rectal temperature of the animals was measured before decapitation. Five milliliters of fresh blood was collected in the tube containing 0.2 mL heparin. Whole brains were dissected within 3 min and homogenized with a Janke and Kunkel KaWerk UltraTurrax homogenizer at 0–4°C in 0.25 M sucrose, 1 mM EDTA and 0.05 M Tris–HCl solution, pH 7.4. The homogenates were sonicated as described by Takada et al. (49) and used to determine the content of AsA, VIT E and GSH 1 GSSG. The remaining sonicates were centrifuged (90 min, 85,000 g, 4°C), and total protein, and enzyme activities were determined in the supernates. Blood glucose concentration, plasma free fatty acid (FFA) level, GSH content in the tissues and GST activity in the blood plasma were determined immediately after the animals were killed. For all other analyses performed successively during a month period upon death, the sonicates and supernates prepared by ultracentrifugation and stored at 220°C were used. Once thawed, samples were not refrozen. Dextrostrix reagent strips were obtained from Ames Divi-
Antioxidant Defense of Ground Squirrel and Rat
sion, Miles Laboratories Ltd. All other chemicals were Sigma (St Louis, MO, U.S.A.) products. SOD activity was determined by the epinephrine method (29), which is a ‘‘negative’’ type method based on the measurement of a degree of epinephrine autoxidation inhibition by SOD contained in the examined samples in 50 mM sodium carbonate buffer, pH 10.2, within a linear range of autoxidation curve. A unit of SOD was defined as the amount of enzyme inhibiting oxidation of epinephrine by 50% under the appropriate reaction conditions (39). CAT activity was assayed as recommended in the Sigma catalogue and activity expressed as µmol H2O2 min21 mg 21 protein. The method is based on the rate of H2O2 degradation by the action of CAT contained in the examined samples followed spectrophotometrically at 230 nm in 5 mM EDTA, Tris–HCl solution, pH 8.0. GSH-Px activity was evaluated by a spectrophotometric method of Paglia and Valentine (36) based on the measurement of NADPH consumption (i.e., NADPH oxidation by GR [106 U/mg protein; a Sigma, type III product] at 340nm). This reaction is preceded by the action of GSH-Px contained in the samples examined on t-butyl hydroperoxide (3 mM) as a substrate in 0.5 phosphate buffer, pH 7.0, at 37°C. The activity was expressed as nmol NADPH oxidized min21 mg 21 protein. GR activity was assayed by the method of Glatzle et al. (16) and expressed as nmol NADPH min21 mg21 protein. This procedure is based on GR catalyzed reduction of oxidized glutathione with NADPH and oxidation of the latter was determined spectrophotometrically at 340nm, using 2 mM GSSG and 0.1 mM NADPH, in phosphate buffer, pH 7.4, at 37°C. GST activity was determined as suggested by Habig et al. (18). The method is based on reaction of 1-chloro2,4-dinitrobenzene with the -SH group of glutathione catalyzed by GST contained in the samples. The reaction proceeded in the presence of 1 mM GSH in phosphate buffer, pH 6.5, at 37°C. The activity was expressed as nmol GSH consumed min21 mg 21 protein. All enzyme assays were performed with two or three dilutions of each homogenate to check that the amount of activity measured was proportional to the amount of homogenate assayed. Total GSH 1 GSSG was measured by an enzyme recycling assay (17). Here, glutathione oxidation by 5,5′-dithio-bis-(2-nitrobenzoic acid) and GR-catalyzed reduction by NADPH occur alternately, and the rate of 2-nitro-5-thiobenzoic acid was measured spectrophotometrically at 412 nm. AsA content was measured by the method of Okamura (34) based on reduction of Fe31 to Fe21 by ascorbic acid in an acid medium and coupling of Fe21 with 2,2′-dipyridyl. The absorbance of the complex formed was spectrophotometrically recorded at 525 nm. VIT E was measured by the procedure of Desai (11) based on the reduction of Fe31 in Fe21 in the presence of tocopherol and production of a colored complex with βphenanthroline. Glucose content in the blood was measured using Dextrostrix reagent strips prepared on the basis
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of the method suggested by and Barnett and Cash (4). Here, the glucose gets oxidized by oxygen in the reaction catalyzed by glucosooxidase, producing gluconic acid and H2O2. The latter oxidizes chromogenes in the reagent strips, and the intensity of change in the reagent strip color corresponds to glucose concentration in the samples examined. Plasma FFA were determined by the method of Ducombe (12) based on the reaction of copper ions present in copper salts of fatty acids extracted with chloroform, with diethyldithiocarbamate affording a colored complex measured spectrophotometrically at 436 nm. Protein content was measured by the method of Lowry et al. (28). Student’s t-test was