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Changes in immunoreactive manganese-superoxide dismutase content in rat tissues under heat or cold environment
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HOPHYSIOL0¢( P ELSEVIER
Pathophysiology 2 (1995) 161-166
Changes in immunoreactive manganese-superoxide dismutase content in rat tissues under heat or cold environment Tomomi Ookawara a, Takako Kizaki a, Hitoshi Yamashita a, Shuji Oh-ishi a, Daizoh Saitoh Keiichiro Suzuki c, Naoyuki Taniguchi c, Hideki Ohno a,,
b,
a Department of Hygiene, National Defense Medical College, 3-2 Namiki Tokorozawa 359, Japan b Department of Traumatology and Emergency Medicine, National Defense Medical College, Tokorozawa 359, Japan c Department of Biochemistry, Osaka University Medical School, Suita 565, Japan Received 14 March 1995; accepted 4 April 1995
Abstract
Forty-one male Wistar rats were kept at 5°C [cold-exposed (CE) rats] or 35°C [heat-exposed (HE) rats] for 1, 7, 14, or 28 days. Seven rats reared at 25°C served as controls. After each exposure animals were sacrificed at the same age of 13 weeks, and blood, liver, kidney and gastrocnemius muscle were collected. Thiobarbituric acid-reactive substances (TBARS) level, an indicator of lipid peroxidation, in plasma was significantly increased on days 7 and 14 after heat exposure but then was remarkably reduced on day 28, whereas it decreased significantly on days 14 and 28 after cold exposure. In the three tissues collected, no significant changes in TBARS level were noted after heat or cold exposure. Immunoreactive Mn-superoxide dismutase (Mn-SOD) level in plasma from HE and CE rats decreased significantly on day 7 but both increased significantly on day 28. Except in the case of liver of HE rats, Mn-SOD level in the three tissues appeared to increase during the experiment. Meanwhile, glutathione peroxidase activity in liver decreased significantly on days 1, 7, and 28 after cold exposure. Conversely, the enzyme activity in kidney increased significantly on days 14 and 28 after heat exposure. These findings suggest that heat exposure imposes oxidative stress on the body, and that exposure to both heat and cold may affect the antioxidant enzyme system.
Keywords: Thiobarbituric acid-reactive substances; Oxidative stress; Glutathione peroxidase; Citrate synthase; Plasma
I. Introduction
Superoxide dismutase (SOD) is one of the most important enzymes in the antioxidant defense system. The enzyme scavenges superoxide anion (O2), which is the first product of oxygen radicals. Mammalian tissues contain two main forms of S O D : M n - S O D is present mostly in the mitochondrial matrix, whereas Cu,ZnSOD is predominantly localized in the cytoplasm (for review see [1,2]). It is currently believed that a vast array of pathologies, such as aging, cancer, artherosclerosis, diabetes, ischemia and cataracta, have possible links to oxygen radicals (for reviews see [1-5]). There is growing evidence, on the other hand, that even the
* Corresponding author. Fax: + 81-429-95-0638. 0928-4680/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 9 2 8 - 4 6 8 0 ( 9 5 ) 0 0 0 2 8 - 3
physiological state, e.g., physical exercise [2,4,6-8] and high altitude [9,10], imposes oxidative stress on the body due to oxygen free radical generation (including an increase in lipid peroxidation in various tissues). Moreover, it has recently been demonstrated that exposure to cold and heat increases lipid peroxidation in several tissues [11-13]. Actually, only limited information, however, could be obtained concerning this phenomenon. In addition, it seems unlikely that only identical observations have been made; for example, spontaneous lipid peroxidation rate was found unchanged in the brown adipose tissue of rats chronically exposed to cold [14]. Further, exposure of rats to heat decreased H 2 0 2 generation in mitochondria of the liver, but not of the kidney or the heart [15]. Chance et al. [16] have reported that mitochondria are one of the main intracellular sources of O 2 and H 2 0 2 even under physiological conditions. Mitochon-
T. Ookawara et al. / Pathophysiology 2 (1995) 161-166
162
Table 1 Changes in body and tissue weights of temperature-exposed rats Group
Body weight
Liver weight
Initial (g)
Final (g)
(g)
257 252 228 194
_+ 7 +_ 6 _+ 3 _+ 4
233 224 206 203
± 5 + 4 _+_5 ± 2
* * * *
9.98 8.98 8.52 8.10
267 248 241 179
± 2 _+ 6 ± 3 ± 7
251 223 206 203
___1 + 6 * ___4 * + 5 *
10.4 9.77 9.21 10.0
Kidney weight (g/lO0 g body weight)
(g)
* * * *
4.29 4.09 4.13 3.99
+ ± ± ±
0.10 0.13 0.10 0.07 *
0.915 0.857 0.814 0.833
± 0.1 _+ 0.36 * ± 0.33 * _+ 0.2 *
4.15 4.37 4.47 4.95
± ± ± ±
0.04 0.05 0.11 0.09 *
0.943 1.05 1.04 1.09
Gastrocnemius muscle weight (g/lO0 g body weight)
(g)
(g/lO0 g body weight)
_+ 0.025 _+ 0.013 ± 0.030 * +_ 0.018 *
0.393 0.383 0.395 0.411
± 0.008 _+ 0.007 ± 0.009 ± 0.007 *
1.26 1.20 1.14 1.16
_+ 0.02 ± 0.04 + 0.03 * _+ 0.03 *
0.541 0.534 0.551 0.571
_+ 0.005 ± 0.007 ± 0.007 * _+ 0.010 *
_+ 0.022 ± 0.04 ± 0.02 _+ 0.02 *
0.375 0.470 0.506 0.539
± ± ± ±
1.33 1.14 1.08 1.03
± ± ± ±
0.530 0.511 0.526 0.508
± 0.014 _+ 0.009 _+ 0.005 ± 0.012
Heat-exposed 1 day (5) 7 days (5) 14 days (5) 28 days (6)
_+ 0.30 ± 0.37 _+ 0.37 ± 0.19
Cold-exposed 1 day (5) 7 days (5) 14 days (5) 28 days (5)
Warm-acclimated (7)
255 ± 3
11.4 +_ 0.4
4.46 ± 0.12
0.947 _+ 0.031
0.009 0.004 * 0.011 * 0.012 *
0.371 ± 0.010
0.03 0.03 * 0.02 * 0.02 *
1.33 ± 0.04
0.519 _+ 0.008
Values are means + SEM. All rats were killed at the same age of 13 weeks. Numbers in parentheses are numbers of animals. * P < 0.05 compared with warm-acclimated rats.
active Mn-SOD level and GPX activity in several tissues of rats.
dria are probably faced with the elevated rate of oxygen utilization under conditions of increased metabolism of the body such as physical exercise, suggesting an increase in oxygen free radical generation in the cell organelle. It has been estimated that 80% of the totally formed superoxide radicals in the mitochondria may be reduced by Mn-SOD [16]. Activity assay generally utilized for SOD measurement may lack specificity and may be subject to interference from other factors. As compared with enzymatic methods, therefore, immunochemical assay methods for SOD appear to be more reliable and reproducible because the determinations are specific to the protein moiety [1,2]. Glutathione peroxidase (GPX), like SOD, is intracellularly located in the cytosol and mitochondrial matrix and detoxifies oxygen-reactive radicals by catalyzing the formation of H 2 0 2 derived from 0 2. The current study was undertaken to investigate the effects of temperature exposure with the passage of time on not only lipid peroxidation but also immunore-
2. Materials and methods
Forty-eight male Wistar rats were divided into three groups. Animals of the first group (n = 21) were reared at an ambient temperature of 35°C and relative humidity of 40% for 1, 7, 14, or 28 days in a climatic room with lighting from 7:00 to 19:00 h daily in individual cage. These rats, which were referred to as heat-exposed (HE) rats, had free access to the standard laboratory diet (Oriental MF, Oriental Yeast Co., Tokyo) and tap water. The animals were cared for in accordance with the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan, based upon the Helsinki Declaration, 1964. The second group (n = 20) was kept at 5°C during the same period of time as for heat expo-
Table 2 Effect of temperature exposure on TBARS level Tissue
Warm-acclimated
Plasma ( n m o l / m l ) Heat-exposed Cold-exposed Liver ( n m o l / m g protein) Heat-exposed Cold-exposed Kidney ( n m o l / m g protein) Heat-exposed Cold-exposed Gastrocnemius muscle ( n m o l / m g protein) Heat-exposed Cold-exposed
47.5 _+ 6.1
Temperature exposure (day) 1
7
14
28
52.4 ± 12.2 28.3 ± 3.9
75.0 ± 9.6 * 52.8 +2.5
97.1 ± 11.3 " 23.1 _+ 4.4 *
8.56 ± 0.94 * 22.4 +_2.2 *
0.74 ± 0.06 0.94 ± 0.06
0.76 ± 0.03 0.73 _+0.04
0.72 _+ 0.04 0.72 _+ 0.06
0.80 ± 0.03 0.80 _+ 0.03
0.74 ± 0.08 1.16 ± 0.23
0.86 +_0.03 0.79 _+0.08
0.95 _+ 0.05 0.70 _+ 0.10
1.04 _+ 0.05 0.92 +_ 0.08
1.78 _+ 0.07 1.09 + 0.05
1.07 ± 0.03 1.38 _+ 0.04
1.38 ± 0.09 1.72 +_ 0.14
1.60 ± 0.17 2.01 _+ 0.14
0.85 ± 0.03
0.85 _+ 0.05
1.47 _+ 0.18
Values are means + SEM. Numbers of rats same as in Table 1. *P < 0.05 compared with warm-acclimated rats.
T. Ookawara et al./ Pathophysiology 2 (1995) 161-166
163
Table 3 Effect of temperature exposure on Mn-SOD level Tissue
Warm-acclimated
Plasma (ng/ml) Heat-exposed Cold-exposed Liver (/zg/mg protein) Heat-exposed Cold-exposed Kidney (/zg/mg protein) Heat-exposed Cold-exposed Gastrocnemius muscle (/zg/mg protein) Heat-exposed Cold-exposed
Temperature exposure (day) 1
7
14
28
1.50 ± 0.09 1.75 ± 0.22
0.94 ± 0.02 * 1.14 ± 0.05 *
1.64 ± 0.34 1.41 ± 0.36
3.30 _+ 0.12 * 2.67 _+ 0.16 *
0.36 ± 0.04 0.41 ± 0.05
0.34 ± 0.03 0.39 ± 0.06
0.33 _+ 0.03 0.53 ± 0.07 *
0.27 _+ 0.03 0.50 ± 0.02 *
0.63 ± 0.03 * 0.61 ± 0.02 *
0.68 + 0.03 ~ 0.72 _+ 0.08 *
0.65 _+ 0.03 * 0.70 ± 0.07 *
0.60 ± 0.04 0.90 ± 0.05 *
0.19 _+ 0.02 0.25 _+ 0.01 *
0.23 ± 0.01 * 0.18 ± 0.01
0.23 ± 0.01 * 0.20 ± 0.01
0.23 ± 0.01 * 0.17 ± 0.01
1.84 ± 0.16
0.27 ± 0.05
0.38 ± 0.08
0.15 ± 0.02
Values are means ± SEM. Numbers of rats same as in Table 1. *P < 0.05 compared with warm-acclimated rats.