used for data comparison between different groups (23). RESULTS Circannual Examinations of the Ground Squirrels Changes in body mass of the ground squirrels kept at 30°C during the whole year and those kept at temperature of thermic neutrality and transferred to 7°C from December to March so that they could hibernate are depicted in Fig. 1, A and B, respectively. As seen, both groups of animals expressed the same seasonal rhythmicity with a maximum in the period September–October and a minimum in February. Rectal temperature of the ground squirrels kept at 30°C was constant throughout a year (Fig. 2); in this way, euthermic (normothermic) (33) animals were obtained for a whole year. Annual Changes in Activity of AD System in the Brain of Ground Squirrels Maintained at the Temperature of Thermoneutrality Figure 3 shows the activity of AD enzymes expressed in units per mg protein (A) and units per gram wet brain tissue (B). Content of low-molecular weight AD components is depicted in Fig. 3, C. As shown, the highest specific activities (per mg protein) of all AD enzymes examined were recorded in spring to be decreasing during the summer, and CAT, GSH-Px and GST activities were the lowest during the winter. When expressed per gram of wet tissue, the activities were about the same in the spring and summer with the exception of SOD and GSH-Px, which were somewhat reduced during the summer. In the winter, although we were dealing with euthermic ground squirrels, the lowest activities of CAT, GSH-Px and GST were recorded, accompanied by the highest levels of AsA and GSH 1 GSSG. Exposure of Ground Squirrels and Rats to Low Environmental Temperature Ground squirrels and rats maintained at temperature of thermoneutrality were exposed to low environmental tem-
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FIG. 1. Body mass of ground squirrels (Citellus citellus) kept at 30°C throughout the entire year (A) or at 30°C and 4°C (B).
FIG. 2. Rectal temperature of ground squirrels (Citellus citel-
lus) kept at 30°C throughout the entire year.
perature of 4°C for 3, 6 or 24 hr. As seen from Table 1 (A and B, respectively), rectal temperature remained unchanged in both animal species. The first differences in response to the exposure to cold between these two mammalian species were observed at the level of blood glucose (Fig. 4, A) and plasma FFA concentration (Fig. 4, B). An increased blood glucose concentration in ground squirrels exposed to low temperature during the summer was recorded only after 6 hr exposure to be returned to control level after 24 hr. In the rats exposed to cold, blood glucose content was increased already after 3 hr exposure to remain at a high level in the two later experimental points (6 and 24 hr). Plasma FFA concentration was remarkably higher in ground squirrels during the summer in comparison with rats. The curve of FFA changes upon various periods of exposure to low temperature was quite different in comparison with the corresponding curve obtained for rats. The level of FFA was
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FIG. 4. Blood glucose (A) and plasma FFA concentrations (B) in ground squirrels (Citellus citellus) and rats exposed to low ambient temperature (4°C). Results are means 6 SEM.
FIG. 3. The activity of antioxidative defense enzymes ex-
pressed in the units per mg protein (A) and per g wet mass (B) and the amount of the low-molecular-weight antioxidative components (C) in the brain of ground squirrels (Citellus citellus) in different seasons. Results are means 6 SEM, n 5 6.
decreased in ground squirrels 6 hr upon exposure to low temperature, whereas in the rats, an increased FFA concentration was recorded after 3 hr exposure to cold. Plasma content of FFA returned to control level in both species of animals 24 hr upon exposure to low temperature. This suggests a species-specific consumption of these substrates (glucose and FFA) during the acute exposure to low temperature. Specific activities of the antioxidative enzymes in the brain of ground squirrels and rats exposed to low environ-
TABLE 1. Rectal temperature of ground squirrels (Citellus citellus) (n 5 6) (A) and rats (n 5 10) (B) exposed to low ambient
temperature (4°C) during the summer
A. Ground squirrels Rectal temp. (°C) before cold exposure Rectal temp. (°C) after cold exposure B. Rats Rectal temp. (°C) before cold exposure Rectal temp. (°C) after cold exposure
Control*
3 hr at 4°C
6 hr at 4°C
24 hr at 4°C
36.7 6 0.3 (0.6)
36.3 6 0.5 (1) 35.6 6 0.6 (1)
36.4 6 0.6 (1.1) 36.4 6 0.4 (0.7)
36.4 6 0.8 (1.3) 35.6 6 0.5 (0.8)
36.7 6 0.2 (0.5)
36.9 6 0.4 (0.7) 36.6 6 0.3 (0.6)
36.6 6 0.5 (0.8) 36.7 6 0.1 (0.3)
36.7 6 0.2 (0.3) 36.3 6 0.2 (0.4)
Results are means 6 SEM; SD is given in parentheses. *Control temperature for ground squirrels was 30 6 2°C and for rats, 28 6 2°C.