sure [cold-exposed (CE) rats]. The third group (n = 7), comprising control [warm-acclimated (WA)] rats, was kept at the thermoneutral temperature of 25°C. After each exposure animals were killed by decapitation at the same age of 13 weeks, and blood, liver, kidney and gastrocnemius muscle were collected. Hematocrit level was measured by a microcapillary tube technique. Each plasma sample was used for measuring Mn-SOD, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS), an indicator of lipid peroxidation. The three tissues were quickly frozen in liquid nitrogen. They were stored at - 8 0 ° C for later analysis. A portion of tissues were thawed in an ice-cold medium containing 0.25 M sucrose, 10 mM 2-amino-2-hydroxymethyl-l,3-propanediol (Tris), and 0.1 mM EDTA (pH 7.4, wt/vol 1:9), minced, and homogenized on ice in brief bursts by a Polytron homogenizer (Kinematika, Luzern). The homogenate was centrifugated at 750 g ~4°C) for 15 min, and the supernatant was used for various assays. Protein content was determined by the method of Lowry et al. [17]. Measurement of immunoreactive Mn-SOD was made by an enzyme-linked immunosorbent assay, using
specific polyclonal antibody against Mn-SOD isoenzyme purified from rat liver, originally developed by our group [18]. GPX activity was assayed spectrophotometrically according to the method of Tappel [19]. Citrate synthase (CS) activity was determined according to Shepherd and Garland [20]. The activities of both transaminases were determined on a kinetic enzyme analyzer at 37°C by the method of Karmen et al. [21]. The TBARS level was measured as described earlier [22]. The statistical significance of the data was assessed by Scheff6's test when significant F-values were obtained in ANOVA. When applicable, Student's t test was used. The significance was set at P < 0.05. Data in the text and tables are given as means + SEM.
3. Results Despite the fact that all the animals used were killed at the same age of 13 weeks, with one exception (on day 1 after cold exposure), the body weights of HE and CE rats were significantly lower than those of WA rats as was anticipated (Table 1). The changes in the
Table 4 Effect of temperature exposure on GPX activity (unit/mg protein) Tissue
Warm-acclimated
Liver Heat-exposed Cold-exposed Kidney Heat-exposed Cold-exposed Gastrocnemius muscle Heat-exposed Cold-exposed
14.6 ± 1.0
Temperature exposure (day) 1
7
14
28
11.6 ± 0.7 8.53±0.14 *
13.3 ± 0.9 11.0±0.6 *
13.7 ± 0.6 12.1 ± 1 . 1
16.7 _+ 0.6 11.3 ± 0 . 4 *
10.7 ± 0.8 10.0 ± 0.5
11.7 ± 0.5 * 9.82 ± 0.65
12.4 _+ 1.0 * 9.62 _+ 0.41
11.6±2.4 18.7 ± 3.9
13.4 ± 1 . 2 21.1 ± 5.1
11.1 _+1.3 16.7 ± 3.8
8.97 ± 0.50 8.22 _+ 0.26 8.89 ± 0.31 14.7 ±
1.1 18.9 ± 2 . 4 11.9 _+ 2.8
Values are means ± SEM. Numbers of rats same as in Table 1. *P < 0.05 compared with warm-acclimated rats.
164
T. Ookawara et al./ Pathophysiology 2 (1995) 161-166
Table 5 Effect of temperature exposure on CS activity(unit/mg protein) Temperature exposure (day) Tissue Warm-acclimated Liver Heat-exposed Cold-exposed Kidney Heat-exposed Cold-exposed Gastrocnemius muscle Heat-exposed Cold-exposed
1
7
14
28
0.053 ± 0.001 * 0.053 ± 0.005
0.048 _+0.003 0.052 +_0.006
0.040 _+0.002 0.088 ± 0.009 *
0.043 ± 0.003 0.090 ± 0.008 *
0.121 + 0.002 * 0.142 ± 0.006 *
0.127 ± 0.005 * 0.151 ± 0.008 *
0.119 ± 0.006 0.151 ± 0.008 *
0.107 ± 0.005 0.168 ± 0.007 *
0.135 +_0.009 0.155 ± 0.002 *
0.098 ± 0.004 * 0.146 _+0.006
0.109 ± 0.004 0.136 ± 0.008
0.067 +_0.005 * 0.138 ± 0.003
0.041 +_0.004 0.084 ± 0.011 0.124 ± 0.008
Values are means ± SEM. Numbers of rats same as in Table 1. *P < 0.05 compared with warm-acclimated rats.
tissue weights of both rats, however, were not always concomitant; that is, the liver weight in terms of weight per unit body was significantly decreased on day 28 after heat exposure, whereas it increased significantly on the same day after cold exposure (Table 1). Conversely, the kidney weight (per unit body weight) increased slightly but significantly on day 28 after heat exposure; marked increases in the tissue weight were noted on days 7, 14, and 28 after cold exposure. There were significant increases in the weight of gastrocnemius muscle on days 14 and 28 after heat exposure, but not after cold exposure (per unit body weight). Hematocrit level in W A rats was 45.3 + 0.9%. With one exception, hematocrit level did not vary substantially throughout the experimental period (data not shown); accordingly, it increased significantly only on day 1 after heat exposure (50.7 + 0.6%), indicating hemoconcentration. On day 1 after heat exposure, however, plasma levels of any parameters measured did not increase definitely. The activity of neither AST nor A L T in plasma seemed to change significantly during the experiment (data not shown), thereby suggesting that no hepatic insufficiency occurred. T B A R S level in plasma was significantly increased on days 7 and 14 after heat exposure but then was remarkably reduced to about one fifth of the W A level on day 28, whereas it decreased significantly on days 14 and 28 after cold exposure (Table 2). In the three tissues collected, no significant changes in T B A R S level were noted after heat or cold exposure. Immunoreactive Mn-SOD level in plasma from H E and CE rats decreased significantly on day 7 but both increased significantly on day 28 (Table 3). While MnSOD content in liver did not change substantially at any time after heat exposure, it increased significantly on days 14 and 28 after cold exposure. Meanwhile, Mn-SOD content in kidney was definitely elevated throughout the experimental period with one exception (although it also tended to increase on day 28 after heat exposure ( P < 0.10)). In gastrocnemius muscle it
increased significantly on days 7, 14, and 28 after heat exposure, and on day 1 after cold exposure, respectively. On the other hand, G P X activity in liver decreased significantly on days 1, 7, and 28 after cold exposure, whereas it did not change during heat exposure (Table 4). Conversely, the enzyme activity in kidney increased significantly on days 14 and 28 after heat exposure, not after cold exposure. No significant changes in G P X activity were found in gastrocnemius muscle. CS activity in liver increased significantly on day 1 in H E rats and on days 14 and 28 in CE rats (Table 5). It also increased significantly on days 1 and 7 in kidney from H E rats, and at any time in that from CE rats, which was in approximate agreement with the findings of Mn-SOD level in the same tissue. CS activity in gastrocnemius muscle decreased significantly on days 7 and 24 after heat exposure but increased on day 1 after cold exposure. 4. D i s c u s s i o n
It is true that rat produces and loses much more heat per unit of body mass than humans. LeBlanc [23] has explained, however, that when the increase in heat production due to cold is expressed as a percentage of the resting metabolic rate, the response is comparable for both species; at 5°C both rats and humans increase their oxygen consumption by about 100% and both are comfortable when the room temperature is about 25°C, that is, both species may be used as models for one another. Meanwhile, whether the same holds for heat remains vague. As described earlier, the three groups have revealed that temperature environment significantly increases lipid peroxidation in several tissues: in the erythrocytes of humans exposed to heat at 42°C for 10 min [13], in the brown adipose tissue of rats exposed to cold at 6°C for 21 days [11], and in the plasma of rabbits exposed to cold at 0°C (iced water) for 20 min [12]. In the
T. Ookawara et al. / Pathophysiology 2 (1995) 161-166
current study, however, although TBARS level in plasma increased significantly on days 7 and 14 after heat exposure, there were no definite changes in the three tissues investigated throughout the experimental period (which was in keeping with the finding of Mory et al. [14] that spontaneous lipid peroxidation rate was unchanged in the brown adipose tissue of rats exposed to cold for 14 days). It seemed, therefore, unlikely that the increases in TBARS level in plasma of HE rats on days 7 and 14 were derived from liver, kidney, or gastrocnemius muscle. These increases were then followed by the remarkable decreases on day 28, being in sharp contrast to the increases in plasma Mn-SOD level. Accordingly, it appeared that increased levels of Mn-SOD could attenuate or eliminate the increased levels of lipid peroxidation seen on day 14. Although the organ(s) where the phenomenon occurred remains obscure, the possibility exists that such Mn-SOD isoenzymes perhaps synthesized in response to the increased level of lipid peroxidation, were derived, in part, from the endothelial cells of blood capillaries. Indeed, MnSOD is localized to the mitochondrial matrix of the capillary vascular endothelial ceils relative to other cell types [24]. Moreover, the main localization of xanthine oxidase known to generate superoxide radical is in the vessel walls of most tissues including cardiac and skeletal muscle [25,26]. The increase in TBARS level in plasma on day 7 after heat exposure might be due to the decrease in Mn-SOD level, whereas the decrease in Mn-SOD level on day 7 after cold exposure did not increase lipid peroxidation. Increased catecholamine values during stress have been reported to be significantly correlated to the severity of myocardial cell necrosis, through formation of strongly cytotoxic free radicals at the intracellular level [27]. Actually, plasma noradrenaline remains elevated as long as rats are kept at 5°C [23]. In the current study, however, we could not observe any increase in TBARS level during cold exposure. Mn-SOD level in the three tissues collected seemed to increase during the experiment. The SOD isoenzyme which can be induced by the oxidative stress is Mn-SOD [28]. Therefore, the increases of Mn-SOD level suggested elevated free radical activity. Enhanced SOD activity accelerates H 2 0 2 formation. Scott et al. [29] have shown that the product of SOD, H 2 0 2 , is at least as hazardous as the substrate, 0 2. So, effective organismal defense against reactive oxygen species may require balanced increments in antioxidant enzymes, namely, increments in GPX as well as SOD in the current study. Except for in kidney of HE rats (on days 14 and 28), however, the GPX activity did not increase or rather decreased significantly. Notwithstanding, such changes in Mn-SOD and GPX did not lead to increased production of lipid peroxidation in the tissues. The same authors have demonstrated that although
165
exposure of rats to heat (39 _ 1°C) for 5 days decreases U 2 0 2 generation in mitochondria of the liver, but not
of the kidney or the heart [15], exposure of the animals to cold (9°C) for 24 h increases the generation of H 2 0 2 in liver mitochondria [30]. In the current study, especially on day 1 after cold exposure, it thus cannot be denied that catalase was an effective alternative as a scavenger of H 2 0 2. The heat conditions used in the current study (35°C) appeared to be relatively lower; nevertheless, our studies raised some interesting points concerning HE rats which developed increased levels of lipid peroxidation in plasma. Except for in the case of gastrocnemius muscle of HE rats, as was anticipated, the activity of CS, which is localized in the mitochondrial matrix like Mn-SOD, showed similar changes to Mn-SOD level. The site of nonshivering thermogenesis would appear to be principally skeletal muscles and brown adipose tissue, so that from day 7 to day 28 after heat exposure the CS activity in gastrocnemius muscle seemed to be decreased in order to economize in energy and heat production.
Acknowledgments The authors thank Mr. Masahiko Segawa for his expert technical assistance. This work was supported in part by a grant from The Chiyoda Life Foundation on Health and Welfare.