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FIG. 6. The activity of antioxidant enzymes expressed in
FIG. 5. Specific activity of AD enzymes in the brain of
ground squirrels (Citellus citellus) ( n 5 6) (A) and rats ( n 5 10) (B) exposed to low temperature (4°C). Results are means 6 SEM, *P , 0.05, **P , 0.02, ***P , 0.005.
mental temperature during the summer are depicted in Fig. 5 (A and B, respectively). In the brain of ground squirrels, SOD activity was increased after 3, 6 and 24 hr of exposure, that of CAT after 3 and 6 hr and that of GR after 6 hr. The same treatment did not result in changes in specific activity of either of the examined enzymes in the rat brain. When enzymatic activities of ground squirrel were expressed in units per gram wet tissue (Fig. 6, A), SOD activity was enhanced in all experimental points, whereas CAT and GSH-Px activities were increased only after 3 hr exposure to low temperature. In the rat brain, only GST activity expressed per gram wet tissue was augmented after 6 hr of exposure to low temperature. The data on the content of total protein and lowmolecular-weight components of AD in the brain of ground squirrels and rats exposed to low temperature and of the corresponding controls are listed in Table 2 (A and B, respectively). The changes in protein content were registered after 6 hr of exposure but they were of opposite direction (i.e., in the rats, an increase was observed, whereas in ground squirrels, a decrease was found in relation to the corresponding controls), although the change in the latter
units per g wet brain mass of ground squirrels (Citellus citellus) ( n 5 6) (A) and rats (n 5 10) (B) exposed to cold. Results are mean 6 SEM; *P , 0.01, **P , 0.02, ***P , 0.005.
was at the limit of statistical significance. Content of lowmolecular antioxidants was unchanged in both animal species after the treatment described above. DISCUSSION Under conditions of thermoneutrality, energetic requirements of homeotherm organisms are reduced to the maintenance of basal metabolic level (15,31,48). As seen from the results presented in this work, a seasonal hibernator, the ground squirrel, although possessing the ability for an inherent endogenous metabolic regulation of body temperature changes (31), remains euthermic during the winter if kept at thermoneutrality throughout a year period, and under these conditions, seasonal rhythmicity of body mass changes remains preserved. It has been reported earlier that alterations of other environmental factors do not affect to a great extent normal rhythmicity (synchronizing behavioral physiology) of the ground squirrel. Based on these findings, it has been believed that the seasonal cycle changes (active and hibernating physiological state, body temperature, body weight, daily food consumption, etc.) are endogenously controlled (37). However, changes in the activity of AD enzymes occurred in the liver and interscapular brown adipose tissue of the ground squirrels kept at 30°C in autumn
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147
TABLE 2. The amount of protein and low-molecular-weight antioxidants in the brain of ground squirrels (Citellus citellus)
(A) and rats (B) exposed to low ambient temperature (4°C) Control (30°C)
3 hr at 4°C
6 hr at 4°C
24 hr at 4°C
A. Ground squirrels Protein (mg/g tissue) GSH (µM /g tissue) AsA (µM /g tissue) VIT E (µg/g tissue)
44.3 6 1.3 (2.9) n 5 6 0.64 6 0.06 (0.14) n 5 6 0.7 6 0.1 (0.28) n 5 6 861 (3.94) n 5 6
43.4 6 2 (3.3) n 5 6 0.5 6 0.1 (0.181) n 5 4 0.71 6 0.04 (0.07) n 5 4 7.2 6 1.3 (3.5) n 5 4
39 6 1* (1.3) n 5 6 0.68 6 0.04 (0.067) n 5 4 0.69 6 0.05 (0.09) n 5 4 7.6 6 1.4 (3.69) n 5 4
45 6 2 (2.7) n 5 6 0.58 6 0.05 (0.086) n 5 4 0.87 6 0.07 (0.1) n 5 4 8 6 1.5 (3.69) n 5 4
B. Rats Protein (mg/g tissue) GSH (µM /g tissue) AsA (µM /g tissue)
26 6 1 (2) n 5 10 0.39 6 0.01 (0.021) n 5 4 0.7 6 0.1 (0.237) n 5 6
25 6 1 (2.7) n 5 10 0.39 6 0.04 (0.039) n 5 2 0.6 6 0.1 (0.247) n 5 4
31 6 1*** (2.7) n 5 10 0.41 6 0.06 (0.059) n 5 2 0.82 6 0.04 (0.08) n 5 4
28 6 2 (4.9) n 5 10 0.4 6 0.01 (0.013) n 5 2 0.66 6 0.17 (0.24) n 5 3
Results are means 6 SEM; SD are given in parentheses. n represents number of examined animals. ***P , 0.005, *P , 0.05.