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HOPHYSIOL0¢( P ELSEVIER
Pathophysiology 2 (1995) 161-166
Changes in immunoreactive manganese-superoxide dismutase content in rat tissues under heat or cold environment Tomomi Ookawara a, Takako Kizaki a, Hitoshi Yamashita a, Shuji Oh-ishi a, Daizoh Saitoh Keiichiro Suzuki c, Naoyuki Taniguchi c, Hideki Ohno a,,
b,
a Department of Hygiene, National Defense Medical College, 3-2 Namiki Tokorozawa 359, Japan b Department of Traumatology and Emergency Medicine, National Defense Medical College, Tokorozawa 359, Japan c Department of Biochemistry, Osaka University Medical School, Suita 565, Japan Received 14 March 1995; accepted 4 April 1995
Abstract
Forty-one male Wistar rats were kept at 5°C [cold-exposed (CE) rats] or 35°C [heat-exposed (HE) rats] for 1, 7, 14, or 28 days. Seven rats reared at 25°C served as controls. After each exposure animals were sacrificed at the same age of 13 weeks, and blood, liver, kidney and gastrocnemius muscle were collected. Thiobarbituric acid-reactive substances (TBARS) level, an indicator of lipid peroxidation, in plasma was significantly increased on days 7 and 14 after heat exposure but then was remarkably reduced on day 28, whereas it decreased significantly on days 14 and 28 after cold exposure. In the three tissues collected, no significant changes in TBARS level were noted after heat or cold exposure. Immunoreactive Mn-superoxide dismutase (Mn-SOD) level in plasma from HE and CE rats decreased significantly on day 7 but both increased significantly on day 28. Except in the case of liver of HE rats, Mn-SOD level in the three tissues appeared to increase during the experiment. Meanwhile, glutathione peroxidase activity in liver decreased significantly on days 1, 7, and 28 after cold exposure. Conversely, the enzyme activity in kidney increased significantly on days 14 and 28 after heat exposure. These findings suggest that heat exposure imposes oxidative stress on the body, and that exposure to both heat and cold may affect the antioxidant enzyme system.
Keywords: Thiobarbituric acid-reactive substances; Oxidative stress; Glutathione peroxidase; Citrate synthase; Plasma
I. Introduction
Superoxide dismutase (SOD) is one of the most important enzymes in the antioxidant defense system. The enzyme scavenges superoxide anion (O2), which is the first product of oxygen radicals. Mammalian tissues contain two main forms of S O D : M n - S O D is present mostly in the mitochondrial matrix, whereas Cu,ZnSOD is predominantly localized in the cytoplasm (for review see [1,2]). It is currently believed that a vast array of pathologies, such as aging, cancer, artherosclerosis, diabetes, ischemia and cataracta, have possible links to oxygen radicals (for reviews see [1-5]). There is growing evidence, on the other hand, that even the
* Corresponding author. Fax: + 81-429-95-0638. 0928-4680/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0 9 2 8 - 4 6 8 0 ( 9 5 ) 0 0 0 2 8 - 3
physiological state, e.g., physical exercise [2,4,6-8] and high altitude [9,10], imposes oxidative stress on the body due to oxygen free radical generation (including an increase in lipid peroxidation in various tissues). Moreover, it has recently been demonstrated that exposure to cold and heat increases lipid peroxidation in several tissues [11-13]. Actually, only limited information, however, could be obtained concerning this phenomenon. In addition, it seems unlikely that only identical observations have been made; for example, spontaneous lipid peroxidation rate was found unchanged in the brown adipose tissue of rats chronically exposed to cold [14]. Further, exposure of rats to heat decreased H 2 0 2 generation in mitochondria of the liver, but not of the kidney or the heart [15]. Chance et al. [16] have reported that mitochondria are one of the main intracellular sources of O 2 and H 2 0 2 even under physiological conditions. Mitochon-
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162
Table 1 Changes in body and tissue weights of temperature-exposed rats Group
Body weight
Liver weight
Initial (g)
Final (g)
(g)
257 252 228 194
_+ 7 +_ 6 _+ 3 _+ 4
233 224 206 203
± 5 + 4 _+_5 ± 2
* * * *
9.98 8.98 8.52 8.10
267 248 241 179
± 2 _+ 6 ± 3 ± 7
251 223 206 203
___1 + 6 * ___4 * + 5 *
10.4 9.77 9.21 10.0
Kidney weight (g/lO0 g body weight)
(g)
* * * *
4.29 4.09 4.13 3.99
+ ± ± ±
0.10 0.13 0.10 0.07 *
0.915 0.857 0.814 0.833
± 0.1 _+ 0.36 * ± 0.33 * _+ 0.2 *
4.15 4.37 4.47 4.95
± ± ± ±
0.04 0.05 0.11 0.09 *
0.943 1.05 1.04 1.09
Gastrocnemius muscle weight (g/lO0 g body weight)
(g)
(g/lO0 g body weight)
_+ 0.025 _+ 0.013 ± 0.030 * +_ 0.018 *
0.393 0.383 0.395 0.411
± 0.008 _+ 0.007 ± 0.009 ± 0.007 *
1.26 1.20 1.14 1.16
_+ 0.02 ± 0.04 + 0.03 * _+ 0.03 *
0.541 0.534 0.551 0.571
_+ 0.005 ± 0.007 ± 0.007 * _+ 0.010 *
_+ 0.022 ± 0.04 ± 0.02 _+ 0.02 *
0.375 0.470 0.506 0.539
± ± ± ±
1.33 1.14 1.08 1.03
± ± ± ±
0.530 0.511 0.526 0.508
± 0.014 _+ 0.009 _+ 0.005 ± 0.012
Heat-exposed 1 day (5) 7 days (5) 14 days (5) 28 days (6)
_+ 0.30 ± 0.37 _+ 0.37 ± 0.19
Cold-exposed 1 day (5) 7 days (5) 14 days (5) 28 days (5)
Warm-acclimated (7)
255 ± 3
11.4 +_ 0.4
4.46 ± 0.12
0.947 _+ 0.031
0.009 0.004 * 0.011 * 0.012 *
0.371 ± 0.010
0.03 0.03 * 0.02 * 0.02 *
1.33 ± 0.04
0.519 _+ 0.008
Values are means + SEM. All rats were killed at the same age of 13 weeks. Numbers in parentheses are numbers of animals. * P < 0.05 compared with warm-acclimated rats.