and winter (8), thus indicating that during the examination periods, the animals were in different metabolic states and consequently characterized by different levels and rates of free radicals production. Armitage and Shulenberger (2) reported that due to circannual cycle of body weight change, a weight-specific oxygen consumption occurs even when there is no metabolic cycle. However, it has been shown that oxygen consumption levels can be only partially evaluated based on the body mass (i.e., the cycle of oxygen consumption is probably endogenously regulated). The results presented here on the ground squirrels kept at 30°C, in the phase of an increasing metabolic rate during the spring, the phase of high metabolic level before the hibernation period (summer) and during the phase of low metabolic rate in the winter (2), revealed the differences in the brain AD system activity. It is possible that the changes of the enzymatic components of the brain AD system coincide with the variations in the central nervous system activity during different seasons. It has been proposed that, in general, thermoregulation of euthermic hibernators does not differ from that of nonhibernators and that a hibernating mammal functions physiologically and behaviorally during the euthermia in the same manner as any non-hibernating mammal such as the rat (43,46). However, we observed the differences at the level of antioxidant defense in the brain between euthermic ground squirrels and the rats exposed to low environmental temperature. Both an increased heat preservation and an enhanced heat production during exposure to cold are thought to be induced by some central nervous system mechanism (48). The intensity of these activities is influenced by thermal input from centrally and peripherally
localized cold receptors (48). Preoptic area in the hypothalamus of the mammals represents a principal thermoregulatory center both in euthermic and hibernating animals (21). Glucose and FFA represent the most important substrates for oxidative processes in the muscles that participate in thermogenesis by shivering as a response to low temperature action. Differences in the level of these substrates in the blood of ground squirrels and rats exposed to cold very probably result from higher FFA and lipid levels in the plasma (14) and tissues (50) of the ground squirrels during the summer in comparison with the rats, so that increased requirements after 3 hr of exposure to cold can be met by using energetic tissue pools. Acute stress evoked by exposure to low temperature results in firing of neural response and activation of hypothalamus and other brain regions. All that together leads to an increase of both metabolic activity and oxygen supply (45,47). Differences in the response of AD system in the brain of both the ground squirrels and the rats after a shortterm exposure to low temperature could be the consequence of a complex variability of brain processes and seasonal variability of their central regulation in the ground squirrel. During the summer, MAO activity was found to be increased, and thus the amount of generated H2O2 should also be increased in the ground squirrels in relation to other seasons (38). These changes may account for the enhanced CAT and GSH-Px activities recorded during the present work. In addition, the possibility for lipid peroxidation is increased during the summer, when higher content of unsaturated fatty acids in the cell membranes of the ground squirrels has been detected (1). The variations at the level of antioxidant defense in the brain of the rat after short-
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term exposure to low environmental temperature were found to be of low intensity, suggesting that this mammalian species is better adapted to rapid changes of metabolic rate during the summer in comparison with the ground squirrel. Some changes registered in the brain of the ground squirrels exposed for a short time to cold were also observed in the brain of the rats maintained at low ambient temperature for 35 days (42) (e.g., GR activity was found to be enhanced). These results support the hypothesis that thermoregulation of euthermic hibernators does not differ from that of nonhibernators (43,46), and the only difference between the ground squirrels and the rats during the summer was higher sensitivity of the former to changes of temperature in comparison with the latter. Differences between the ground squirrel and the rat brain were recorded at the level of somatostatin binding sites. Namely, in the ground squirrel, a single population of somatostatin binding sites was recorded during both hibernation and non-hibernation phases, whereas in the rat brain cortex, two types of somatostatin binding sites occur (27). In relation to the rat, ground squirrels possess a capability for seasonal modification of thermoregulation and regression of brain activity accompanied by seasonal fluctuations in the content of individual transmitters (24), as well as sensitivity of some brain regions toward these transmitters (51). Also, a total regulation of sensitivity and reactivity level of brain regions to certain stimuli was found to change (9). Variation in brain SOD activity of the ground squirrels exposed to low temperature during the summer could lead to alterations in free radical equilibrium. Bearing in mind interactions of superoxide anion radicals with nitric oxide (5,25,40), as well as the fact that a neurotransmitter role of nitric oxide has been confirmed (35,44), these changes could be connected to both alterations in oxygen metabolism and regulatory changes at the level of central nervous system. Seasonal changes of AD in the brain of ground squirrel may be the cause of the species-specific differences in variation in brain AD between rat and ground squirrel after short exposure to low temperature. This work was supported by Ministry for Science and Technology of Serbia, Grant No.03E18.
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