active Mn-SOD level and GPX activity in several tissues of rats.
dria are probably faced with the elevated rate of oxygen utilization under conditions of increased metabolism of the body such as physical exercise, suggesting an increase in oxygen free radical generation in the cell organelle. It has been estimated that 80% of the totally formed superoxide radicals in the mitochondria may be reduced by Mn-SOD [16]. Activity assay generally utilized for SOD measurement may lack specificity and may be subject to interference from other factors. As compared with enzymatic methods, therefore, immunochemical assay methods for SOD appear to be more reliable and reproducible because the determinations are specific to the protein moiety [1,2]. Glutathione peroxidase (GPX), like SOD, is intracellularly located in the cytosol and mitochondrial matrix and detoxifies oxygen-reactive radicals by catalyzing the formation of H 2 0 2 derived from 0 2. The current study was undertaken to investigate the effects of temperature exposure with the passage of time on not only lipid peroxidation but also immunore-
2. Materials and methods
Forty-eight male Wistar rats were divided into three groups. Animals of the first group (n = 21) were reared at an ambient temperature of 35°C and relative humidity of 40% for 1, 7, 14, or 28 days in a climatic room with lighting from 7:00 to 19:00 h daily in individual cage. These rats, which were referred to as heat-exposed (HE) rats, had free access to the standard laboratory diet (Oriental MF, Oriental Yeast Co., Tokyo) and tap water. The animals were cared for in accordance with the Guiding Principles for the Care and Use of Animals approved by the Council of the Physiological Society of Japan, based upon the Helsinki Declaration, 1964. The second group (n = 20) was kept at 5°C during the same period of time as for heat expo-
Table 2 Effect of temperature exposure on TBARS level Tissue
Warm-acclimated
Plasma ( n m o l / m l ) Heat-exposed Cold-exposed Liver ( n m o l / m g protein) Heat-exposed Cold-exposed Kidney ( n m o l / m g protein) Heat-exposed Cold-exposed Gastrocnemius muscle ( n m o l / m g protein) Heat-exposed Cold-exposed
47.5 _+ 6.1
Temperature exposure (day) 1
7
14
28
52.4 ± 12.2 28.3 ± 3.9
75.0 ± 9.6 * 52.8 +2.5
97.1 ± 11.3 " 23.1 _+ 4.4 *
8.56 ± 0.94 * 22.4 +_2.2 *
0.74 ± 0.06 0.94 ± 0.06
0.76 ± 0.03 0.73 _+0.04
0.72 _+ 0.04 0.72 _+ 0.06
0.80 ± 0.03 0.80 _+ 0.03
0.74 ± 0.08 1.16 ± 0.23
0.86 +_0.03 0.79 _+0.08
0.95 _+ 0.05 0.70 _+ 0.10
1.04 _+ 0.05 0.92 +_ 0.08
1.78 _+ 0.07 1.09 + 0.05
1.07 ± 0.03 1.38 _+ 0.04
1.38 ± 0.09 1.72 +_ 0.14
1.60 ± 0.17 2.01 _+ 0.14
0.85 ± 0.03
0.85 _+ 0.05
1.47 _+ 0.18
Values are means + SEM. Numbers of rats same as in Table 1. *P < 0.05 compared with warm-acclimated rats.
T. Ookawara et al./ Pathophysiology 2 (1995) 161-166
163
Table 3 Effect of temperature exposure on Mn-SOD level Tissue
Warm-acclimated
Plasma (ng/ml) Heat-exposed Cold-exposed Liver (/zg/mg protein) Heat-exposed Cold-exposed Kidney (/zg/mg protein) Heat-exposed Cold-exposed Gastrocnemius muscle (/zg/mg protein) Heat-exposed Cold-exposed
Temperature exposure (day) 1
7
14
28
1.50 ± 0.09 1.75 ± 0.22
0.94 ± 0.02 * 1.14 ± 0.05 *
1.64 ± 0.34 1.41 ± 0.36
3.30 _+ 0.12 * 2.67 _+ 0.16 *
0.36 ± 0.04 0.41 ± 0.05
0.34 ± 0.03 0.39 ± 0.06
0.33 _+ 0.03 0.53 ± 0.07 *
0.27 _+ 0.03 0.50 ± 0.02 *
0.63 ± 0.03 * 0.61 ± 0.02 *
0.68 + 0.03 ~ 0.72 _+ 0.08 *
0.65 _+ 0.03 * 0.70 ± 0.07 *
0.60 ± 0.04 0.90 ± 0.05 *
0.19 _+ 0.02 0.25 _+ 0.01 *
0.23 ± 0.01 * 0.18 ± 0.01
0.23 ± 0.01 * 0.20 ± 0.01
0.23 ± 0.01 * 0.17 ± 0.01
1.84 ± 0.16
0.27 ± 0.05
0.38 ± 0.08
0.15 ± 0.02
Values are means ± SEM. Numbers of rats same as in Table 1. *P < 0.05 compared with warm-acclimated rats.
sure [cold-exposed (CE) rats]. The third group (n = 7), comprising control [warm-acclimated (WA)] rats, was kept at the thermoneutral temperature of 25°C. After each exposure animals were killed by decapitation at the same age of 13 weeks, and blood, liver, kidney and gastrocnemius muscle were collected. Hematocrit level was measured by a microcapillary tube technique. Each plasma sample was used for measuring Mn-SOD, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS), an indicator of lipid peroxidation. The three tissues were quickly frozen in liquid nitrogen. They were stored at - 8 0 ° C for later analysis. A portion of tissues were thawed in an ice-cold medium containing 0.25 M sucrose, 10 mM 2-amino-2-hydroxymethyl-l,3-propanediol (Tris), and 0.1 mM EDTA (pH 7.4, wt/vol 1:9), minced, and homogenized on ice in brief bursts by a Polytron homogenizer (Kinematika, Luzern). The homogenate was centrifugated at 750 g ~4°C) for 15 min, and the supernatant was used for various assays. Protein content was determined by the method of Lowry et al. [17]. Measurement of immunoreactive Mn-SOD was made by an enzyme-linked immunosorbent assay, using
specific polyclonal antibody against Mn-SOD isoenzyme purified from rat liver, originally developed by our group [18]. GPX activity was assayed spectrophotometrically according to the method of Tappel [19]. Citrate synthase (CS) activity was determined according to Shepherd and Garland [20]. The activities of both transaminases were determined on a kinetic enzyme analyzer at 37°C by the method of Karmen et al. [21]. The TBARS level was measured as described earlier [22]. The statistical significance of the data was assessed by Scheff6's test when significant F-values were obtained in ANOVA. When applicable, Student's t test was used. The significance was set at P < 0.05. Data in the text and tables are given as means + SEM.
3. Results Despite the fact that all the animals used were killed at the same age of 13 weeks, with one exception (on day 1 after cold exposure), the body weights of HE and CE rats were significantly lower than those of WA rats as was anticipated (Table 1). The changes in the
Table 4 Effect of temperature exposure on GPX activity (unit/mg protein) Tissue
Warm-acclimated
Liver Heat-exposed Cold-exposed Kidney Heat-exposed Cold-exposed Gastrocnemius muscle Heat-exposed Cold-exposed
14.6 ± 1.0
Temperature exposure (day) 1
7
14
28
11.6 ± 0.7 8.53±0.14 *
13.3 ± 0.9 11.0±0.6 *
13.7 ± 0.6 12.1 ± 1 . 1
16.7 _+ 0.6 11.3 ± 0 . 4 *
10.7 ± 0.8 10.0 ± 0.5
11.7 ± 0.5 * 9.82 ± 0.65
12.4 _+ 1.0 * 9.62 _+ 0.41
11.6±2.4 18.7 ± 3.9
13.4 ± 1 . 2 21.1 ± 5.1
11.1 _+1.3 16.7 ± 3.8
8.97 ± 0.50 8.22 _+ 0.26 8.89 ± 0.31 14.7 ±
1.1 18.9 ± 2 . 4 11.9 _+ 2.8
Values are means ± SEM. Numbers of rats same as in Table 1. *P < 0.05 compared with warm-acclimated rats.
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T. Ookawara et al./ Pathophysiology 2 (1995) 161-166
Table 5 Effect of temperature exposure on CS activity(unit/mg protein) Temperature exposure (day) Tissue Warm-acclimated Liver Heat-exposed Cold-exposed Kidney Heat-exposed Cold-exposed Gastrocnemius muscle Heat-exposed Cold-exposed
1
7
14
28
0.053 ± 0.001 * 0.053 ± 0.005
0.048 _+0.003 0.052 +_0.006
0.040 _+0.002 0.088 ± 0.009 *
0.043 ± 0.003 0.090 ± 0.008 *
0.121 + 0.002 * 0.142 ± 0.006 *
0.127 ± 0.005 * 0.151 ± 0.008 *
0.119 ± 0.006 0.151 ± 0.008 *
0.107 ± 0.005 0.168 ± 0.007 *
0.135 +_0.009 0.155 ± 0.002 *
0.098 ± 0.004 * 0.146 _+0.006
0.109 ± 0.004 0.136 ± 0.008
0.067 +_0.005 * 0.138 ± 0.003
0.041 +_0.004 0.084 ± 0.011 0.124 ± 0.008
Values are means ± SEM. Numbers of rats same as in Table 1. *P < 0.05 compared with warm-acclimated rats.
tissue weights of both rats, however, were not always concomitant; that is, the liver weight in terms of weight per unit body was significantly decreased on day 28 after heat exposure, whereas it increased significantly on the same day after cold exposure (Table 1). Conversely, the kidney weight (per unit body weight) increased slightly but significantly on day 28 after heat exposure; marked increases in the tissue weight were noted on days 7, 14, and 28 after cold exposure. There were significant increases in the weight of gastrocnemius muscle on days 14 and 28 after heat exposure, but not after cold exposure (per unit body weight). Hematocrit level in W A rats was 45.3 + 0.9%. With one exception, hematocrit level did not vary substantially throughout the experimental period (data not shown); accordingly, it increased significantly only on day 1 after heat exposure (50.7 + 0.6%), indicating hemoconcentration. On day 1 after heat exposure, however, plasma levels of any parameters measured did not increase definitely. The activity of neither AST nor A L T in plasma seemed to change significantly during the experiment (data not shown), thereby suggesting that no hepatic insufficiency occurred. T B A R S level in plasma was significantly increased on days 7 and 14 after heat exposure but then was remarkably reduced to about one fifth of the W A level on day 28, whereas it decreased significantly on days 14 and 28 after cold exposure (Table 2). In the three tissues collected, no significant changes in T B A R S level were noted after heat or cold exposure. Immunoreactive Mn-SOD level in plasma from H E and CE rats decreased significantly on day 7 but both increased significantly on day 28 (Table 3). While MnSOD content in liver did not change substantially at any time after heat exposure, it increased significantly on days 14 and 28 after cold exposure. Meanwhile, Mn-SOD content in kidney was definitely elevated throughout the experimental period with one exception (although it also tended to increase on day 28 after heat exposure ( P < 0.10)). In gastrocnemius muscle it
increased significantly on days 7, 14, and 28 after heat exposure, and on day 1 after cold exposure, respectively. On the other hand, G P X activity in liver decreased significantly on days 1, 7, and 28 after cold exposure, whereas it did not change during heat exposure (Table 4). Conversely, the enzyme activity in kidney increased significantly on days 14 and 28 after heat exposure, not after cold exposure. No significant changes in G P X activity were found in gastrocnemius muscle. CS activity in liver increased significantly on day 1 in H E rats and on days 14 and 28 in CE rats (Table 5). It also increased significantly on days 1 and 7 in kidney from H E rats, and at any time in that from CE rats, which was in approximate agreement with the findings of Mn-SOD level in the same tissue. CS activity in gastrocnemius muscle decreased significantly on days 7 and 24 after heat exposure but increased on day 1 after cold exposure. 4. D i s c u s s i o n
It is true that rat produces and loses much more heat per unit of body mass than humans. LeBlanc [23] has explained, however, that when the increase in heat production due to cold is expressed as a percentage of the resting metabolic rate, the response is comparable for both species; at 5°C both rats and humans increase their oxygen consumption by about 100% and both are comfortable when the room temperature is about 25°C, that is, both species may be used as models for one another. Meanwhile, whether the same holds for heat remains vague. As described earlier, the three groups have revealed that temperature environment significantly increases lipid peroxidation in several tissues: in the erythrocytes of humans exposed to heat at 42°C for 10 min [13], in the brown adipose tissue of rats exposed to cold at 6°C for 21 days [11], and in the plasma of rabbits exposed to cold at 0°C (iced water) for 20 min [12]. In the
T. Ookawara et al. / Pathophysiology 2 (1995) 161-166
current study, however, although TBARS level in plasma increased significantly on days 7 and 14 after heat exposure, there were no definite changes in the three tissues investigated throughout the experimental period (which was in keeping with the finding of Mory et al. [14] that spontaneous lipid peroxidation rate was unchanged in the brown adipose tissue of rats exposed to cold for 14 days). It seemed, therefore, unlikely that the increases in TBARS level in plasma of HE rats on days 7 and 14 were derived from liver, kidney, or gastrocnemius muscle. These increases were then followed by the remarkable decreases on day 28, being in sharp contrast to the increases in plasma Mn-SOD level. Accordingly, it appeared that increased levels of Mn-SOD could attenuate or eliminate the increased levels of lipid peroxidation seen on day 14. Although the organ(s) where the phenomenon occurred remains obscure, the possibility exists that such Mn-SOD isoenzymes perhaps synthesized in response to the increased level of lipid peroxidation, were derived, in part, from the endothelial cells of blood capillaries. Indeed, MnSOD is localized to the mitochondrial matrix of the capillary vascular endothelial ceils relative to other cell types [24]. Moreover, the main localization of xanthine oxidase known to generate superoxide radical is in the vessel walls of most tissues including cardiac and skeletal muscle [25,26]. The increase in TBARS level in plasma on day 7 after heat exposure might be due to the decrease in Mn-SOD level, whereas the decrease in Mn-SOD level on day 7 after cold exposure did not increase lipid peroxidation. Increased catecholamine values during stress have been reported to be significantly correlated to the severity of myocardial cell necrosis, through formation of strongly cytotoxic free radicals at the intracellular level [27]. Actually, plasma noradrenaline remains elevated as long as rats are kept at 5°C [23]. In the current study, however, we could not observe any increase in TBARS level during cold exposure. Mn-SOD level in the three tissues collected seemed to increase during the experiment. The SOD isoenzyme which can be induced by the oxidative stress is Mn-SOD [28]. Therefore, the increases of Mn-SOD level suggested elevated free radical activity. Enhanced SOD activity accelerates H 2 0 2 formation. Scott et al. [29] have shown that the product of SOD, H 2 0 2 , is at least as hazardous as the substrate, 0 2. So, effective organismal defense against reactive oxygen species may require balanced increments in antioxidant enzymes, namely, increments in GPX as well as SOD in the current study. Except for in kidney of HE rats (on days 14 and 28), however, the GPX activity did not increase or rather decreased significantly. Notwithstanding, such changes in Mn-SOD and GPX did not lead to increased production of lipid peroxidation in the tissues. The same authors have demonstrated that although
165
exposure of rats to heat (39 _ 1°C) for 5 days decreases U 2 0 2 generation in mitochondria of the liver, but not
of the kidney or the heart [15], exposure of the animals to cold (9°C) for 24 h increases the generation of H 2 0 2 in liver mitochondria [30]. In the current study, especially on day 1 after cold exposure, it thus cannot be denied that catalase was an effective alternative as a scavenger of H 2 0 2. The heat conditions used in the current study (35°C) appeared to be relatively lower; nevertheless, our studies raised some interesting points concerning HE rats which developed increased levels of lipid peroxidation in plasma. Except for in the case of gastrocnemius muscle of HE rats, as was anticipated, the activity of CS, which is localized in the mitochondrial matrix like Mn-SOD, showed similar changes to Mn-SOD level. The site of nonshivering thermogenesis would appear to be principally skeletal muscles and brown adipose tissue, so that from day 7 to day 28 after heat exposure the CS activity in gastrocnemius muscle seemed to be decreased in order to economize in energy and heat production.
Acknowledgments The authors thank Mr. Masahiko Segawa for his expert technical assistance. This work was supported in part by a grant from The Chiyoda Life Foundation on Health and Welfare.
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